Hindawi Publishing CorporationEnzyme ResearchVolume 2013 Article ID 869062 9 pageshttpdxdoiorg1011552013869062

Research ArticleLaccase Production from a Temperature and pH Tolerant FungalStrain of Trametes hirsuta (MTCC 11397)

Kusum Dhakar and Anita Pandey

G B Pant Institute of Himalayan Environment and Development Kosi-Katarmal Almora Uttarakhand 263 643 India

Correspondence should be addressed to Anita Pandey anitagbpihednicin

Received 7 February 2013 Revised 19 March 2013 Accepted 19 March 2013

Academic Editor Jose Miguel Palomo

Copyright copy 2013 K Dhakar and A Pandey This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Laccase production by a temperature and pH tolerant fungal strain (GBPI-CDF-03) isolated from a glacial site in IndianHimalayanRegion (IHR) has been investigated The fungus developed white cottony mass on potato dextrose agar and revealed thread-like mycelium under microscope ITS region analysis of fungus showed its 100 similarity with Trametes hirsuta The fungustolerated temperature from 4 to 48∘Cplusmn 2 (25∘C opt) and pH 3ndash13 (5ndash7 opt) Molecular weight of laccase was determinedapproximately 45 kDa by native PAGE Amplification of laccase gene fragment (corresponding to the copper-binding conserveddomain) contained 200 bp The optimum pH for laccase production at optimum growth temperature was determined between55 and 75 In optimization experiments fructose and ammonium sulfate were found to be the best carbon and nitrogen sourcesrespectively for enhancing the laccase production Production of laccase was favored by high carbonnitrogen ratio Addition ofCuSO

4

(up to 10mM) induced laccase production up to 2-fold in case of 04mM concentration Addition of organic solvents alsoinduced the production of laccase acetone showed the highest (2-fold) induction The study has implications in bioprospecting ofecologically resilient microbial strains

1 Introduction

Laccases (phenol oxidases EC 11032) also known asmulticopper blue oxidases belong to the oxidoreductasegroup of enzymes Biochemically they are glycoproteinscarrying molecular mass between 50 kDa and 130 kDa [1]Fungi belonging to ascomycetes deuteromycetes and basid-iomycetes are known to produce laccases of ecological aswell as biotechnological importance such as biodegradationand bioremediation [1ndash4] In addition laccases are alsoresponsible for various physiological functions in fungi [5]Due to their broad specificity toward substrate they canoxidize a range of chemical compounds leading to variousindustrial applications [6]

Enhancement of laccase production by modifying thenutritional and physiological conditions during cultivationof promising fungi is a prerequisite for their optimum uti-lization at industrial scale Besides nutritional supplementsinducers like organic solvents and metal ions also playimportant role in production of laccases [3 7 8] Isolation ofnew microbial strains of biotechnological applications from

various ecological habitats is a prerequisite for industrialgrowth The fungi capable of producing laccase at widertemperature and pH range are likely to play important rolein biodegradation under low temperature environments Inthe present study a temperature and pH tolerant fungusisolated from a glacial site in IndianHimalayan Region (IHR)has been investigated for production of laccase at differentphysicochemical and nutritional conditions

2 Materials and Methods

21 Fungal Strain (GBPI-CDF-03) The fungus was originallyisolated from the soil collected from alpine zone of Pindariglacier region (33∘51015840-30∘101015840N to 70∘481015840-79∘521015840E) coveringaltitudes between 3000 and 4500m amsl (above mean sealevel) of IHRThemean monthly temperature at the site hasbeen reported between 55∘C (January) and 201∘C (August)the soil pH ranged from 45 to 51 The description of sitehas been reported earlier [9ndash11] The soil was serially dilutedand appropriate dilution was plated using potato dextroseagar (PDA) The pure fungus obtained was maintained on

2 Enzyme Research

PDA slants following subculturing (2 months interval) at4∘C in the Microbial Culture Collection established in theMicrobiology Laboratory of the Institute Fresh culture wasraised for the experimentation

22 Identification andCharacterization of the Fungus Colonymorphology of the fungus was recorded on 5 days old culturegrown on PDA at 25∘C Four other media namely Myco-logical agar Czapek dox agar Sabouraud dextrose maltoseagar and Vegetable juice 8 agar (all from Hi Media) werealso used for recording the observations on the growth offungus Microscopic observations were recorded followinglactophenol cotton blue staining (Nikon-Eclipse 50i Japan)Molecular identification was based on ITS (ITS1-58S-ITS2)region analysis using ITS1 and ITS4 primers [12] The fungalisolate and the gene sequence have been accessioned inMicrobial Type Culture Collection and Gene Bank Instituteof Microbial Technology Chandigarh India and NCBIrespectively The phylogenetic tree was made by using NJmethod with the bootstrap value 1000 MEGA 40 was usedfor the phylogenetic analyses (courtesy A Sharma MCCNCCS Pune India)

Temperature tolerance was determined on PDA by incu-bating the fungal strain between 4 and 50∘C (4 9 14 2535 45 and 50∘C) up to two weeks The pH tolerance wasdetermined between 1 to 14 pH (with 05 pH interval) byincubating the fungus at 25∘C for oneweek on PD agarbroth

23 Medium and Culture Condition for Laccase ProductionLaccase production was carried out using modified Kirk andFarrell [13] medium containing (gL) 20 g malt extract 20 gglucose 20 g NH

4NO3 026 g Na

2HPO4 026 g KH

2PO4

05 g MgSO4(7H2O) 001 g CuSO

4(5H2O) 0006 g CaCl

2

(2H2O) 0005 g FeSO

4(7H2O) 00005 g ZnSO

4(7H2O)

000002 g Na2MoO4 000009 g MnSO

4sdotH2O and 000007 g

H3BO3 Qualitative plate based estimation was done on

the aforesaid medium supplemented with 030 g ABTS (221015840-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid) Theplate assays were carried out at 6 different temperatures (4 915 25 35 and 45∘C) Ligninolytic efficiency was determinedas ABTS zone to fungal colony diameter ratio lowast 100 DMPsyringaldazine and guaiacol (separately) were also used inplate assays for determination of the oxidizing ability oflaccase In quantitative estimations 50mL medium was pre-pared in 250mL Erlenmeyer flasks without supplementingABTS The pH of the medium was set at 55 plusmn 02 beforeautoclaving Autoclaved medium was inoculated with 5mmdisc (per flask) of 6-day old fungus culture The experimentswere conducted under static conditions

24 Determination ofMolecularWeight of Laccase The crudeenzyme (filtrate) was precipitated with ammonium sulfateup to 70 saturation and centrifuged at 15000 lowast g RCF for10min at 4∘C Pellets were resuspended in citrate-phosphate(pH = 26) buffer PAGE without SDS was carried outfollowing standard method described by Laemmli [14] using15 separating and 4 stacking gel Polyacrylamide gel was

stained with 10 of ABTS solution prepared in citrate-phosphate buffer (pH 26) following 1 h incubation at roomtemperature Pre-stained protein marker (Puregene) wasused for determination of molecular weight of the enzyme

25 Molecular Studies on Partial Sequence of Laccase GeneLaccase gene fragment (corresponding to conserved copperbinding domain) was amplified by PCR (BIO-RAD USA)DNA isolation was done by the method given by Voigt et al[15] PCR amplification was carried out using basidiomycetesspecific laccase primers Cu1F and Cu2R [16] 25 120583L PCRreaction contained 2X reaction buffer 125120583L sterile water62 120583L dNTP (10mM of each dNTP) 25120583L MgCl

2(25mM)

15 120583L forward and reverse primer 10 120583L (10 pmole120583L each)and Taq DNA polymerase (3U120583L) 03120583L (dNTP MgCl

2

and Taq DNA polymerase) from Fermentas Life ScienceThe PCR conditions were initial denaturation 94∘C for3min denaturation 94∘C for 30 s annealing 48∘C for 45 sextension 72∘C for 3min final extension 72∘C for 10minand total cycles 35 Molecular weight of amplified productwas analyzed on agarose gel (08)

26 Effect of Physicochemical and Nutritional Parameters andOther Supplements on Laccase Production Production oflaccase was determined at 4 temperatures (15 25 35 and45∘C) at every 3rd day up to 15 days of incubation Laccaseproductionwith respect to pHwas determined by inoculatingthe broth set at different pH ranging from 35 to 115 (with aninterval of 2 units)

Six carbon glucose (control) fructose maltose sucrosestarch and cellulose 5 nitrogen sources (ammonium nitrate(control) ammonium sulfate ammonium ferrous sulfatepotassium nitrate and urea) and one set without inorganicnitrogen at 02 levelwere used for enhancing the productionof laccase All the carbon and nitrogen sources were from HiMedia Effect of the best carbon and nitrogen sources wasfurther determined by increasing the concentration level upto 10

In other supplements category 4 organic solvents namelymethanol ethanol isopropanol and acetone (02supple-mented at day 4 of incubation) were tested as inducers Effectof CuSO

4concentration (02 to 10mMsupplemented at day

4 of incubation) was also determinedThese experiments (effect of pH carbon sources nitrogen

sources CuSO4 and solvents) were carried out at 25∘C

following 12 days of incubation under static conditions

27 Enzyme Assay Laccase activity was determined in termsof oxidation of ABTS using spectrophotometer (AmershamBioscience Ultrospec 2100 pro) [17] The fungal culture wasfiltered with Whatman No 1 filter paper and used as crudeenzyme Mycelium collected and dried at 65∘C for 72 h wasused for determination of biomass Reaction mixture wasprepared with citrate-phosphate buffer (pH = 26) 20mMABTS and the crude enzyme Absorbance was recorded at420 nm following 2min of incubation at room temperatureLaccase activity was calculated using extinction coefficient =36000Mminus1 cmminus1 [18] 1 unit of enzyme activity is defined as

Enzyme Research 3

1 120583Mof ABTS oxidized per min Protein was also determinedfollowing the method of Lowry et al [19]

3 Results

The fungus (GBPI-CDF-03) developed white cottony masson all the 5 media used with growth being more compacton Vegetable juice 8 agar Microscopy revealed presenceof thread-like septate mycelium without any reproductivestructures (Figure 1(a)) The fungus tolerated wide rangeof temperature (4ndash48 plusmn 2∘C optimum 25 plusmn 2∘C) and pH(30ndash130 pH optimum between 5 and 7) ITS region basedmolecular identification revealed its maximum similaritywith Trametes hirsuta Growth requirements along with thephenotypic and genotypic characters and the phylogeneticrelationship of the fungus are presented in Table 1 andFigure 2 The accessions given to fungus and the nucleotidesequence are MTCC 11397 and JX910367 respectively

In plate assays conducted at optimumgrowth conditionsthe fungus oxidized ABTS and developed green colouraround the colony indicating the production of laccase(Figure 1(b)) The fungus also produced laccase through oxi-dation of other three substrates namely guaiacol syringal-dazine and DMP ABTS however was found to exhibithighest sensitivity towards laccase activity At suboptimaltemperatures (4 9 15 35 and 45∘C) maximum ligninolyticefficiency (ABTS zone to colony dia) was observed at4∘C The efficiency for production of ligninolytic enzymeswas found to be inversely proportional to the temperature(Table 2) Molecular weight of laccase was determined to beapproximately 45 kDa (Figure 3) while the PCR amplifiedsequence of laccase gene fragment (corresponding to copperbinding domain) was obtained approximately of 200 bp sizeIn quantitative estimations conducted at 15 25 35 and 45∘Cproduction of laccase fungal biomass and total proteincontent varied with culture conditions Maximum laccaseproduction (3945UL) was recorded at 35∘C on day 12 ofincubation In this case the highest biomass (228mg50mL)and maximum protein (511 120583gmL) were recorded on day12 and 15 respectively Laccase production was recordedminimum (2697UL) at 25∘Cwithmaximum fungal biomass(256mg50mL) and protein concentration (419120583gmL)at day 12 of incubation Slightly increased production oflaccase (272UL) was recorded at 15∘C with fungal biomass(144mg50mL) and protein content (662120583gmL) at day12 of incubation (Table 3 Figures 4(a) 4(b) and 4(c)) Theactivity at 45∘C was estimated to be 2821 UL with very littlebiomass (80mg50mL) and protein content (168 120583gmL) atday 12 of incubation (not presented in the Figures)While theproduction of laccase was recorded between pH 35 and 115the maximum production (270UL) along with biomass andthe protein content was favoured at pH between 55 and 75(Table 4 Figure 5)

Among 6 carbon sources (02) fructose was foundto increase the production of laccase along with higherprotein content and reduced fungal biomass in comparisonto control (Table 5 Figure 6(a)) The laccase productionincreased with the increase of fructose concentration upto 10 maximum being at 04 concentration of fructose

Table 1 Morphological and molecular characteristics of the fungus(GBPI-CDF-03)

Character DescriptionColony morphology(on PD agar)

White cottony mass with noexudation or pigmentation

Microscopic featuresThread like mycelium with septashowing the characteristic feature ofupper fungi

Growth characters

Temperature requirement between4 and 48∘C (optimum 25∘C) pHrequirement between 3 and 13(optimum 5ndash7)

Phylogenetic relationship(ITS region analysis)Culture accession noNucleotide sequenceaccession no

Maximum similarity (100) withTrametes hirsuta (JF439511)MTCC 11397JX910367

Table 2 Ligninolytic efficiency of the fungus (GBPI-CDF-03) atdifferent temperatures

Temperature(∘C)

Incubationtime(days)

ABTS zonedia (mm)(ZD)

Fungalcolony

dia (mm)(CD)

Ligninolyticefficiency =ZDCD lowast 100

4 21 22 5 4409 21 19 7 27115 14 15 10 15025 7 10 50 2035 7 12 46 2645 7 8 15 53Dia diameter

(2 1 carbonnitrogen ratio) (Table 6) However the increasein biomass and protein continued up to 10 fructose concen-tration Other carbon sources (maltose sucrose starch andcellulose) were found to be inhibitory for laccase productionas well as the production of fungal biomass Cellulose wasfound to be the most inhibitory carbon source followed bystarch sucrose and maltose Among five nitrogen sourcesammonium sulfate was found to be the best at 02 forproduction of laccase and the protein content (Table 5Figure 6(b)) Further increment in ammonium sulfate con-centration was found to be inhibitory for laccase production(Table 6) Effect of ammonium ferrous sulfate was found to beat par to the control (ammonium nitrate) Presence of othernitrogen sources (urea and potassium nitrate) and also thecase without any nitrogen source were found to reduce theproduction of laccase

Addition of CuSO4increased laccase production along

with production of protein content up to 2-fold in compari-son to control maximum being in case of 04mM concentra-tion Further increment in CuSO

4(up to 10mM) resulted in

decline of laccase production although the decreased valueswere higher in comparison to control (Figure 6(c) Table 7)Other inducers the low molecular weight organic solventsat 02 concentration were also found to enhance the

4 Enzyme Research

(a) (b)

Figure 1 (a) Microscopic features of the fungus (Bar = 2 120583m) (b) laccase production on ABTS plate

HM595571 Trametes spFJ550367 Trametes hirsutaAB638346 Trametes hirsutaJF439511 Trametes hirsuta

GBPI-CDF-03

HM004553 Stereum hirsutumJN164953 Trametes hirsutaGU062274 Trametes hirsutaJN164942 Trametes hirsuta

JN198490 Trametes versicolorJQ673021 Trametes versicolorJX501313 Trametes versicolorJQ673022 Trametes versicolor

JQ781852 Ganoderma lucidumJQ781851 Ganoderma lucidumJQ520188 Ganoderma lucidum

AB735969 Neofavolus alveolarisAB735967 Neofavolus alveolarisAB735973 Favolus acervatusAB735971 Favolus acervatusAB735975 Favolus roseus

AB735977 Favolus brasiliensisJQ403610 Irpex lacteusJX077039 Irpex lacteusJX559574 Irpex lacteusJN588552 Irpex lacteus

EU232218 Laetiporus sulphureusAY089742 Laetiporus sulphureus

GU203516 Laetiporus sulphureusEF088657 Laetiporus sulphureus

JX501310 Laetiporus sulphureus

4247

100

100

100

99

99

10070

100

65

41

5897

95

92

5872

50

93

64

10067

51

005

AB735979 Neofavolus cremeoalbidusAB735981 Neofavolus cremeoalbidus

Figure 2 Phylogenetic relationship of the fungus

Enzyme Research 5

Table 3 Production of fungal biomass and protein content at different temperatures

Incubation time(days)

Temperature (∘C)15 25 35

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

3 41 plusmn 05 364 plusmn 31 86 plusmn 16 344 plusmn 66 53 plusmn 11 346 plusmn 50

6 76 plusmn 08 446 plusmn 107 130 plusmn 32 369 plusmn 82 136 plusmn 20 394 plusmn 40

9 115 plusmn 20 546 plusmn 44 179 plusmn 62 329 plusmn 41 187 plusmn 44 429 plusmn 80

12 144 plusmn 22 662 plusmn 57 256 plusmn 42 419 plusmn 67 228 plusmn 28 436 plusmn 55

15 179 plusmn 34 683 plusmn 58 198 plusmn 40 338 plusmn 55 193 plusmn 24 511 plusmn 88

Values are mean plusmn SD (119899 = 3)

Table 4 Production of fungal biomass and protein content atdifferent pH

pH Biomass (mg50mL) Protein (120583gmL)35 153 plusmn 35 196 plusmn 39

55 243 plusmn 45 280 plusmn 21

75 213 plusmn 30 351 plusmn 44

95 140 plusmn 20 309 plusmn 28

115 84 plusmn 26 1112 plusmn 29

Values are mean plusmn SD (119899 = 3)

production of laccase in comparison to control (Figure 6(d)Table 7) Among these acetone favoured the production oflaccase (up to 2 fold induction) with higher protein contentThe induction effect in this case was recorded up to 06(Table 8) Addition of acetone did not favor the productionof fungal biomass

4 Discussion

The growth characters such as tolerance to wider range oftemperature and pH are indicative of the ecological resiliencepossessed by the fungus Isolation of new fungal strains fromlow temperature environments possessing similar growthcharacteristics along a range of biotechnological applicationshas been reported in recent years [20ndash25] Temperature andpH are two of the most important factors in productionof enzymes Role of suboptimal temperatures both towardshigher as well as lower (15 and 35∘C resp) in enhancingthe production of laccase was clearly demonstrated in thepresent investigation In both the instances it coincided withdecrease in production of fungal biomass Varying resultsbased on the organisms as well as the growth conditionshave been reported by different workers on production ofligninolytic enzymes Snajdr and Baldrian [26] reportedproduction of laccase by T versicolor between 25 and 30∘Cwhile in a recent study laccase production has been reportedto decrease beyond 28∘C [27] Generally a bell-shaped curvehas been reported for laccase activity with respect to pH [3]Janusz et al [28] have reported pH 75 optimum for produc-tion of laccase byRhizoctonia praticola as an exceptional caseIn a recent study on Trametes trogii pH 3 was found to beoptimum for laccase production [29]

L1 L2 Protein marker245kDa

100 kDa75kDa

63kDa

48kDa

35kDa

25kDa

sim45kDa

Figure 3 Zymogram of laccase (L1 and L2) stained with 1 ABTSfollowing native PAGE M-prestained protein marker

Optimization for enzyme production can be regulatedby modifying the nutritional sources carbon and nitrogenin particular Generally glucose is considered to be the bestcarbon source for production of enzymes [7] Production oflaccase favoured by replacing glucose with fructose in thepresent study can be attributed to the specific preference forthe carbon source by the fungus under study Similar obser-vation in case of Agaricus sp has been reported recently byManimozhi and Kaviyarasan [30] Among nitrogen sourcesammonium sulfate at 02 concentration gave maximumproduction of laccase in the present investigation Reduc-tion in laccase production on increasing the ammoniumsulfate concentration beyond 1 1 carbonnitrogen ratio canbe attributed to the fact that nitrogen depletion is likelyto favour the production of laccase [31] In general highcarbon nitrogen ratio favors the production of laccase asobserved in the present investigation Contrary to theseobservations reverse conditions have also been reported [32]

CuSO4and some organic solvents are known as inducers

for enhancing the laccase activity In the present investigationCuSO

4induced laccase production almost up to 2 fold over

control Inducing effect of CuSO4has also been reported

6 Enzyme Research

Table 5 Production of fungal biomass and protein content in presence of different carbon and nitrogen sources

Carbon source (02) Biomass (mg50mL) Protein (120583gmL) Nitrogen source (02) Biomass (mg50mL) Protein (120583gmL)Glucose 249 plusmn 40 270 plusmn 55 Absence of nitrogen 210 plusmn 22 286 plusmn 49

Fructose 193 plusmn 30 423 plusmn 40 Ammonium nitrate 280 plusmn 45 346 plusmn 45

Maltose 156 plusmn 40 336 plusmn 55 Ammonium sulfate 230 plusmn 36 400 plusmn 50

Sucrose 113 plusmn 26 390 plusmn 65 Ammonium ferrous sulfate 185 plusmn 25 383 plusmn 45

Starch 105 plusmn 18 316 plusmn 51 Potassium nitrate 251 plusmn 45 196 plusmn 32

Cellulose 95 plusmn 10 261 plusmn 76 Urea 164 plusmn 41 416 plusmn 40

Values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(a) 15∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(b) 25∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(c) 35∘C

Figure 4 Effect of different temperatures with respective time on laccase production (a) 15∘C (b) 25∘C (c) 35∘C

0

100

200

300

400

500

35 55 75 95 115

Enzy

me a

ctiv

ity (U

L)

pHFigure 5 Effect of pH on laccase production

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Signal TransductionJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Enzyme Research

PDA slants following subculturing (2 months interval) at4∘C in the Microbial Culture Collection established in theMicrobiology Laboratory of the Institute Fresh culture wasraised for the experimentation

22 Identification andCharacterization of the Fungus Colonymorphology of the fungus was recorded on 5 days old culturegrown on PDA at 25∘C Four other media namely Myco-logical agar Czapek dox agar Sabouraud dextrose maltoseagar and Vegetable juice 8 agar (all from Hi Media) werealso used for recording the observations on the growth offungus Microscopic observations were recorded followinglactophenol cotton blue staining (Nikon-Eclipse 50i Japan)Molecular identification was based on ITS (ITS1-58S-ITS2)region analysis using ITS1 and ITS4 primers [12] The fungalisolate and the gene sequence have been accessioned inMicrobial Type Culture Collection and Gene Bank Instituteof Microbial Technology Chandigarh India and NCBIrespectively The phylogenetic tree was made by using NJmethod with the bootstrap value 1000 MEGA 40 was usedfor the phylogenetic analyses (courtesy A Sharma MCCNCCS Pune India)

Temperature tolerance was determined on PDA by incu-bating the fungal strain between 4 and 50∘C (4 9 14 2535 45 and 50∘C) up to two weeks The pH tolerance wasdetermined between 1 to 14 pH (with 05 pH interval) byincubating the fungus at 25∘C for oneweek on PD agarbroth

23 Medium and Culture Condition for Laccase ProductionLaccase production was carried out using modified Kirk andFarrell [13] medium containing (gL) 20 g malt extract 20 gglucose 20 g NH

4NO3 026 g Na

2HPO4 026 g KH

2PO4

05 g MgSO4(7H2O) 001 g CuSO

4(5H2O) 0006 g CaCl

2

(2H2O) 0005 g FeSO

4(7H2O) 00005 g ZnSO

4(7H2O)

000002 g Na2MoO4 000009 g MnSO

4sdotH2O and 000007 g

H3BO3 Qualitative plate based estimation was done on

the aforesaid medium supplemented with 030 g ABTS (221015840-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid) Theplate assays were carried out at 6 different temperatures (4 915 25 35 and 45∘C) Ligninolytic efficiency was determinedas ABTS zone to fungal colony diameter ratio lowast 100 DMPsyringaldazine and guaiacol (separately) were also used inplate assays for determination of the oxidizing ability oflaccase In quantitative estimations 50mL medium was pre-pared in 250mL Erlenmeyer flasks without supplementingABTS The pH of the medium was set at 55 plusmn 02 beforeautoclaving Autoclaved medium was inoculated with 5mmdisc (per flask) of 6-day old fungus culture The experimentswere conducted under static conditions

24 Determination ofMolecularWeight of Laccase The crudeenzyme (filtrate) was precipitated with ammonium sulfateup to 70 saturation and centrifuged at 15000 lowast g RCF for10min at 4∘C Pellets were resuspended in citrate-phosphate(pH = 26) buffer PAGE without SDS was carried outfollowing standard method described by Laemmli [14] using15 separating and 4 stacking gel Polyacrylamide gel was

stained with 10 of ABTS solution prepared in citrate-phosphate buffer (pH 26) following 1 h incubation at roomtemperature Pre-stained protein marker (Puregene) wasused for determination of molecular weight of the enzyme

25 Molecular Studies on Partial Sequence of Laccase GeneLaccase gene fragment (corresponding to conserved copperbinding domain) was amplified by PCR (BIO-RAD USA)DNA isolation was done by the method given by Voigt et al[15] PCR amplification was carried out using basidiomycetesspecific laccase primers Cu1F and Cu2R [16] 25 120583L PCRreaction contained 2X reaction buffer 125120583L sterile water62 120583L dNTP (10mM of each dNTP) 25120583L MgCl

2(25mM)

15 120583L forward and reverse primer 10 120583L (10 pmole120583L each)and Taq DNA polymerase (3U120583L) 03120583L (dNTP MgCl

2

and Taq DNA polymerase) from Fermentas Life ScienceThe PCR conditions were initial denaturation 94∘C for3min denaturation 94∘C for 30 s annealing 48∘C for 45 sextension 72∘C for 3min final extension 72∘C for 10minand total cycles 35 Molecular weight of amplified productwas analyzed on agarose gel (08)

26 Effect of Physicochemical and Nutritional Parameters andOther Supplements on Laccase Production Production oflaccase was determined at 4 temperatures (15 25 35 and45∘C) at every 3rd day up to 15 days of incubation Laccaseproductionwith respect to pHwas determined by inoculatingthe broth set at different pH ranging from 35 to 115 (with aninterval of 2 units)

Six carbon glucose (control) fructose maltose sucrosestarch and cellulose 5 nitrogen sources (ammonium nitrate(control) ammonium sulfate ammonium ferrous sulfatepotassium nitrate and urea) and one set without inorganicnitrogen at 02 levelwere used for enhancing the productionof laccase All the carbon and nitrogen sources were from HiMedia Effect of the best carbon and nitrogen sources wasfurther determined by increasing the concentration level upto 10

In other supplements category 4 organic solvents namelymethanol ethanol isopropanol and acetone (02supple-mented at day 4 of incubation) were tested as inducers Effectof CuSO

4concentration (02 to 10mMsupplemented at day

4 of incubation) was also determinedThese experiments (effect of pH carbon sources nitrogen

sources CuSO4 and solvents) were carried out at 25∘C

following 12 days of incubation under static conditions

27 Enzyme Assay Laccase activity was determined in termsof oxidation of ABTS using spectrophotometer (AmershamBioscience Ultrospec 2100 pro) [17] The fungal culture wasfiltered with Whatman No 1 filter paper and used as crudeenzyme Mycelium collected and dried at 65∘C for 72 h wasused for determination of biomass Reaction mixture wasprepared with citrate-phosphate buffer (pH = 26) 20mMABTS and the crude enzyme Absorbance was recorded at420 nm following 2min of incubation at room temperatureLaccase activity was calculated using extinction coefficient =36000Mminus1 cmminus1 [18] 1 unit of enzyme activity is defined as

Enzyme Research 3

1 120583Mof ABTS oxidized per min Protein was also determinedfollowing the method of Lowry et al [19]

3 Results

The fungus (GBPI-CDF-03) developed white cottony masson all the 5 media used with growth being more compacton Vegetable juice 8 agar Microscopy revealed presenceof thread-like septate mycelium without any reproductivestructures (Figure 1(a)) The fungus tolerated wide rangeof temperature (4ndash48 plusmn 2∘C optimum 25 plusmn 2∘C) and pH(30ndash130 pH optimum between 5 and 7) ITS region basedmolecular identification revealed its maximum similaritywith Trametes hirsuta Growth requirements along with thephenotypic and genotypic characters and the phylogeneticrelationship of the fungus are presented in Table 1 andFigure 2 The accessions given to fungus and the nucleotidesequence are MTCC 11397 and JX910367 respectively

In plate assays conducted at optimumgrowth conditionsthe fungus oxidized ABTS and developed green colouraround the colony indicating the production of laccase(Figure 1(b)) The fungus also produced laccase through oxi-dation of other three substrates namely guaiacol syringal-dazine and DMP ABTS however was found to exhibithighest sensitivity towards laccase activity At suboptimaltemperatures (4 9 15 35 and 45∘C) maximum ligninolyticefficiency (ABTS zone to colony dia) was observed at4∘C The efficiency for production of ligninolytic enzymeswas found to be inversely proportional to the temperature(Table 2) Molecular weight of laccase was determined to beapproximately 45 kDa (Figure 3) while the PCR amplifiedsequence of laccase gene fragment (corresponding to copperbinding domain) was obtained approximately of 200 bp sizeIn quantitative estimations conducted at 15 25 35 and 45∘Cproduction of laccase fungal biomass and total proteincontent varied with culture conditions Maximum laccaseproduction (3945UL) was recorded at 35∘C on day 12 ofincubation In this case the highest biomass (228mg50mL)and maximum protein (511 120583gmL) were recorded on day12 and 15 respectively Laccase production was recordedminimum (2697UL) at 25∘Cwithmaximum fungal biomass(256mg50mL) and protein concentration (419120583gmL)at day 12 of incubation Slightly increased production oflaccase (272UL) was recorded at 15∘C with fungal biomass(144mg50mL) and protein content (662120583gmL) at day12 of incubation (Table 3 Figures 4(a) 4(b) and 4(c)) Theactivity at 45∘C was estimated to be 2821 UL with very littlebiomass (80mg50mL) and protein content (168 120583gmL) atday 12 of incubation (not presented in the Figures)While theproduction of laccase was recorded between pH 35 and 115the maximum production (270UL) along with biomass andthe protein content was favoured at pH between 55 and 75(Table 4 Figure 5)

Among 6 carbon sources (02) fructose was foundto increase the production of laccase along with higherprotein content and reduced fungal biomass in comparisonto control (Table 5 Figure 6(a)) The laccase productionincreased with the increase of fructose concentration upto 10 maximum being at 04 concentration of fructose

Table 1 Morphological and molecular characteristics of the fungus(GBPI-CDF-03)

Character DescriptionColony morphology(on PD agar)

White cottony mass with noexudation or pigmentation

Microscopic featuresThread like mycelium with septashowing the characteristic feature ofupper fungi

Growth characters

Temperature requirement between4 and 48∘C (optimum 25∘C) pHrequirement between 3 and 13(optimum 5ndash7)

Phylogenetic relationship(ITS region analysis)Culture accession noNucleotide sequenceaccession no

Maximum similarity (100) withTrametes hirsuta (JF439511)MTCC 11397JX910367

Table 2 Ligninolytic efficiency of the fungus (GBPI-CDF-03) atdifferent temperatures

Temperature(∘C)

Incubationtime(days)

ABTS zonedia (mm)(ZD)

Fungalcolony

dia (mm)(CD)

Ligninolyticefficiency =ZDCD lowast 100

4 21 22 5 4409 21 19 7 27115 14 15 10 15025 7 10 50 2035 7 12 46 2645 7 8 15 53Dia diameter

(2 1 carbonnitrogen ratio) (Table 6) However the increasein biomass and protein continued up to 10 fructose concen-tration Other carbon sources (maltose sucrose starch andcellulose) were found to be inhibitory for laccase productionas well as the production of fungal biomass Cellulose wasfound to be the most inhibitory carbon source followed bystarch sucrose and maltose Among five nitrogen sourcesammonium sulfate was found to be the best at 02 forproduction of laccase and the protein content (Table 5Figure 6(b)) Further increment in ammonium sulfate con-centration was found to be inhibitory for laccase production(Table 6) Effect of ammonium ferrous sulfate was found to beat par to the control (ammonium nitrate) Presence of othernitrogen sources (urea and potassium nitrate) and also thecase without any nitrogen source were found to reduce theproduction of laccase

Addition of CuSO4increased laccase production along

with production of protein content up to 2-fold in compari-son to control maximum being in case of 04mM concentra-tion Further increment in CuSO

4(up to 10mM) resulted in

decline of laccase production although the decreased valueswere higher in comparison to control (Figure 6(c) Table 7)Other inducers the low molecular weight organic solventsat 02 concentration were also found to enhance the

4 Enzyme Research

(a) (b)

Figure 1 (a) Microscopic features of the fungus (Bar = 2 120583m) (b) laccase production on ABTS plate

HM595571 Trametes spFJ550367 Trametes hirsutaAB638346 Trametes hirsutaJF439511 Trametes hirsuta

GBPI-CDF-03

HM004553 Stereum hirsutumJN164953 Trametes hirsutaGU062274 Trametes hirsutaJN164942 Trametes hirsuta

JN198490 Trametes versicolorJQ673021 Trametes versicolorJX501313 Trametes versicolorJQ673022 Trametes versicolor

JQ781852 Ganoderma lucidumJQ781851 Ganoderma lucidumJQ520188 Ganoderma lucidum

AB735969 Neofavolus alveolarisAB735967 Neofavolus alveolarisAB735973 Favolus acervatusAB735971 Favolus acervatusAB735975 Favolus roseus

AB735977 Favolus brasiliensisJQ403610 Irpex lacteusJX077039 Irpex lacteusJX559574 Irpex lacteusJN588552 Irpex lacteus

EU232218 Laetiporus sulphureusAY089742 Laetiporus sulphureus

GU203516 Laetiporus sulphureusEF088657 Laetiporus sulphureus

JX501310 Laetiporus sulphureus

4247

100

100

100

99

99

10070

100

65

41

5897

95

92

5872

50

93

64

10067

51

005

AB735979 Neofavolus cremeoalbidusAB735981 Neofavolus cremeoalbidus

Figure 2 Phylogenetic relationship of the fungus

Enzyme Research 5

Table 3 Production of fungal biomass and protein content at different temperatures

Incubation time(days)

Temperature (∘C)15 25 35

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

3 41 plusmn 05 364 plusmn 31 86 plusmn 16 344 plusmn 66 53 plusmn 11 346 plusmn 50

6 76 plusmn 08 446 plusmn 107 130 plusmn 32 369 plusmn 82 136 plusmn 20 394 plusmn 40

9 115 plusmn 20 546 plusmn 44 179 plusmn 62 329 plusmn 41 187 plusmn 44 429 plusmn 80

12 144 plusmn 22 662 plusmn 57 256 plusmn 42 419 plusmn 67 228 plusmn 28 436 plusmn 55

15 179 plusmn 34 683 plusmn 58 198 plusmn 40 338 plusmn 55 193 plusmn 24 511 plusmn 88

Values are mean plusmn SD (119899 = 3)

Table 4 Production of fungal biomass and protein content atdifferent pH

pH Biomass (mg50mL) Protein (120583gmL)35 153 plusmn 35 196 plusmn 39

55 243 plusmn 45 280 plusmn 21

75 213 plusmn 30 351 plusmn 44

95 140 plusmn 20 309 plusmn 28

115 84 plusmn 26 1112 plusmn 29

Values are mean plusmn SD (119899 = 3)

production of laccase in comparison to control (Figure 6(d)Table 7) Among these acetone favoured the production oflaccase (up to 2 fold induction) with higher protein contentThe induction effect in this case was recorded up to 06(Table 8) Addition of acetone did not favor the productionof fungal biomass

4 Discussion

The growth characters such as tolerance to wider range oftemperature and pH are indicative of the ecological resiliencepossessed by the fungus Isolation of new fungal strains fromlow temperature environments possessing similar growthcharacteristics along a range of biotechnological applicationshas been reported in recent years [20ndash25] Temperature andpH are two of the most important factors in productionof enzymes Role of suboptimal temperatures both towardshigher as well as lower (15 and 35∘C resp) in enhancingthe production of laccase was clearly demonstrated in thepresent investigation In both the instances it coincided withdecrease in production of fungal biomass Varying resultsbased on the organisms as well as the growth conditionshave been reported by different workers on production ofligninolytic enzymes Snajdr and Baldrian [26] reportedproduction of laccase by T versicolor between 25 and 30∘Cwhile in a recent study laccase production has been reportedto decrease beyond 28∘C [27] Generally a bell-shaped curvehas been reported for laccase activity with respect to pH [3]Janusz et al [28] have reported pH 75 optimum for produc-tion of laccase byRhizoctonia praticola as an exceptional caseIn a recent study on Trametes trogii pH 3 was found to beoptimum for laccase production [29]

L1 L2 Protein marker245kDa

100 kDa75kDa

63kDa

48kDa

35kDa

25kDa

sim45kDa

Figure 3 Zymogram of laccase (L1 and L2) stained with 1 ABTSfollowing native PAGE M-prestained protein marker

Optimization for enzyme production can be regulatedby modifying the nutritional sources carbon and nitrogenin particular Generally glucose is considered to be the bestcarbon source for production of enzymes [7] Production oflaccase favoured by replacing glucose with fructose in thepresent study can be attributed to the specific preference forthe carbon source by the fungus under study Similar obser-vation in case of Agaricus sp has been reported recently byManimozhi and Kaviyarasan [30] Among nitrogen sourcesammonium sulfate at 02 concentration gave maximumproduction of laccase in the present investigation Reduc-tion in laccase production on increasing the ammoniumsulfate concentration beyond 1 1 carbonnitrogen ratio canbe attributed to the fact that nitrogen depletion is likelyto favour the production of laccase [31] In general highcarbon nitrogen ratio favors the production of laccase asobserved in the present investigation Contrary to theseobservations reverse conditions have also been reported [32]

CuSO4and some organic solvents are known as inducers

for enhancing the laccase activity In the present investigationCuSO

4induced laccase production almost up to 2 fold over

control Inducing effect of CuSO4has also been reported

6 Enzyme Research

Table 5 Production of fungal biomass and protein content in presence of different carbon and nitrogen sources

Carbon source (02) Biomass (mg50mL) Protein (120583gmL) Nitrogen source (02) Biomass (mg50mL) Protein (120583gmL)Glucose 249 plusmn 40 270 plusmn 55 Absence of nitrogen 210 plusmn 22 286 plusmn 49

Fructose 193 plusmn 30 423 plusmn 40 Ammonium nitrate 280 plusmn 45 346 plusmn 45

Maltose 156 plusmn 40 336 plusmn 55 Ammonium sulfate 230 plusmn 36 400 plusmn 50

Sucrose 113 plusmn 26 390 plusmn 65 Ammonium ferrous sulfate 185 plusmn 25 383 plusmn 45

Starch 105 plusmn 18 316 plusmn 51 Potassium nitrate 251 plusmn 45 196 plusmn 32

Cellulose 95 plusmn 10 261 plusmn 76 Urea 164 plusmn 41 416 plusmn 40

Values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(a) 15∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(b) 25∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(c) 35∘C

Figure 4 Effect of different temperatures with respective time on laccase production (a) 15∘C (b) 25∘C (c) 35∘C

0

100

200

300

400

500

35 55 75 95 115

Enzy

me a

ctiv

ity (U

L)

pHFigure 5 Effect of pH on laccase production

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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BioinformaticsAdvances in

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Signal TransductionJournal of

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Enzyme Research 3

1 120583Mof ABTS oxidized per min Protein was also determinedfollowing the method of Lowry et al [19]

3 Results

The fungus (GBPI-CDF-03) developed white cottony masson all the 5 media used with growth being more compacton Vegetable juice 8 agar Microscopy revealed presenceof thread-like septate mycelium without any reproductivestructures (Figure 1(a)) The fungus tolerated wide rangeof temperature (4ndash48 plusmn 2∘C optimum 25 plusmn 2∘C) and pH(30ndash130 pH optimum between 5 and 7) ITS region basedmolecular identification revealed its maximum similaritywith Trametes hirsuta Growth requirements along with thephenotypic and genotypic characters and the phylogeneticrelationship of the fungus are presented in Table 1 andFigure 2 The accessions given to fungus and the nucleotidesequence are MTCC 11397 and JX910367 respectively

In plate assays conducted at optimumgrowth conditionsthe fungus oxidized ABTS and developed green colouraround the colony indicating the production of laccase(Figure 1(b)) The fungus also produced laccase through oxi-dation of other three substrates namely guaiacol syringal-dazine and DMP ABTS however was found to exhibithighest sensitivity towards laccase activity At suboptimaltemperatures (4 9 15 35 and 45∘C) maximum ligninolyticefficiency (ABTS zone to colony dia) was observed at4∘C The efficiency for production of ligninolytic enzymeswas found to be inversely proportional to the temperature(Table 2) Molecular weight of laccase was determined to beapproximately 45 kDa (Figure 3) while the PCR amplifiedsequence of laccase gene fragment (corresponding to copperbinding domain) was obtained approximately of 200 bp sizeIn quantitative estimations conducted at 15 25 35 and 45∘Cproduction of laccase fungal biomass and total proteincontent varied with culture conditions Maximum laccaseproduction (3945UL) was recorded at 35∘C on day 12 ofincubation In this case the highest biomass (228mg50mL)and maximum protein (511 120583gmL) were recorded on day12 and 15 respectively Laccase production was recordedminimum (2697UL) at 25∘Cwithmaximum fungal biomass(256mg50mL) and protein concentration (419120583gmL)at day 12 of incubation Slightly increased production oflaccase (272UL) was recorded at 15∘C with fungal biomass(144mg50mL) and protein content (662120583gmL) at day12 of incubation (Table 3 Figures 4(a) 4(b) and 4(c)) Theactivity at 45∘C was estimated to be 2821 UL with very littlebiomass (80mg50mL) and protein content (168 120583gmL) atday 12 of incubation (not presented in the Figures)While theproduction of laccase was recorded between pH 35 and 115the maximum production (270UL) along with biomass andthe protein content was favoured at pH between 55 and 75(Table 4 Figure 5)

Among 6 carbon sources (02) fructose was foundto increase the production of laccase along with higherprotein content and reduced fungal biomass in comparisonto control (Table 5 Figure 6(a)) The laccase productionincreased with the increase of fructose concentration upto 10 maximum being at 04 concentration of fructose

Table 1 Morphological and molecular characteristics of the fungus(GBPI-CDF-03)

Character DescriptionColony morphology(on PD agar)

White cottony mass with noexudation or pigmentation

Microscopic featuresThread like mycelium with septashowing the characteristic feature ofupper fungi

Growth characters

Temperature requirement between4 and 48∘C (optimum 25∘C) pHrequirement between 3 and 13(optimum 5ndash7)

Phylogenetic relationship(ITS region analysis)Culture accession noNucleotide sequenceaccession no

Maximum similarity (100) withTrametes hirsuta (JF439511)MTCC 11397JX910367

Table 2 Ligninolytic efficiency of the fungus (GBPI-CDF-03) atdifferent temperatures

Temperature(∘C)

Incubationtime(days)

ABTS zonedia (mm)(ZD)

Fungalcolony

dia (mm)(CD)

Ligninolyticefficiency =ZDCD lowast 100

4 21 22 5 4409 21 19 7 27115 14 15 10 15025 7 10 50 2035 7 12 46 2645 7 8 15 53Dia diameter

(2 1 carbonnitrogen ratio) (Table 6) However the increasein biomass and protein continued up to 10 fructose concen-tration Other carbon sources (maltose sucrose starch andcellulose) were found to be inhibitory for laccase productionas well as the production of fungal biomass Cellulose wasfound to be the most inhibitory carbon source followed bystarch sucrose and maltose Among five nitrogen sourcesammonium sulfate was found to be the best at 02 forproduction of laccase and the protein content (Table 5Figure 6(b)) Further increment in ammonium sulfate con-centration was found to be inhibitory for laccase production(Table 6) Effect of ammonium ferrous sulfate was found to beat par to the control (ammonium nitrate) Presence of othernitrogen sources (urea and potassium nitrate) and also thecase without any nitrogen source were found to reduce theproduction of laccase

Addition of CuSO4increased laccase production along

with production of protein content up to 2-fold in compari-son to control maximum being in case of 04mM concentra-tion Further increment in CuSO

4(up to 10mM) resulted in

decline of laccase production although the decreased valueswere higher in comparison to control (Figure 6(c) Table 7)Other inducers the low molecular weight organic solventsat 02 concentration were also found to enhance the

4 Enzyme Research

(a) (b)

Figure 1 (a) Microscopic features of the fungus (Bar = 2 120583m) (b) laccase production on ABTS plate

HM595571 Trametes spFJ550367 Trametes hirsutaAB638346 Trametes hirsutaJF439511 Trametes hirsuta

GBPI-CDF-03

HM004553 Stereum hirsutumJN164953 Trametes hirsutaGU062274 Trametes hirsutaJN164942 Trametes hirsuta

JN198490 Trametes versicolorJQ673021 Trametes versicolorJX501313 Trametes versicolorJQ673022 Trametes versicolor

JQ781852 Ganoderma lucidumJQ781851 Ganoderma lucidumJQ520188 Ganoderma lucidum

AB735969 Neofavolus alveolarisAB735967 Neofavolus alveolarisAB735973 Favolus acervatusAB735971 Favolus acervatusAB735975 Favolus roseus

AB735977 Favolus brasiliensisJQ403610 Irpex lacteusJX077039 Irpex lacteusJX559574 Irpex lacteusJN588552 Irpex lacteus

EU232218 Laetiporus sulphureusAY089742 Laetiporus sulphureus

GU203516 Laetiporus sulphureusEF088657 Laetiporus sulphureus

JX501310 Laetiporus sulphureus

4247

100

100

100

99

99

10070

100

65

41

5897

95

92

5872

50

93

64

10067

51

005

AB735979 Neofavolus cremeoalbidusAB735981 Neofavolus cremeoalbidus

Figure 2 Phylogenetic relationship of the fungus

Enzyme Research 5

Table 3 Production of fungal biomass and protein content at different temperatures

Incubation time(days)

Temperature (∘C)15 25 35

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

3 41 plusmn 05 364 plusmn 31 86 plusmn 16 344 plusmn 66 53 plusmn 11 346 plusmn 50

6 76 plusmn 08 446 plusmn 107 130 plusmn 32 369 plusmn 82 136 plusmn 20 394 plusmn 40

9 115 plusmn 20 546 plusmn 44 179 plusmn 62 329 plusmn 41 187 plusmn 44 429 plusmn 80

12 144 plusmn 22 662 plusmn 57 256 plusmn 42 419 plusmn 67 228 plusmn 28 436 plusmn 55

15 179 plusmn 34 683 plusmn 58 198 plusmn 40 338 plusmn 55 193 plusmn 24 511 plusmn 88

Values are mean plusmn SD (119899 = 3)

Table 4 Production of fungal biomass and protein content atdifferent pH

pH Biomass (mg50mL) Protein (120583gmL)35 153 plusmn 35 196 plusmn 39

55 243 plusmn 45 280 plusmn 21

75 213 plusmn 30 351 plusmn 44

95 140 plusmn 20 309 plusmn 28

115 84 plusmn 26 1112 plusmn 29

Values are mean plusmn SD (119899 = 3)

production of laccase in comparison to control (Figure 6(d)Table 7) Among these acetone favoured the production oflaccase (up to 2 fold induction) with higher protein contentThe induction effect in this case was recorded up to 06(Table 8) Addition of acetone did not favor the productionof fungal biomass

4 Discussion

The growth characters such as tolerance to wider range oftemperature and pH are indicative of the ecological resiliencepossessed by the fungus Isolation of new fungal strains fromlow temperature environments possessing similar growthcharacteristics along a range of biotechnological applicationshas been reported in recent years [20ndash25] Temperature andpH are two of the most important factors in productionof enzymes Role of suboptimal temperatures both towardshigher as well as lower (15 and 35∘C resp) in enhancingthe production of laccase was clearly demonstrated in thepresent investigation In both the instances it coincided withdecrease in production of fungal biomass Varying resultsbased on the organisms as well as the growth conditionshave been reported by different workers on production ofligninolytic enzymes Snajdr and Baldrian [26] reportedproduction of laccase by T versicolor between 25 and 30∘Cwhile in a recent study laccase production has been reportedto decrease beyond 28∘C [27] Generally a bell-shaped curvehas been reported for laccase activity with respect to pH [3]Janusz et al [28] have reported pH 75 optimum for produc-tion of laccase byRhizoctonia praticola as an exceptional caseIn a recent study on Trametes trogii pH 3 was found to beoptimum for laccase production [29]

L1 L2 Protein marker245kDa

100 kDa75kDa

63kDa

48kDa

35kDa

25kDa

sim45kDa

Figure 3 Zymogram of laccase (L1 and L2) stained with 1 ABTSfollowing native PAGE M-prestained protein marker

Optimization for enzyme production can be regulatedby modifying the nutritional sources carbon and nitrogenin particular Generally glucose is considered to be the bestcarbon source for production of enzymes [7] Production oflaccase favoured by replacing glucose with fructose in thepresent study can be attributed to the specific preference forthe carbon source by the fungus under study Similar obser-vation in case of Agaricus sp has been reported recently byManimozhi and Kaviyarasan [30] Among nitrogen sourcesammonium sulfate at 02 concentration gave maximumproduction of laccase in the present investigation Reduc-tion in laccase production on increasing the ammoniumsulfate concentration beyond 1 1 carbonnitrogen ratio canbe attributed to the fact that nitrogen depletion is likelyto favour the production of laccase [31] In general highcarbon nitrogen ratio favors the production of laccase asobserved in the present investigation Contrary to theseobservations reverse conditions have also been reported [32]

CuSO4and some organic solvents are known as inducers

for enhancing the laccase activity In the present investigationCuSO

4induced laccase production almost up to 2 fold over

control Inducing effect of CuSO4has also been reported

6 Enzyme Research

Table 5 Production of fungal biomass and protein content in presence of different carbon and nitrogen sources

Carbon source (02) Biomass (mg50mL) Protein (120583gmL) Nitrogen source (02) Biomass (mg50mL) Protein (120583gmL)Glucose 249 plusmn 40 270 plusmn 55 Absence of nitrogen 210 plusmn 22 286 plusmn 49

Fructose 193 plusmn 30 423 plusmn 40 Ammonium nitrate 280 plusmn 45 346 plusmn 45

Maltose 156 plusmn 40 336 plusmn 55 Ammonium sulfate 230 plusmn 36 400 plusmn 50

Sucrose 113 plusmn 26 390 plusmn 65 Ammonium ferrous sulfate 185 plusmn 25 383 plusmn 45

Starch 105 plusmn 18 316 plusmn 51 Potassium nitrate 251 plusmn 45 196 plusmn 32

Cellulose 95 plusmn 10 261 plusmn 76 Urea 164 plusmn 41 416 plusmn 40

Values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(a) 15∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(b) 25∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(c) 35∘C

Figure 4 Effect of different temperatures with respective time on laccase production (a) 15∘C (b) 25∘C (c) 35∘C

0

100

200

300

400

500

35 55 75 95 115

Enzy

me a

ctiv

ity (U

L)

pHFigure 5 Effect of pH on laccase production

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Enzyme Research

(a) (b)

Figure 1 (a) Microscopic features of the fungus (Bar = 2 120583m) (b) laccase production on ABTS plate

HM595571 Trametes spFJ550367 Trametes hirsutaAB638346 Trametes hirsutaJF439511 Trametes hirsuta

GBPI-CDF-03

HM004553 Stereum hirsutumJN164953 Trametes hirsutaGU062274 Trametes hirsutaJN164942 Trametes hirsuta

JN198490 Trametes versicolorJQ673021 Trametes versicolorJX501313 Trametes versicolorJQ673022 Trametes versicolor

JQ781852 Ganoderma lucidumJQ781851 Ganoderma lucidumJQ520188 Ganoderma lucidum

AB735969 Neofavolus alveolarisAB735967 Neofavolus alveolarisAB735973 Favolus acervatusAB735971 Favolus acervatusAB735975 Favolus roseus

AB735977 Favolus brasiliensisJQ403610 Irpex lacteusJX077039 Irpex lacteusJX559574 Irpex lacteusJN588552 Irpex lacteus

EU232218 Laetiporus sulphureusAY089742 Laetiporus sulphureus

GU203516 Laetiporus sulphureusEF088657 Laetiporus sulphureus

JX501310 Laetiporus sulphureus

4247

100

100

100

99

99

10070

100

65

41

5897

95

92

5872

50

93

64

10067

51

005

AB735979 Neofavolus cremeoalbidusAB735981 Neofavolus cremeoalbidus

Figure 2 Phylogenetic relationship of the fungus

Enzyme Research 5

Table 3 Production of fungal biomass and protein content at different temperatures

Incubation time(days)

Temperature (∘C)15 25 35

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

3 41 plusmn 05 364 plusmn 31 86 plusmn 16 344 plusmn 66 53 plusmn 11 346 plusmn 50

6 76 plusmn 08 446 plusmn 107 130 plusmn 32 369 plusmn 82 136 plusmn 20 394 plusmn 40

9 115 plusmn 20 546 plusmn 44 179 plusmn 62 329 plusmn 41 187 plusmn 44 429 plusmn 80

12 144 plusmn 22 662 plusmn 57 256 plusmn 42 419 plusmn 67 228 plusmn 28 436 plusmn 55

15 179 plusmn 34 683 plusmn 58 198 plusmn 40 338 plusmn 55 193 plusmn 24 511 plusmn 88

Values are mean plusmn SD (119899 = 3)

Table 4 Production of fungal biomass and protein content atdifferent pH

pH Biomass (mg50mL) Protein (120583gmL)35 153 plusmn 35 196 plusmn 39

55 243 plusmn 45 280 plusmn 21

75 213 plusmn 30 351 plusmn 44

95 140 plusmn 20 309 plusmn 28

115 84 plusmn 26 1112 plusmn 29

Values are mean plusmn SD (119899 = 3)

production of laccase in comparison to control (Figure 6(d)Table 7) Among these acetone favoured the production oflaccase (up to 2 fold induction) with higher protein contentThe induction effect in this case was recorded up to 06(Table 8) Addition of acetone did not favor the productionof fungal biomass

4 Discussion

The growth characters such as tolerance to wider range oftemperature and pH are indicative of the ecological resiliencepossessed by the fungus Isolation of new fungal strains fromlow temperature environments possessing similar growthcharacteristics along a range of biotechnological applicationshas been reported in recent years [20ndash25] Temperature andpH are two of the most important factors in productionof enzymes Role of suboptimal temperatures both towardshigher as well as lower (15 and 35∘C resp) in enhancingthe production of laccase was clearly demonstrated in thepresent investigation In both the instances it coincided withdecrease in production of fungal biomass Varying resultsbased on the organisms as well as the growth conditionshave been reported by different workers on production ofligninolytic enzymes Snajdr and Baldrian [26] reportedproduction of laccase by T versicolor between 25 and 30∘Cwhile in a recent study laccase production has been reportedto decrease beyond 28∘C [27] Generally a bell-shaped curvehas been reported for laccase activity with respect to pH [3]Janusz et al [28] have reported pH 75 optimum for produc-tion of laccase byRhizoctonia praticola as an exceptional caseIn a recent study on Trametes trogii pH 3 was found to beoptimum for laccase production [29]

L1 L2 Protein marker245kDa

100 kDa75kDa

63kDa

48kDa

35kDa

25kDa

sim45kDa

Figure 3 Zymogram of laccase (L1 and L2) stained with 1 ABTSfollowing native PAGE M-prestained protein marker

Optimization for enzyme production can be regulatedby modifying the nutritional sources carbon and nitrogenin particular Generally glucose is considered to be the bestcarbon source for production of enzymes [7] Production oflaccase favoured by replacing glucose with fructose in thepresent study can be attributed to the specific preference forthe carbon source by the fungus under study Similar obser-vation in case of Agaricus sp has been reported recently byManimozhi and Kaviyarasan [30] Among nitrogen sourcesammonium sulfate at 02 concentration gave maximumproduction of laccase in the present investigation Reduc-tion in laccase production on increasing the ammoniumsulfate concentration beyond 1 1 carbonnitrogen ratio canbe attributed to the fact that nitrogen depletion is likelyto favour the production of laccase [31] In general highcarbon nitrogen ratio favors the production of laccase asobserved in the present investigation Contrary to theseobservations reverse conditions have also been reported [32]

CuSO4and some organic solvents are known as inducers

for enhancing the laccase activity In the present investigationCuSO

4induced laccase production almost up to 2 fold over

control Inducing effect of CuSO4has also been reported

6 Enzyme Research

Table 5 Production of fungal biomass and protein content in presence of different carbon and nitrogen sources

Carbon source (02) Biomass (mg50mL) Protein (120583gmL) Nitrogen source (02) Biomass (mg50mL) Protein (120583gmL)Glucose 249 plusmn 40 270 plusmn 55 Absence of nitrogen 210 plusmn 22 286 plusmn 49

Fructose 193 plusmn 30 423 plusmn 40 Ammonium nitrate 280 plusmn 45 346 plusmn 45

Maltose 156 plusmn 40 336 plusmn 55 Ammonium sulfate 230 plusmn 36 400 plusmn 50

Sucrose 113 plusmn 26 390 plusmn 65 Ammonium ferrous sulfate 185 plusmn 25 383 plusmn 45

Starch 105 plusmn 18 316 plusmn 51 Potassium nitrate 251 plusmn 45 196 plusmn 32

Cellulose 95 plusmn 10 261 plusmn 76 Urea 164 plusmn 41 416 plusmn 40

Values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(a) 15∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(b) 25∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(c) 35∘C

Figure 4 Effect of different temperatures with respective time on laccase production (a) 15∘C (b) 25∘C (c) 35∘C

0

100

200

300

400

500

35 55 75 95 115

Enzy

me a

ctiv

ity (U

L)

pHFigure 5 Effect of pH on laccase production

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Enzyme Research 5

Table 3 Production of fungal biomass and protein content at different temperatures

Incubation time(days)

Temperature (∘C)15 25 35

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

Biomass(mg50mL)

Protein(120583gmL)

3 41 plusmn 05 364 plusmn 31 86 plusmn 16 344 plusmn 66 53 plusmn 11 346 plusmn 50

6 76 plusmn 08 446 plusmn 107 130 plusmn 32 369 plusmn 82 136 plusmn 20 394 plusmn 40

9 115 plusmn 20 546 plusmn 44 179 plusmn 62 329 plusmn 41 187 plusmn 44 429 plusmn 80

12 144 plusmn 22 662 plusmn 57 256 plusmn 42 419 plusmn 67 228 plusmn 28 436 plusmn 55

15 179 plusmn 34 683 plusmn 58 198 plusmn 40 338 plusmn 55 193 plusmn 24 511 plusmn 88

Values are mean plusmn SD (119899 = 3)

Table 4 Production of fungal biomass and protein content atdifferent pH

pH Biomass (mg50mL) Protein (120583gmL)35 153 plusmn 35 196 plusmn 39

55 243 plusmn 45 280 plusmn 21

75 213 plusmn 30 351 plusmn 44

95 140 plusmn 20 309 plusmn 28

115 84 plusmn 26 1112 plusmn 29

Values are mean plusmn SD (119899 = 3)

production of laccase in comparison to control (Figure 6(d)Table 7) Among these acetone favoured the production oflaccase (up to 2 fold induction) with higher protein contentThe induction effect in this case was recorded up to 06(Table 8) Addition of acetone did not favor the productionof fungal biomass

4 Discussion

The growth characters such as tolerance to wider range oftemperature and pH are indicative of the ecological resiliencepossessed by the fungus Isolation of new fungal strains fromlow temperature environments possessing similar growthcharacteristics along a range of biotechnological applicationshas been reported in recent years [20ndash25] Temperature andpH are two of the most important factors in productionof enzymes Role of suboptimal temperatures both towardshigher as well as lower (15 and 35∘C resp) in enhancingthe production of laccase was clearly demonstrated in thepresent investigation In both the instances it coincided withdecrease in production of fungal biomass Varying resultsbased on the organisms as well as the growth conditionshave been reported by different workers on production ofligninolytic enzymes Snajdr and Baldrian [26] reportedproduction of laccase by T versicolor between 25 and 30∘Cwhile in a recent study laccase production has been reportedto decrease beyond 28∘C [27] Generally a bell-shaped curvehas been reported for laccase activity with respect to pH [3]Janusz et al [28] have reported pH 75 optimum for produc-tion of laccase byRhizoctonia praticola as an exceptional caseIn a recent study on Trametes trogii pH 3 was found to beoptimum for laccase production [29]

L1 L2 Protein marker245kDa

100 kDa75kDa

63kDa

48kDa

35kDa

25kDa

sim45kDa

Figure 3 Zymogram of laccase (L1 and L2) stained with 1 ABTSfollowing native PAGE M-prestained protein marker

Optimization for enzyme production can be regulatedby modifying the nutritional sources carbon and nitrogenin particular Generally glucose is considered to be the bestcarbon source for production of enzymes [7] Production oflaccase favoured by replacing glucose with fructose in thepresent study can be attributed to the specific preference forthe carbon source by the fungus under study Similar obser-vation in case of Agaricus sp has been reported recently byManimozhi and Kaviyarasan [30] Among nitrogen sourcesammonium sulfate at 02 concentration gave maximumproduction of laccase in the present investigation Reduc-tion in laccase production on increasing the ammoniumsulfate concentration beyond 1 1 carbonnitrogen ratio canbe attributed to the fact that nitrogen depletion is likelyto favour the production of laccase [31] In general highcarbon nitrogen ratio favors the production of laccase asobserved in the present investigation Contrary to theseobservations reverse conditions have also been reported [32]

CuSO4and some organic solvents are known as inducers

for enhancing the laccase activity In the present investigationCuSO

4induced laccase production almost up to 2 fold over

control Inducing effect of CuSO4has also been reported

6 Enzyme Research

Table 5 Production of fungal biomass and protein content in presence of different carbon and nitrogen sources

Carbon source (02) Biomass (mg50mL) Protein (120583gmL) Nitrogen source (02) Biomass (mg50mL) Protein (120583gmL)Glucose 249 plusmn 40 270 plusmn 55 Absence of nitrogen 210 plusmn 22 286 plusmn 49

Fructose 193 plusmn 30 423 plusmn 40 Ammonium nitrate 280 plusmn 45 346 plusmn 45

Maltose 156 plusmn 40 336 plusmn 55 Ammonium sulfate 230 plusmn 36 400 plusmn 50

Sucrose 113 plusmn 26 390 plusmn 65 Ammonium ferrous sulfate 185 plusmn 25 383 plusmn 45

Starch 105 plusmn 18 316 plusmn 51 Potassium nitrate 251 plusmn 45 196 plusmn 32

Cellulose 95 plusmn 10 261 plusmn 76 Urea 164 plusmn 41 416 plusmn 40

Values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(a) 15∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(b) 25∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(c) 35∘C

Figure 4 Effect of different temperatures with respective time on laccase production (a) 15∘C (b) 25∘C (c) 35∘C

0

100

200

300

400

500

35 55 75 95 115

Enzy

me a

ctiv

ity (U

L)

pHFigure 5 Effect of pH on laccase production

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Enzyme Research

Table 5 Production of fungal biomass and protein content in presence of different carbon and nitrogen sources

Carbon source (02) Biomass (mg50mL) Protein (120583gmL) Nitrogen source (02) Biomass (mg50mL) Protein (120583gmL)Glucose 249 plusmn 40 270 plusmn 55 Absence of nitrogen 210 plusmn 22 286 plusmn 49

Fructose 193 plusmn 30 423 plusmn 40 Ammonium nitrate 280 plusmn 45 346 plusmn 45

Maltose 156 plusmn 40 336 plusmn 55 Ammonium sulfate 230 plusmn 36 400 plusmn 50

Sucrose 113 plusmn 26 390 plusmn 65 Ammonium ferrous sulfate 185 plusmn 25 383 plusmn 45

Starch 105 plusmn 18 316 plusmn 51 Potassium nitrate 251 plusmn 45 196 plusmn 32

Cellulose 95 plusmn 10 261 plusmn 76 Urea 164 plusmn 41 416 plusmn 40

Values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(a) 15∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(b) 25∘C

0

100

200

300

400

500

3 6 9 12 15

Enzy

me a

ctiv

ity (U

L)

Incubation time (days)

(c) 35∘C

Figure 4 Effect of different temperatures with respective time on laccase production (a) 15∘C (b) 25∘C (c) 35∘C

0

100

200

300

400

500

35 55 75 95 115

Enzy

me a

ctiv

ity (U

L)

pHFigure 5 Effect of pH on laccase production

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Enzyme Research 7

Table 6 Laccase activity fungal biomass and protein content with different concentrations of fructose and ammonium sulfate

Fructose()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Ammoniumsulfate ()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 2480 plusmn 65 224 plusmn 68 316 plusmn 40 Control 2600 plusmn 89 261 plusmn 44 223 plusmn 4902 (1 1) 3350 plusmn 143 287 plusmn 15 452 plusmn 66 02 (1 1) 3440 plusmn 141 296 plusmn 45 383 plusmn 8504 (2 1) 4350 plusmn 355 318 plusmn 36 653 plusmn 87 04 (1 2) 1940 plusmn 236 272 plusmn 44 336 plusmn 7706 (3 1) 3740 plusmn 312 343 plusmn 40 696 plusmn 45 06 (1 3) 1540 plusmn 47 255 plusmn 22 323 plusmn 3708 (4 1) 3360 plusmn 118 365 plusmn 34 843 plusmn 100 08 (1 4) 1330 plusmn 71 246 plusmn 50 306 plusmn 3510 (5 1) 3100 plusmn 94 373 plusmn 45 1086 plusmn 133 10 (1 5) 1300 plusmn 33 258 plusmn 49 260 plusmn 43Values given in parentheses depict the carbonnitrogen ratio values are mean plusmn SD (119899 = 3)

0

100

200

300

400

500

600

700

G F M S St C

Enzy

me a

ctiv

ity (U

L)

Carbon source

(a)

0

100

200

300

400

500

600

700

Enzy

me a

ctiv

ity (U

L)

AbN AN AS AFS KN UreaNitrogen source

(b)

0

100

200

300

400

500

600

700

C 02 04 06 08 1

Enzy

me a

ctiv

ity (U

L)

CuSO4 (mM)

(c)

0

100

200

300

400

500

600

700

C M E A I

Enzy

me a

ctiv

ity (U

L)

Solvent(d)

Figure 6 Effect of nutritional parameters on laccase production (a) carbon sources G (glucose) F (fructose) M (maltose) S (sucrose)St (starch) C (cellulose) (b) nitrogen sources AbN (absence of inorganic nitrogen) AN (ammonium nitrate) AS (ammonium sulfate)AFS (ammonium ferrous sulfate) KN (potassium nitrate) (c) CuSO

4

concentration (02 to 10mM) (d) organic solvents C (control) M(methanol) E (ethanol) A (acetone) I (iso-propanol)

in earlier studies in T hirsuta CuSO4is a constituent of

the catalytic center of the laccase hence it is important forthe synthesis of the respective enzyme [33] Importance ofcopper in regulation of laccase production at transcriptionallevels has also been reported [7] Enhancement in laccaseproduction in the present investigation due to addition of

organic solvents such as methanol ethanol acetone andisopropanol is also an important finding The increase inlaccase production by ethanol has been observed in earlierstudies [34]The purpose of using simple organic solvents forinduction is to increase the ecological and economical valueof the process

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 Enzyme Research

Table 7 Production of fungal biomass and protein content in presence of different concentrations of CuSO4 and different solvents (02)

CuSO4 (mM) Biomass (mg50mL) Protein (120583gmL) Solvent (02) Biomass (mg50mL) Protein (120583gmL)02 168 plusmn 25 356 plusmn 66 Control 253 plusmn 30 239 plusmn 3404 131 plusmn 33 440 plusmn 65 Methanol 161 plusmn 36 381 plusmn 4006 117 plusmn 09 466 plusmn 70 Ethanol 166 plusmn 20 418 plusmn 5708 83 plusmn 30 540 plusmn 55 Acetone 184 plusmn 22 452 plusmn 6110 71 plusmn 25 551 plusmn 72 Isopropanol 130 plusmn 25 383 plusmn 56Control value for biomass = 240 plusmn 38 and protein = 312 plusmn 25 values are mean plusmn SD (119899 = 3)

Table 8 Laccase activity fungal biomass and protein content withdifferent concentrations of acetone

Acetone()

Laccase(UL)

Biomass(mg50mL)

Protein(120583gmL)

Control 263 plusmn 94 218 plusmn 38 251 plusmn 36

02 542 plusmn 16 177 plusmn 15 454 plusmn 59

04 397 plusmn 14 170 plusmn 30 411 plusmn 60

06 349 plusmn 10 163 plusmn 40 410 plusmn 60

08 227 plusmn 11 133 plusmn 35 357 plusmn 45

10 201 plusmn 17 110 plusmn 30 315 plusmn 40Values are mean plusmn SD (119899 = 3)

5 Conclusion

It can be concluded that increased production of laccaseat suboptimal conditions as recorded in the present studyis likely to be advantageous from ecological as well asbiotechnological prospects Temperature tolerant organismspsychrotolerant ones in particular can be presumed fortheir contributions in biodegradation in low temperatureenvironments High yield consistent production and sus-tainability of secondary metabolites under stress conditionswill be important parameters in such investigationsThe fun-gus (GBPI-CDF-03) produced laccase under wide range oftemperature covering psychrophilic to mesophilic range alsotouching the thermophilic range The present investigationemphasizes the importance of isolation and identificationof new and efficient microbial strains from the extremeenvironments of IHR for production of industrially desiredbiomolecules [35] The findings of the present study basedon the identification of a laccase producing fungal strainfollowed by the optimization for nutritional and physiologicalconditions are likely to be useful in further upgrade of theprocess

Acknowledgments

The director of G B Pant Institute of Himalayan Environ-ment and Development Almora is gratefully acknowledgedfor extending the facilities Ministry of Environment andForests Government of India is thanked for financial sup-port Senior author is thankful to the Indian Council ofMedical Research (ICMR) Government of India NewDelhifor awarding the research fellowship

References

[1] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquoBlue laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

[2] S S Desai and C Nityanand ldquoMicrobial laccases and theirapplications a reviewrdquoAsian Journal of Biotechnology vol 3 no2 pp 98ndash124 2011

[3] Shraddha R Shekher S Sehgal M Kamthania and A KumarldquoLaccase microbial sources production purification andpotential biotechnological applicationsrdquo Enzyme Research vol2011 Article ID 217861 11 pages 2011

[4] A M Mayer and R C Staples ldquoLaccase new functions for anold enzymerdquo Phytochemistry vol 60 no 6 pp 551ndash565 2002

[5] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[6] A Kunamneni A Ballesteros F J Plou and M AlcaldeldquoFungal laccase-a versatile enzyme for biotechnological appli-cationsrdquo in CommunicatIng Current Research and EducationalTopics and Trends In Applied Microbiology A Mendez-VilasEd pp 233ndash245 Formex Badajoz Spain 2007

[7] A Piscitelli P Giardina V Lettera C Pezzella G Sannia andV Faraco ldquoInduction and transcriptional regulation of laccasesin fungirdquo Current Genomics vol 12 no 2 pp 104ndash112 2011

[8] K Brijwani A Rigdon and P V Vadlani ldquoFungal laccasesproduction function and applications in food processingrdquoEnzyme Research vol 2010 Article ID 149748 10 pages 2010

[9] B Chaurasia A Pandey and L M S Palni ldquoDistributioncolonization and diversity of arbuscularmycorrhizal fungi asso-ciated with central Himalayan rhododendronsrdquo Forest Ecologyand Management vol 207 no 3 pp 315ndash324 2005

[10] A Pandey and L M S Palni ldquoThe rhizosphere effect in trees ofthe Indian Central Himalaya with special reference to altituderdquoApplied Ecology and Environmental Research vol 5 no 1 pp93ndash102 2007

[11] M K Malviya A Pandey P Trivedi G Gupta and B KumarldquoChitinolytic activity of cold tolerant antagonistic species ofstreptomyces isolated from glacial sites of Indian HimalayardquoCurrent Microbiology vol 59 no 5 pp 502ndash508 2009

[12] T JWhite TD Bruns S B Lee and JW Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide To Methods andApplications M A Innis D H Gelfand J J Sninsky and TJ White Eds pp 315ndash322 Academic Press San Diego CalifUSA 1990

[13] T K Kirk and R L Farrell ldquoEnzymatic ldquocombustionrdquo themicrobial degradation of ligninrdquo Annual Review of Microbiol-ogy vol 41 pp 465ndash505 1987

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Enzyme Research 9

[14] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[15] K Voigt E Cigelnik and K OrsquoDonnell ldquoPhylogeny and PCRidentification of clinically important zygomycetes based onnuclear ribosomal-DNA sequence datardquo Journal of ClinicalMicrobiology vol 37 no 12 pp 3957ndash3964 1999

[16] P Luis G Walther H Kellner F Martin and F BuscotldquoDiversity of laccase genes from basidiomycetes in a forest soilrdquoSoil Biology and Biochemistry vol 36 no 7 pp 1025ndash1036 2004

[17] M L Niku-Paavola E Karhunen P Salola and V RaunioldquoLigninolytic enzymes of the white-rot fungus Phlebia radiatardquoBiochemical Journal vol 254 no 3 pp 877ndash884 1988

[18] M J Han H T Choi and H G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[19] H Lowry N J Rosebrough A L Farr and R L RandallldquoProtein measurement with the folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[20] A Pandey L M S Palni and D Bisht ldquoDominant fungi in therhizosphere of established tea bushes and their interaction withthe dominant bacteria under in situ conditionsrdquoMicrobiologicalResearch vol 156 no 4 pp 377ndash382 2001

[21] A Ghildiyal and A Pandey ldquoIsolation of cold tolerant anti-fungal strains of Trichoderma sp from glacial sites of IndianHimalayan regionrdquo Research Journal of Microbiology vol 3 no8 pp 559ndash564 2008

[22] A PandeyNDas B Kumar K Rinu andP Trivedi ldquoPhosphatesolubilization by Penicillium spp isolated from soil samples ofIndian Himalayan regionrdquo World Journal of Microbiology andBiotechnology vol 24 no 1 pp 97ndash102 2008

[23] K Rinu and A Pandey ldquoTemperature-dependent phosphatesolubilization by cold- and pH-tolerant species of Aspergillusisolated from Himalayan soilrdquo Mycoscience vol 51 no 4 pp263ndash271 2010

[24] K Rinu and A Pandey ldquoSlow and steady phosphate solubiliza-tion by a psychrotolerant strain of Paecilomyces hepiali (MTCC9621)rdquoWorld Journal of Microbiology and Biotechnology vol 27no 5 pp 1055ndash1062 2011

[25] K Rinu A Pandey and L M S Palni ldquoUtilization of psychro-tolerant phosphate solubilizing fungi under low temperatureconditions of the mountain ecosystemrdquo in Microorganisms inSustainable Agriculture and Biotechnology T Satyanarayana BN Johri and A Prakash Eds pp 77ndash90 Springer ScienceBuisiness Media 2012

[26] J Snajdr and P Baldrian ldquoTemperature affects the productionactivity and stability of ligninolytic enzymes in Pleurotus ostrea-tus and Trametes versicolorrdquo Folia Microbiologica vol 52 no 5pp 498ndash502 2007

[27] M A Elsayed M M Hassan A M Elshafei B M Harounand A M Othman ldquoOptimization of cultural and nutritionalparameters for the production of laccase by Pleurotus ostreatusARC280rdquoBritish Biotechnology Journal vol 2 no 3 pp 115ndash1322012

[28] G Janusz J Rogalski M Barwinska and J Szczodrak ldquoEffectsof culture conditions on production of extracellular laccase byRhizoctonia praticolardquoPolish Journal ofMicrobiology vol 55 no4 pp 309ndash319 2006

[29] AKocyigitM B Pazarbasi I YasaGOzdemir and I KarabozldquoProduction of laccase from Trametes trogii TEM H2 a newly

isolated white-rot fungus by air samplingrdquo Journal of BasicMicrobiology vol 52 pp 661ndash669 2012

[30] M Manimozhi and V Kaviyarasan ldquoScreening the effect ofnutritional parameters on biomass and laccase productionin submerged medium by litter decomposing basidiomyceteAgaricus heterocystisrdquo International Journal of Pharmacy andPharmaceutical Sciences vol 4 no 3 pp 592ndash599 2012

[31] P Keyser T K Kirk and J G Zeikus ldquoLigninolytic enzymesystem of Phanerochaete chrysosporium synthesized in theabsence of lignin in response to nitrogen starvationrdquo Journal ofBacteriology vol 135 no 3 pp 790ndash797 1978

[32] J A Buswell Y Cai and S T Chang ldquoEffect of nutrientnitrogen and manganese on manganese peroxidase and laccaseproduction by Lentinula (Lentinus) edodesrdquo FEMSMicrobiologyLetters vol 128 no 1 pp 81ndash87 1995

[33] E Rosales S R Couto and M A Sanroman ldquoIncreasedlaccase production byTrametes hirsuta grownon groundorangepeelingsrdquo Enzyme and Microbial Technology vol 40 no 5 pp1286ndash1290 2007

[34] I Y Lee K H Jung C H Lee and Y H Park ldquoEnhancedproduction of laccase in Trametes vesicolor by the addition ofethanolrdquo Biotechnology Letters vol 21 no 11 pp 965ndash968 1999

[35] A Pandey P Trivedi B Kumar B Chaurasia and L MS Palni Soil Microbial Diversity From the Himalaya NeedFor Documentation and Conservation vol 5 of NBA ScientificBulletin National Biodiversity Authority Tamil Nadu India2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology