Research Article Unraveling the Lipolytic Activity of...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 703130 13 pageshttpdxdoiorg1011552013703130

Research ArticleUnraveling the Lipolytic Activity of Thermophilic BacteriaIsolated from a Volcanic Environment

Panagiota M Stathopoulou Alexander L SavvidesAmalia D Karagouni and Dimitris G Hatzinikolaou

Microbiology Group Sector of Botany Department of Biology National and Kapodistrian University of AthensZografou Campus Zografou 15784 Attica Greece

Correspondence should be addressed to Dimitris G Hatzinikolaou dhatzinibioluoagr

Received 9 February 2013 Accepted 25 March 2013

Academic Editor George Tsiamis

Copyright copy 2013 Panagiota M Stathopoulou et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In a bioprospecting effort towards novel thermostable lipases we assessed the lipolytic profile of 101 bacterial strains isolated fromthe volcanic area of Santorini Aegean Sea Greece Screening of lipase activity was performed both in agar plates and liquid culturesusing olive oil as carbon source Significant differences were observed between the two screeningmethods with no clear correlationbetween them While the percentage of lipase producing strains identified in agar plates was only 17 lipolytic activity in liquidculture supernatants was detected for 74 of them Nine strains exhibiting elevated extracellular lipase activities were selectedfor lipase production and biochemical characterization The majority of lipase producers revealed high phylogenetic similaritywith Geobacillus species and related genera whilst one of them was identified as Aneurinibacillus sp Lipase biosynthesis stronglydepended on the carbon source that supplemented the culture medium Olive oil induced lipase production in all strains butmaximum enzyme yields for some of the strains were also obtained with Tween-80 mineral oil and glycerol Partially purifiedlipases revealed optimal activity at 70ndash80∘C and pH 8-9 Extensive thermal stability studies revealed marked thermostability for themajority of the lipases as well as a two-step thermal deactivation pattern

1 Introduction

Thermophilic microorganisms unequivocally represent avaluable source of highly thermostable enzymes with numer-ous advantages towards biotechnological applications dueto their overall inherent stability and high reaction ratesat elevated temperatures [1 2] Among them lipases (EC3113) the enzymes that catalyze both the synthesis andhydrolysis of long chain fatty acid esters (depending on wateravailability) constitute one of the most versatile and widelyused biocatalytical group [3] They are used in numerousdiverse biotechnological applications ranging from biodieseland biopolymers production to the synthesis of fine chem-icals for medical agrochemical and cosmetic applications[3ndash5] Due to this fact novel thermostable lipases are incontinuous demand for commercial applications especially indetergent food and pulp and paper industries [6] As a resultseveral thermophilic microbial strains able to produce ther-mostable lipases have been isolated [7] and the corresponding

enzymes have been purified either from the wild-type culturesupernatants [8ndash10] or following cloning and expression inmesophilic hosts [11ndash14]

Volcanic areas represent ecological niches of rich meta-bolic diversity especially among bacteria and archaea [15ndash17]and the isolation of several thermophilic microorganisms hasbeen described from such natural sources The vast majorityof these studies though is focused on the phylogeneticdiversity of the corresponding microbial communities andonly few reports are available in the literature assessing theenzymatic potential of the microbial strains isolated eitherfrom volcanic [18] or other extreme environments [19] withculture dependent or metagenomic approaches

Pursuant to the above mentioned statements screeningof thermophilic microorganisms for lipolytic activitiescould facilitate the discovery for industrial purposes ofnovel lipases that are stable and function optimally at hightemperatures In this work we examined the enzymaticlipolytic potential of 101 bacterial strains which had

2 BioMed Research International

previously been isolated from the volcanic habitat of theSantorini volcano at South Aegean Sea [20] A thoroughbiochemical screening for extracellular lipase activity wasperformed both in solid and liquid media in an attempt toestablish benchmark criteria for such an analysis The mostactive strains were selected and characterized with respect totheir 16S rDNA sequences and the effect of various carbonsources on the production of extracellular lipolytic activityFinally the pH and temperature-activity optima as well as thecorresponding thermal stability kinetics were determinedfor the partially purified and concentrated lipases from allselected isolates

2 Materials and Methods

21 Microbial Strains The 101 thermophilic bacterial strainsused in this study have been previously isolated from theseawater and sediment near the active volcano of Santoriniisland at Aegean Sea Greece [20] All isolates were preservedin 30 (wv) sterile glycerol solution at minus80∘C

22 Growth Media and Conditions All isolates were prelim-inary screened for their lipolytic activity both in solid andliquid cultures A standard inoculum was prepared for eachstrain from glycerol stocks on Nutrient Agar (Nutrient brothplus 3 wv agar) plates Following incubation for 24 h at60∘C bacterial biomass collected in sterile water and dilutedto an OD

600of 03 was served as inoculum

Solid cultures were conducted in sterile 12-well platesfilled with Rhodamine BmdashOlive Oil agar medium containing10 gsdotLminus1 olive oil (ROA) [8] Twenty 120583L of standard inoculumwas placed in the middle of each well (3 wells per strain)and the plates were incubated at 60∘C up to 48 hoursLipase production was evaluated from the diameter of theorange fluorescent halo (reaction of liberated fatty acids withRhodamine B) observed under UV exposure [21]

Liquid cultures were conducted in 100mL Erlenmeyerflasks with 20mL working volume The basal medium usedconsisted of (gsdotLminus1) NaNO

3 3 K2HPO4 1 KCl 05 CaCl

2

01 MgSO4sdot7H2O 05 yeast extract 1 trace elements solu-

tion 1mLsdotLminus1 supplemented with 20 gsdotLminus1 olive oil as solecarbon and energy source Triplicate flasks were inoculatedfor each strain with 05mL standard inoculum and all flaskswere incubated in a thermostated orbital shaker (SANYOBiomedical UK) at 60∘C and 180 rpm Evaporation was dailychecked gravimetrically and compensated with the additionof sterile water Samples were collected every 24 h for thedetermination of biomass concentration and extracellularlipolytic activity The effect of other carbon sources onlipase production was determined in a similar manner bysubstituting olive oil with either glucose mineral oil corncob wheat bran eicosane Tween-80 or glycerol at an initialconcentration of 20 gsdotLminus1 Lipase production was followed byassaying of the lipase activity in the supernatant as describedin enzyme assays All lipolytic activities were determined intriplicate and reported as averages

23 Molecular Characterization of Isolates Bacterial identifi-cation of the nine selected isolates was based on 16S rDNAsequence analysis Genomic DNA extraction from liquidcultures was performed according to Haught et al [22] andthe quantity of the isolated DNA was determined photomet-rically [23]The 16S rDNAwas amplified by polymerase chainreaction (PCR) using two universal primers [24 25]

pA (51015840-AGA GTT TGA TCC TGG CTC AG-31015840) and

R1492 (51015840-TACGGYTACCTTGTTACGACTT-31015840)

Amplification reactions were performed in volumes of50120583L containing 40 ng templateDNA 04 120583Mof each primer1X buffer with Mg2+ 1 U KAPA Taq DNA polymerase(Kapa Biosystems Woburn MA USA) and 02mM dNTPsNucleases free water was used to bring the reaction vol-ume to 50 120583L After initial denaturation at 95∘C for 2minsamples were cycled for 30 PCR cycles using the followingcycle profile 95∘C denaturation for 30 s primer annealingat 53∘C for 30 s and primer extension at 72∘C for 2minplus a final 2min elongation step at 72∘C Amplified PCRproducts were separated by gel electrophoresis on 12 (wv)agarose gel and then purified using NucleoSpin ExtractPCR kit (MACHEREY-NAGEL Germany) The 16S rDNAfragment (gt1400 bp) was fully sequenced in both direc-tions (Macrogen Republic of Korea) and the correspond-ing gene sequences have been submitted to the GenBankdatabases under accession numbers JQ808132 to JQ808140(httpwwwncbinlmnihgov) The 16S sequences wereused for the determination of the evolutionary history (phy-logenetic tree) of the nine isolates using theNeighbor-Joiningmethod [26] in MEGA5 software [27] The percentage ofreplicate trees in which the associated taxa clustered togetherwas calculated by the bootstrap test (1000 replicates) [28]Evolutionary distances were computed using the MaximumComposite Likelihood method [29]

24 Preparation of Partially Purified Lipase Samples Liquidcultures of the nine selected strains were conducted in 2 LErlenmeyer flasks of 400mL working volume (3 flasks perstrain) as described above Biomass was removed by cen-trifugation (6000 rpm 5min) and the resulting supernatantwas passed through Whatman number 1 filter paper inorder to remove the residual olive oil The filtrate was thensubjected to ammonium sulphate precipitation Optimumprecipitation range was determined by employing a gradualammonium sulphate procedure (5 saturation step) andmeasuring specific lipase activity on the resulting super-natants and precipitates The optimum precipitation condi-tions were subsequently applied to each culture supernatantand the collected precipitates were resuspended in 20mMpotassium phosphate buffer (pH 8) and desalted using PD-10 gel filtration columns (GE Healthcare) using the abovebuffer for elution The concentrated and desalted enzymesolutionswere used for the determination of the optimumpHand temperature and thermal stability of the correspondinglipolytic activities

BioMed Research International 3

Table 1 Comparison of the lipolytic activities among the 101thermophilic bacterial isolates in Rhodamine olive oil agar (ROA)and olive oil liquid (OLM) cultures

Strain ROA OLM24 h 48 h 48 h 72 h

SP1 minus minus minus minus

SP2 minus minus 0040 0063SP3 minus minus minus minus

SP4 minus + 0062 0102SP5 minus minus minus minus

SP6 + + 0058 0073SP7 minus minus 0250 0085SP8 minus minus 0250 0270SP9 minus minus 0307 0167SP10 minus minus 0500 0200SP11 minus minus 0358 0217SP12 minus minus 0350 0142SP13 minus minus 0583 0175SP14 minus minus 1070 2367SP15 minus minus 0108 0147SP16 minus minus 0022 0065SP17 minus minus minus minus

SP18 minus minus 0200 0067SP19 minus minus 0217 0248SP20 minus minus 0153 0055SP21 minus minus 0550 0427SP22 minus minus 1618 0450SP23 minus minus 0567 0192SP24 minus minus 0217 0133SP25 minus + 0044 0092SP26 minus minus minus minus

SP27 + + 0150 0288SP28 minus minus minus minus

SP29 minus minus 0967 0620SP30 minus + 0422 1027SP31 minus minus minus minus







SP42 minus minus 0450 0192SP43 minus minus 0120 0072SP44 minus minus 0120 0123SP45 minus minus 0153 0088SP46 minus minus minus minus

SP47 minus minus 0038 0045

Table 1 Continued







SP63 minus minus 0038 0053SP64 minus minus 0057 0000SP65 minus minus 0045 0043SP66 minus minus 0115 0047SP67 minus minus 0053 0075SP68 minus minus minus minus


SP70 minus minus 0043 0070SP71 + + 0043 0052SP72 minus minus 0208 0244SP73 + ++ 1083 0740SP74 minus minus 0088 0000SP75 + ++ 1540 1667SP76 minus minus 1297 1317SP77 minus minus 0483 0228SP78 minus minus 0220 0150SP79 minus minus 1167 1245SP80 + + 0137 0110SP81 minus minus 0543 0293SP82 minus + 0235 0250SP83 + + 0625 1433SP84 minus + 0167 0133SP85 minus minus 0077 0000SP86 minus minus 0063 0070SP87 minus + 0058 0000SP88 minus minus minus minus


SP90 minus + 0593 0233SP91 minus minus 0517 0767SP92 + + 0517 0522SP93 ++ ++ 0610 0633SP94 minus minus 0550 0257SP95 minus minus 1027 0600SP96 minus minus 0073 0085


Table 1 Continued





SP100 minus minus 0400 0642SP101 minus minus 0000 0122InROAcultures qualitative evaluationwas performed based on fluorescencehalo production (minus no halo + partial coverage of the plate ++ fullcoverage of the plate) In OLM cultures values represent the extracellularlipolytic activity in nkatsdotmLminus1 (average of triplicate flasks max SD plusmn 94)Bolditalicized lines represent those strains selected for further study

25 Enzyme Assays Lipase activity assays in liquid sampleswere routinely performed using p-nitro phenyl palmitate(pNPP) as substrate in 50mM Tris-HCl buffer pH 8essentially as described by Vorderwulbecke et al [30] withminor modifications pNPP was dissolved in propane-2-olat a final concentration of 75mgsdotmLminus1 The final substratesolution (950 120583L) was prepared by dropwise addition undercontinuous vortexing of 95120583L pNPP solution into 855120583Lof buffer solution (prepared by dissolving 04 g of Triton X-100 and 01 g gum arabic in 90mL of buffer) The reactionwas initiated by adding 50120583L sample into the above substratesolution followed by incubation in a water bath at 60∘Cfor 15min The reaction was terminated by placing thereaction mixture on ice for 10min p-nitro phenol (pNP)concentration was determined by measuring the absorbanceat 410 nm using a calibration curve constructed at the assaybuffer (pH 8) For blank we used heat sterilized (15min121∘C) samples undergone the same procedure Activity wasexpressed as nkatsdotmLminus1

26 Determination of Lipase Thermal Stability and ActivityOptima Optimum temperature for lipase activity was deter-mined under the above described assay conditions at pH 8using an incubation temperature range from 50 to 100∘COptimum pH values were determined in the same mannerat 60∘C using an assay buffer range from 6 to 10 A differentcalibration curve was constructed and used for pNP at eachpH value used For temperature stability enzyme sampleswere incubated in sealed screw-cap vials at pH 7 (phosphatebuffer 100mM) at various temperatures sample aliquotswere withdrawn periodically and remaining activity wasdetermined at regular assay conditions All enzyme activitieswere determined in triplicate and reported as averages

3 Results and Discussion

31 Detection of Lipolytic Activity The lipolytic activities of allisolates were assessed both qualitatively (agar plate cultures)and quantitatively (liquid cultures) using olive oil as sole car-bon and energy source The corresponding results are sum-marized inTable 1Only 17 out of 101 (168) strains producedclear fluorescent halos designating free fatty acid release dur-ing growth on ROA plates On the contrary in liquid cultures

significant lipolytic activity (gt005 nkatmL) was detected inthe culture supernatant of 56 strains (554) while for 19strains (189) the corresponding average activities were verylow (lt005 nkatmL) but detectable For 26 strains (257) noactivity was detected No clear correlation could be identifiedamong the two screening methods used As a result strainssuch as SP6 and SP71 yielded clear fluorescent halos duringgrowth on plates while the same strains produced only lowlevels of lipase in liquid cultures in addition strains like SP14SP22 SP29 SP76 and SP79 revealed the opposite lipolyticphenotype (no halo on ROA high extracellular lipolyticactivity in OLM) Only four out of the 101 strains namelySP73 SP75 SP83 and SP93 revealed an equivalent lipolyticprofile both on solid and liquid cultures In addition wecould not descry a single strain yielding fluorescence haloson Rhodamine B agar plates without displaying even a smallamount of extracellular lipolytic activity in liquid cultures

The Rhodamine B plate assay has been widely used inthe past by many researchers as the sole screening methodfor the identification of lipase producing microorganismsfrom various environmental samples [31] including extremeenvironments such as hot springs and soda lakes [32ndash34]Our results clearly demonstrate the fact that the solid-phase Rhodamine system has failed to detect all the lipaseproducing microorganisms even though olive oil (a truelipase inducer) was employed as sole carbon and energysource Liquid cultures on olive oil combined with an extra-cellular enzyme assay proved more reliable in identifying thelipolytic potential of the strains under consideration Thelower sensitivity of Rhodamine assay that is sometimes usedto explain the discrepancy between the two approaches canbe easily overruled in our case by the fact that we were ableto identify strains that yielded very clear halos with onlytraces of lipase activity in liquid culturesThese results clearlyshow that liquid culture screening on various lipase inducersyieldsmore reliable results and should be preferred especiallytoday where several high throughput liquid enzymatic assayscreening methods are being available [35 36]

32 Phylogeny of Lipase Producers Following the resultsof this primary screening we decided to proceed withour work by selecting those strains that either excretedhigh average extracellular lipolytic activity in liquid cultures(gt075 nkatsdotmLminus1) or produced intense fluorescent halosin Rhodamine media The nine strains that fulfill thesecriteria are in bold italics in Table 1 The selected strainswere subjected to 16S rDNA sequencing (NCBI Accessionnumbers JQ808132 to JQ808140) and phylogenetic analysisA phylogenetic tree constructed using the correspondingsequences along with those from annotated Geobacillus andother related genera is given in Figure 1 All nine lipolyticstrains revealed great phylogenetic similarity (gt99) withknownGeobacillus and other closely related speciesThis high16S rDNA similarity is characteristic of this relatively recentlycharacterized genus [37] that includes species with promisingbiotechnological potential [38] Members of geobacilli havebeen reported to represent the majority of the species thatinhabit a variety of diverse ldquohotrdquo environments such as


SP73 (JQ808135)

SP93 (JQ808140)

SP75 (JQ808136)

SP22 (JQ808133)

SP79 (JQ808138)

SP76 (JQ808137)

SP29 (JQ808134)

SP14 (JQ808132)

SP83 (JQ808139)

93100

91

88

42

52

45

41

54

75

85

83

100

100

99

87

74

100

001

Geobacillus kaustophilus NBRC 102445 (AB681787)

Geobacillus kaustophilus L17 (FJ823106)

Geobacillus lituanicus N-3 (AY044055)

Geobacillus zalihae NBRC 101842 (AB681570)

Geobacillus stearothermophilus NBRC 100862 (AB681268)

Geobacillus jurassicus DS1 (AY312404)

Geobacillus subterraneus 34 (T) (AF276306)

Geobacillus kaue BGSC W9A78 (AY608975)

Geobacillus thermoglucosidasius ATCC43742 (AB021197)

Anoxybacillus amylolyticus MR3C (AJ618979)

Aeribacillus pallidus DSM 3670 (Z26930)

Aeribacillus pallidus NCCP-131 (AB543491)

Bacillus subtilis DSM10 (AJ276351)

Aneurinibacillus danicus NBRC 102444 (AB681786)

Aneurinibacillus thermoaerophilus DSM 10154T (AB112726)

Geobacillus vulcani BCRC 17563 (EU484349)

Geobacillus kaustophilus HTA426 (NC 006510)

Geobacillus thermoleovorans NP33 (JQ343209)

Figure 1 Evolutionary relationships of the selected strains using the Neighbor-Joining method The bootstrap consensus tree was inferredfrom 1000 replicates Branches corresponding to partitions reproduced in less than 50 bootstrap replicates are collapsed The percentageof replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches Thetree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic treeThe evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of basesubstitutions per site All positions containing gaps and missing data were eliminated

marine thermal vents [39 40] high temperature oil fields[37] hot springs [41] geothermal regions [42] and sugarrefinery wastewaters [43]

In our case three of the strains namely SP73 SP75 andSP93 have been clustered together with known Geobacilluskaustophilus strainsmost probably representing strains of thisspecies To our knowledge there are no published reportson lipolytic activities from G kaustophilus related strains

although it was possible to identify a provisional putativelipase gene in the G kaustophilusHTA426 complete genomesequence (NCBI Accession number NC 006510 Gene ID3185865) Isolates SP22 and SP79 that revealed identical 16SrDNA partial sequences clustered together with close phylo-genetic similarity to the G kaustophilus group while isolatesSP76 and SP29 can clearly be characterized as members ofthe Geobacillus group closely related to G thermoleovorans


SP14 SP22 SP29 SP73 SP75 SP76 SP79 SP83 SP93

Glycerol

Eicosane

Wheat bran

Tween-80

Mineral oil

Olive oil

Glucose

118 11 143 34

2861872413822

488 113

11562156121128185

165 159 103 348 123 28

158116148461129161205264

178 192 412

141

Figure 2 Effect of carbon source type on maximum extracellular lipase production (nkatsdotmLminus1) in liquid cultures achieved between 48 and72 h of growth depending on the source used Circle areas are proportional to enzyme activities while only activities above 1 nkatsdotmLminus1 areshown with numerical values Black circles denote no detection of lipolytic activity Values represent the mean of triplicate flasks MaximumSD plusmn 182

and G kaue respectively G thermoleovorans is a knownlipase producer among the geobacilli and the correspondingenzymes from various strains of this species have beenpurified and characterized either from wild-type cultures[9 44] or following heterologous expression in E coli [1245 46] On the contrary there is no information in theliterature concerning lipolytic activity by G kaue strainsIsolate SP14 clustered togetherwith the recently characterizedAeribacillus pallidus species the only characterized memberin the Aeribacillus taxonomic group [47] Concerning lipaseproduction only an A pallidus strain isolated from thermalsites in Mexico has been reported to express lipolytic activityin ROA plates [48] although this was not the case for strainSP14 that showed high extracellular lipase activity only inliquid cultures Finally strain SP83 revealed an identical16S rDNA sequence with Aneurinibacillus thermoaerophilusDSM 10154 representing the most phylogenetically distantmember among the lipase producers in our study TheAneurinibacillus genus does not belong to the Bacillaceaefamily (like Geobacillus and Aeribacillus) since is a memberof the Paenibacillaceae family [49] Strains related to thisgroup have been isolated from geothermal soils throughoutthe globe [50 51] while lipase production has been optimizedin submerged cultures of an A thermoaerophilus strainisolated from a Malaysian hot spring [52]

33 Lipase Production Pattern Theeffect of carbon source onlipase production by the nine selected isolates was studiedusing the minimal basal medium supplemented with aninitial concentration of 20 gsdotLminus1 of various carbon sourcesSamples were withdrawn twice a day and the lipase activitywas determined in the cell free supernatant Growth ofthe microorganisms was observed in all cases as verifiedby either OD

600or cfu measurements (data not shown)

Figure 2 summarizes the corresponding lipase productionpattern which appeared to be quite diverse among the nine

isolates Olive oil a well-known lipase inducer was able toinduce significant lipase production levels in all nine strainsalthough it proved to be the optimal carbon source onlyfor strain SP75 These results justify its use as the preferredcarbon source in various screening studies for lipolyticactivity in thermophilic bacteria [7] or in studies concerningthe optimization of lipase production using wild type strains[53] Tween-80 an oleic acid ester (Polyoxyethylene sorbitanmonooleate) yielded maximum lipase titers in strains SP79and SP83 but it completely inhibited lipase production instrains SP22 and SP75 Its addition in culture media for lipaseproduction by members of the Bacillaceae family has oftenbeen evaluated exhibiting either inducing [54ndash56] or inhibit-ing effect [8 53 57 58] Optimum lipase production withTween-80 as sole carbon source has been also achieved with aG stearothermophilus strain following medium optimization[59] All but one strains were able to produce at least somelipase activity in the presence of pure alkanes (eicosane)or alkane mixtures (mineral oil) as sole carbon sourcesStimulation of lipase production by aliphatic hydrocarbonsin bacteria although documented several decades ago [60]has been reported only once for members of the Bacillaceaefamily [44] It is also noteworthy that eicosane and mineraloil revealed very similar lipase production patterns amongthe nine strains (Figure 2) suggesting that lipase biosynthesisby alkanes in thermophilic bacteria is induced in a mannerindependent of their chain length

The two low molecular weight compounds examinedglycerol and glucose showed limited lipase induction abilitywith two notable exceptions of an apparent inducing effectthat of glycerol for strain SP93 and that of glucose for strainSP79 A repressing effect of glucose [55 61] or glycerol [62] onlipase production by members of the Bacillaceae family hasbeen reported in several studies but this does not seem to be auniversal phenomenon since both glucose and glycerol werefound to be excellent carbon sources for lipase productionby the G stearothermophilus strain-5 [59 63] Wheat bran


a complex agricultural byproduct was also examined on thebasis of its use in several reports as an inducer of lipaseactivity by various fungi [64 65] despite the fact that there areno references concerning its use as carbon source for lipaseproduction in bacteria Wheat bran was indeed a poor lipaseinducer for the majority of the bacterial strains but quitesurprisingly it was proved to be the optimal carbon sourcefor the Aeribacillus sp strain SP14 The latter isolate was alsoamong the three strains that were able to produce lipase in allcarbon sources examined the other two being strains SP76and SP79

Lipase production among the nine isolates did not revealany specific correlations with their evolutionary relation-ships Phylogenetically close isolates such as SP22 and SP79showed quite diverse lipolytic activity patterns while theoppositewas observedwith several other pairs of isolates suchas SP14 and SP79Theonlyweak relation involved the threeGkaustophilus strains of our study (SP73 SP75 and SP93) thatproved to be the poorest lipase producers both in substratediversity and maximum observed lipolytic activities It isnoteworthy though to point out that the maximum lipaseactivities obtained in this experimental set are comparableto those reported in studies concerning the optimization ofmediumculture parameters for lipase production by variousthermophilic Bacillus andGeobacillus strains [53] despite thefact that we did not perform any optimization efforts

34 Assessment of Lipolytic Activities Following partialpurification from the corresponding culture supernatants weperformed a biochemical characterization of the nine lipasesby determining their temperature and pH-activity optima(Figure 3) as well as their thermostability kinetics (Figure 4)All lipases had a relatively basic pH optimum between 8 and9 The functional pH range was relatively narrow between 7and 95 with most of the enzymes showing a sharp decreasein the activity at pH 10 The lipases from strains SP22 andSP83 were the most alkalophilic showing optimum activityat pH 9 and maintaining more than 30 of their activityat pH 10 These pH optima are similar to those reportedfor several Bacillus species [8 11 66 67] although the pH-activity profiles for the majority of mesophilic Bacilli arewider probably because pH effects are being determined atmoderate temperatures between 20 and 30∘C As far as thelipases from other geobacilli are concerned the reported pHoptima are also 8 [32 63 68] or 9 [12 13 69 70] indicating auniversal lipolytic pH-activity pattern within the genus

The temperature optima determined for the nine partiallypurified enzymes were relatively high and reached the levelof either 70 (4 isolates) or 80∘C (5 isolates) The lipases forstrains SP14 (A pallidus) SP22 SP73 SP79 (G kaustophilus)and SP29 (G kaue) had the highest temperature-activityoptimamaintaining (with the exception of isolate SP79)morethan 80 of their optimum activity even at the temperatureof 90∘C These lipolytic activity optima are significantlyhigher than those reported for other geobacilli More specif-ically the optimum reported activity temperatures were55∘C for the G stearothermophilus JC [13] and Geobacillussp TW1 [32] lipases 60∘C for Geobacillus sp SBS-4S [69]

and G thermoleovorans CCR11 [12] 65∘C for Geobacillus spstrain ARM [68] G stearothermophilus strain-5 [63] and Gthermoleovorans [45 71] and 70∘C for the lipase ofGeobacillussp T1 [70] and G zalihae [7] These results indicate that theSantorini volcanic habitat is most probably an environmentthat hosts bacterial species that harbor enzymatic activitiesof exceptional thermophilicity

The above promising results concerning the temperatureoptima of the nine partially purified lipases led us to pursuea thorough study on their thermal stability properties in 10∘Cincrements In agreement with their high optimum tempera-tures the lipases from the Santorini geobacilli revealed verysignificant thermal stability properties With the exception ofthe lipases from isolates SP75 and SP76 that maintained 82and 75 of their activity respectively after 1 h incubation at60∘C all other lipases maintained over 90 of their activity atthis temperature and incubation time (data not shown) Forthe temperature range from 70 to 100∘C thermal deactivationcurves are given in Figure 4 Analysis of the time point datafor all enzymes using SigmaPlot software revealed that firstorder deactivation was not able to adequately describe thedeactivation kinetics of the lipases at various temperaturesIn an effort to find the best fit model we applied the approachof Henley and Sadana [72] where lipase thermal inactivationis sought to take place following a two-step deactivationprocess through an intermediate state as follows

119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864

2are the specific activities of the initial

intermediate and final state respectively and 1198961and 119896

2are

first-order deactivation constants By defining 1205721= 119864

1119864

0

and 1205722= 119864

2119864

0and assuming that the 119864

2state is completely

inactive (1198862= 0) the observed residual enzyme activity as a

function of time 120572 is given by (2) following integration overtime [72]

120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)

The fitting of the experimental data into (2) for each lipaseand temperature was performed using the nonlinear regres-sion routines of Sigmaplot software and the results arepresented in Figure 4 and Table 2 Regression was allowedto converge freely into the least-squares value and the onlyrestriction that was applied concerned the 120572

1specific activity

ratio that was confined between the values of 0 and 1All lipases seemed to follow the two-step deactiva-

tion scheme since we observed an excellent fitting of theexperimental data into (2) This means that the enzymesupon the effect of temperature undergo a relatively rapidinactivation into the 119864

1intermediate form which has lower

activity compared to the native enzyme (1198640)Thedeactivation

rate though from the intermediate form to the completelyinactive enzyme (119864

2) is significantly slower advocating for a

more compact and heat resistant character for the1198641enzyme


40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

Figure 3 Effect of pH () and temperature (I) on the activity of the partially purified lipases from the nine selected strains Error barsrepresent the SD from triplicate determinations


0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400

Figure 4 Thermal stability profiles of the nine partially purified lipases 998779 70∘C 998771 80∘C ◻ 90∘C ◼ 100∘C Straight lines represent thenon-linear fitting of (2) in the experimental data


Table 2 Values of the kinetic constants for the thermal deactivation kinetics as determined through nonlinear regression fitting of theexperimental data in (2) Activation energies for k1 and k2 were determined from the Arrhenius equation through linear regression

SP14 SP22 SP29 SP73 SP75

120572

1

70∘C 0647 plusmn 0021 0589 plusmn 0060 0662 plusmn 0055 0740 plusmn 0021 0267 plusmn 003080∘C 0759 plusmn 0007 0879 plusmn 0035 0673 plusmn 0040 0726 plusmn 0028 0459 plusmn 004490∘C 0690 plusmn 0026 0687 plusmn 0026 0566 plusmn 0031 0562 plusmn 0059 0335 plusmn 0005100∘C 0328 plusmn 0027 0432 plusmn 0006 0500 plusmn 0011 0399 plusmn 0034 0302 plusmn 0009

119896

1

(minminus1)

70∘C 224 plusmn 037 times 10minus2 506 plusmn 114 times 10minus3 143 plusmn 044 times 10minus2 321 plusmn 074 times 10minus2 254 plusmn 030 times 10minus1



100∘C 359 plusmn 040 times 10minus1 881 plusmn 037 times 10minus1 647 plusmn 054 times 10minus1 627 plusmn 127 times 10minus1 142 plusmn 008 times 100

119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)

70∘C 182 plusmn 027 times 10minus4 942 times 149 times 10minus5 826 plusmn 113 times 10minus4 706 plusmn 062 times 10minus4 359 plusmn 133 times 10minus4




119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1

70∘C 0493 plusmn 0055 0639 plusmn 0013 0627 plusmn 0026 0662 plusmn 000680∘C 0377 plusmn 0008 0384 plusmn 0047 0458 plusmn 0029 0380 plusmn 001190∘C 0270 plusmn 0048 0330 plusmn 0022 0254 plusmn 0053 0366 plusmn 0083100∘C 0176 plusmn 0054 0313 plusmn 0013 0491 plusmn 0052 0271 plusmn 0065

119896

1

(minminus1)

70∘C 536 plusmn 121 times 10minus2 723 plusmn 163 times 10minus2 412 plusmn 053 times 10minus2 257 plusmn 012 times 10minus2



100∘C 368 plusmn 058 times 10minus1 505 plusmn 027 times 10minus1 119 plusmn 047 times 101 187 plusmn 046 times 100

119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559

This was verified by the fact that for all enzymes and at alltemperatures examined the values of the deactivation con-stant 119896

2were on average two orders of magnitude lower than

the corresponding 1198961levels (Table 2) With the exceptions of

the lipases from isolates SP75 and SP76 above mentionedall other enzymes showed exceptional thermal stabilities thatallowed clear detection of activity even after 1 h incubation at100∘C The most thermostable enzymes were those of strainsSP14 SP22 SP29 and SP73 The most significant differenti-ation of these four lipases that seemed to contribute in theirobserved high thermal stability was the fact that the specificactivity ratio 120572

1for these enzymes was markedly higher

than the other five lipases and more importantly its valuewas practically stable in the temperature range from 70 to90∘C As was the case with their temperature-activity optimathe lipases from the Santorini thermophilic bacilli were alsomore thermostable than the majority of other Geobacillussp lipases reported in the literature Although this is thefirst extensive study on the thermal stabilities for Geobacilluslipases literature data fully support our claim since completeloss of activity has been reported for the G zalihae lipase

following 30min incubation at 80∘C [7] the Geobacillus spSBS-4S lipase when exposed for 100min at 60∘C [69] and thelipase fromG stearothermophilus JC after 30min at 70∘C [13]while the Geobacillus sp strain ARM lipase had half lives ofonly few minutes at 60 and 70∘C [68]

4 Conclusions

An extensive assessment of the lipolytic potential in a set ofthermophilic bacteria isolated from the Santorini volcanichabitat was performed By combining both solid and liquidculture techniques a widespread lipolytic phenotype of vari-able intensity was revealed that involved almost 75 of theisolates The genus Geobacillus was the dominant one withinthe subset of nine strains selected for their high extracellularlipase production Enzyme levels were strongly dependent onthe type of carbon source but we were not able to clearlyidentify a universal phenotype for lipase induction among thestrains The biochemical characterization of the nine lipasesmarked a significant differentiation tomost otherGeobacillus


lipases with respect to the effect of temperature on the activityand stability of the corresponding enzymes The majorityof lipases from the Santorini thermophilic bacteria revealedexceptional thermostability with high optimum activitytemperatures thus representing very promising candidateenzymes for a variety of high temperature industrial lipolyticapplications Such an endeavor would probably require theirefficient cloning and overexpression in mesophilic hostseven though that for some of the strains lipase productionwas at relatively high levels compared to other wild-typethermophilic bacterial strains

Conflict of Interests

All the authors of the paper do not have a direct financialrelation with any identity mentioned in the paper that mightlead to a conflict of interests

References

[1] C Vieille and G J Zeikus ldquoHyperthermophilic enzymessources uses and molecular mechanisms for thermostabilityrdquoMicrobiology and Molecular Biology Reviews vol 65 no 1 pp1ndash43 2001

[2] P Turner G Mamo and E N Karlsson ldquoPotential and utiliza-tion of thermophiles and thermostable enzymes in biorefiningrdquoMicrobial Cell Factories vol 6 p 9 2007

[3] R Gupta N Gupta and P Rathi ldquoBacterial lipases an overviewof production purification and biochemical propertiesrdquoApplied Microbiology and Biotechnology vol 64 no 6 pp763ndash781 2004

[4] K-E Jaeger and T Eggert ldquoLipases for biotechnologyrdquo CurrentOpinion in Biotechnology vol 13 no 4 pp 390ndash397 2002

[5] H Treichel D de Oliveira M A Mazutti M Di Luccio andJ V Oliveira ldquoA review on microbial lipases productionrdquo Foodand Bioprocess Technology vol 3 no 2 pp 182ndash196 2010

[6] M Salameh and J Wiegel ldquoLipases from extremophiles andpotential for industrial applicationsrdquo Advances in AppliedMicrobiology vol 61 pp 253ndash283 2007

[7] R N Abd Rahman T C Leow A B Salleh and M BasrildquoGeobacillus zalihae sp nov a thermophilic lipolytic bacteriumisolated from palm oil mill effluent in Malaysiardquo BMCMicrobi-ology vol 7 p 77 2007

[8] C Schmidt-Dannert H Sztajer W Stocklein U Menge andR D Schmid ldquoScreening purification and properties of a ther-mophilic lipase from Bacillus thermocatenulatusrdquo Biochimica etBiophysica Acta vol 1214 no 1 pp 43ndash53 1994

[9] L D Castro-Ochoa C Rodrıguez-Gomez G Valerio-Alfaroand R Oliart Ros ldquoScreening purification and characterizationof the thermoalkalophilic lipase produced by Bacillus ther-moleovorans CCR11rdquo Enzyme and Microbial Technology vol 37no 6 pp 648ndash654 2005

[10] M A Salameh and JWiegel ldquoPurification and characterizationof two highly thermophilic alkaline lipases from Thermosyn-tropha lipolyticardquo Applied and Environmental Microbiology vol73 no 23 pp 7725ndash7731 2007

[11] S Sinchaikul B Sookkheo S Phutrakul F M Pan and ST Chen ldquoOptimization of a thermostable lipase from Bacillusstearothermophilus p1 overexpression purification and charac-terizationrdquo Protein Expression and Purification vol 22 no 3 pp388ndash398 2001

[12] RQuintana-Castro P Dıaz G Valerio-AlfaroH S Garcıa andR Oliart-Ros ldquoGene cloning expression and characterizationof the Geobacillus thermoleovorans CCR11 thermoalkaliphiliclipaserdquoMolecular Biotechnology vol 42 no 1 pp 75ndash83 2009

[13] Y Jiang X Zhou and Z Chen ldquoCloning expression andbiochemical characterization of a thermostable lipase fromGeobacillus stearothermophilus JCrdquoWorld Journal of Microbiol-ogy and Biotechnology vol 26 no 4 pp 747ndash751 2010

[14] T C Leow R N Abd RahmanM Basri andA B Salleh ldquoHighlevel expression of thermostable lipase from Geobacillus spstrain T1rdquo Bioscience Biotechnology and Biochemistry vol 68no 1 pp 96ndash103 2004

[15] R J Martinez H J Mills S Story and P A Sobecky ldquoProkary-otic diversity and metabolically active microbial populations insediments from an active mud volcano in the Gulf of MexicordquoEnvironmental Microbiology vol 8 no 10 pp 1783ndash1796 2006

[16] M G Pachiadaki V Lykousis E G Stefanou and K AKormas ldquoProkaryotic community structure and diversity in thesediments of an active submarine mud volcano (Kazan mudvolcano EastMediterranean Sea)rdquo FEMSMicrobiology Ecologyvol 72 no 3 pp 429ndash444 2010

[17] M I Prokofeva I V Kublanov O Nercessian et al ldquoCultivatedanaerobic acidophilicacidotolerant thermophiles from terres-trial and deep-sea hydrothermal habitatsrdquo Extremophiles vol 9no 6 pp 437ndash448 2005

[18] I V Kublanov A A Perevalova G B Slobodkina et al ldquoBiodi-versity of thermophilic prokaryotes with hydrolytic activities inhot springs of Uzon caldera Kamchatka (Russia)rdquo Applied andEnvironmental Microbiology vol 75 no 1 pp 286ndash291 2009

[19] G F da Cruz C F F Angolini L G De Oliveira et al ldquoSearch-ing for monooxygenases and hydrolases in bacteria from anextreme environmentrdquoAppliedMicrobiology and Biotechnologyvol 87 no 1 pp 319ndash329 2010

[20] CMeintanis K I Chalkou K A Kormas andAD KaragounildquoBiodegradation of crude oil by thermophilic bacteria isolatedfrom a volcano islandrdquo Biodegradation vol 17 no 2 pp 105ndash1112006

[21] G Kouker and K E Jaeger ldquoSpecific and sensitive plate assayfor bacterial lipasesrdquo Applied and Environmental Microbiologyvol 53 no 1 pp 211ndash213 1987

[22] C Haught D L Wilkinson K Zgafas and R G HarrisonldquoA method to insert a DNA fragment into a double-strandedplasmidrdquo BioTechniques vol 16 no 1 pp 46ndash48 1994

[23] J Sambrook E F Fritsch and T Maniatis Molecular CloningA Laboratory Manual Cold Spring Harbor Laboratory ColdSpring Harbor NY USA 1989

[24] U Edwars T Rogall H Blocker M Emde and E C BottgerldquoIsolation and direct complete nucleotide determination ofentire genes Characterization of a gene coding for 16S riboso-mal RNArdquoNucleic Acids Research vol 17 no 19 pp 7843ndash78531989

[25] D J Lane ldquo16S23S rRNA Sequencingrdquo in Nucleic AcidTechniques in Bacterial Systematics E Stackebrandt and MGoodfellow Eds pp 115ndash147 John Wiley amp Sons New YorkNY USA 1991

[26] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[27] K Tamura D Peterson N Peterson et al ldquoMEGA5 molecu-lar evolutionary genetics analysis using maximum likelihoodevolutionary distance and maximum parsimony methodsrdquo


Molecular Biology and Evolution vol 28 no 10 pp 2731ndash27392011

[28] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 no 4 pp 783ndash791 1985

[29] K Tamura M Nei and S Kumar ldquoProspects for inferringvery large phylogenies by using the neighbor-joining methodrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 30 pp 11030ndash11035 2004

[30] T Vorderwulbecke K Kieslich andH Erdmann ldquoComparisonof lipases by different assaysrdquoEnzyme andMicrobial Technologyvol 14 no 8 pp 631ndash639 1992

[31] Z Shu R Lin H Jiang Y Zhang M Wang and J HuangldquoA rapid and efficient method for directed screening of lipase-producing Burkholderia cepacia complex strains with organicsolvent tolerance from rhizosphererdquo Journal of Bioscience andBioengineering vol 107 no 6 pp 658ndash661 2009

[32] H Li and X Zhang ldquoCharacterization of thermostable lipasefrom thermophilicGeobacillus sp TW1rdquo Protein Expression andPurification vol 42 no 1 pp 153ndash159 2005

[33] N S AbdulHamidH B ZenO B Tein YMHalifahN Saariand F Abu Bakar ldquoScreening and identification of extracellularlipase-producing thermophilic bacteria from a Malaysian hotspringrdquo World Journal of Microbiology and Biotechnology vol19 no 9 pp 961ndash968 2003

[34] V A Vargas O D Delgado R Hatti-Kaul and B MattiassonldquoLipase-producing microorganisms from a Kenyan alkalinesoda lakerdquo Biotechnology Letters vol 26 no 2 pp 81ndash86 2004

[35] J-L Reymond and P Babiak ldquoScreening systemsrdquo in WhiteBiotechnology R Ulber and D Sell Eds pp 31ndash58 SpringerBerlin Germany 2007

[36] D Navarro M Couturier G G D da Silva et al ldquoAutomatedassay for screening the enzymatic release of reducing sugarsfrommicronized biomassrdquoMicrobial Cell Factories vol 9 p 582010

[37] T N Nazina T P Tourova A B Poltaraus et al ldquoTaxo-nomic study of aerobic thermophilic bacilli descriptions ofGeobacillus subterraneus gen nov sp nov and Geobacillusuzenensis sp nov from petroleum reservoirs and transfer ofBacillus stearothermophilus Bacillus thermocatenulatus Bacil-lus thermoleovoransrdquo International Journal of Systematic andEvolutionary Microbiology vol 51 no 2 pp 433ndash446 2001

[38] G McMullan J M Christie T J Rahman I M Banat N GTernan and R Marchant ldquoHabitat applications and genomicsof the aerobic thermophilic genus Geobacillusrdquo BiochemicalSociety Transactions vol 32 no 2 pp 214ndash217 2004

[39] T L Maugeri C Gugliandolo D Caccamo and E Stacke-brandt ldquoA polyphasic taxonomic study of thermophilic bacillifrom shallow marine ventsrdquo Systematic and Applied Microbiol-ogy vol 24 no 4 pp 572ndash587 2001

[40] T L Maugeri C Gugliandolo D Caccamo and E Stacke-brandt ldquoThree novel halotolerant and thermophilicGeobacillusstrains from shallow marine ventsrdquo Systematic and AppliedMicrobiology vol 25 no 3 pp 450ndash455 2002

[41] S Canakci K Inan M Kacagan and A O Belduz ldquoEvaluationof arabinofuranosidase and xylanase activities of Geobacillusspp isolated from some hot springs in Turkeyrdquo Journal ofMicrobiology and Biotechnology vol 17 no 8 pp 1262ndash12702007

[42] A Cihan B Ozcan N Tekin et al ldquoPhylogenetic diversityof isolates belonging to genera Geobacillus and Aeribacillusisolated from different geothermal regions of Turkeyrdquo World

Journal of Microbiology and Biotechnology vol 27 no 11 pp2683ndash2696 2011

[43] S-K Tai H-P Lin J Kuo and J K Liu ldquoIsolation and charac-terization of a cellulolyticGeobacillus thermoleovoransT4 strainfrom sugar refinery wastewaterrdquo Extremophiles vol 8 no 5 pp345ndash349 2004

[44] D-W Lee Y-S Koh K-J Kim et al ldquoIsolation and character-ization of a thermophilic lipase from Bacillus thermoleovoransID-1rdquo FEMS Microbiology Letters vol 179 no 2 pp 393ndash4001999

[45] Y R Abdel-Fattah and A A Gaballa ldquoIdentification andover-expression of a thermostable lipase from Geobacillusthermoleovorans Toshki in Escherichia colirdquo MicrobiologicalResearch vol 163 no 1 pp 13ndash20 2008

[46] A R Cho S K Yoo and E J Kim ldquoCloning sequencing andexpression in Escherichia coli of a thermophilic lipase fromBacillus thermoleovorans ID-1rdquo FEMS Microbiology Letters vol186 no 2 pp 235ndash238 2000

[47] DMinana-Galbis D L Pinzon J G Loren AManresa and RM Oliart-Ros ldquoReclassification of Geobacillus pallidus (Scholzet al 1988) Banat et al 2004 as Aeribacillus pallidus gen novcomb novrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 60 no 7 pp 1600ndash1604 2010

[48] D L Pinzon-Martınez C Rodrıguez-Gomez D Minana-Galbis et al ldquoThermophilic bacteria from Mexican thermalenvironments isolation and potential applicationsrdquo Environ-mental Technology vol 31 no 8-9 pp 957ndash966 2010

[49] M Heyndrickx L Lebbe M Vancanneyt et al ldquoA polyphasicreassessment of the genus Aneurinibacillus reclassificationof Bacillus thermoaerophilus (Meier-Stauffer et al 1996) asAneurinibacillus thermoaerophilus comb nov and emendeddescriptions of A aneurinilyticus corrig A migulanus and Athermoaerophilusrdquo International Journal of Systematic Bacteriol-ogy vol 47 no 3 pp 808ndash817 1997

[50] RN Allan L Lebbe J Heyrman P DeVos C J Buchanan andN A Logan ldquoBrevibacillus levickii sp nov andAneurinibacillusterranovensis sp nov two novel thermoacidophiles isolatedfrom geothermal soils of northern Victoria Land AntarcticardquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 55 no 3 pp 1039ndash1050 2005

[51] M B Stott M A Crowe B W Mountain et al ldquoIsolation ofnovel bacteria including a candidate division from geothermalsoils in New Zealandrdquo Environmental Microbiology vol 10 no8 pp 2030ndash2041 2008

[52] M Masomian R N Abd Rahman A B Salleh and M BasrildquoA unique thermostable and organic solvent tolerant lipasefrom newly isolated Aneurinibacillus thermoaerophilus strainHZ physical factor studiesrdquoWorld Journal of Microbiology andBiotechnology vol 26 no 9 pp 1693ndash1701 2010

[53] Y R Abdel-Fattah ldquoOptimization of thermostable lipaseproduction from a thermophilic Geobacillus sp using Box-Behnken experimental designrdquo Biotechnology Letters vol 24no 14 pp 1217ndash1222 2002

[54] S Kumar K Kikon A Upadhyay S S Kanwar and R GuptaldquoProduction purification and characterization of lipase fromthermophilic and alkaliphilic Bacillus coagulans BTS-3rdquo ProteinExpression and Purification vol 41 no 1 pp 38ndash44 2005

[55] R Kumar S Mahajan A Kumar and D Singh ldquoIdentificationof variables and value optimization for optimum lipase produc-tion by Bacillus pumilus RK31 using statistical methodologyrdquoNew Biotechnology vol 28 no 1 pp 65ndash71 2011


[56] S Ertugrul G Donmez and S Takac ldquoIsolation of lipase pro-ducing Bacillus sp from olivemill wastewater and improving itsenzyme activityrdquo Journal of Hazardous Materials vol 149 no 3pp 720ndash724 2007

[57] F M Shariff T C Leow A D Mukred A B Salleh M Basriand R N Abd Rahman ldquoProduction of L2 lipase by Bacillussp strain L2 nutritional and physical factorsrdquo Journal of BasicMicrobiology vol 47 no 5 pp 406ndash412 2007

[58] A Ebrahimpour R N Abd Rahman D H Ean Chrsquong MBasri and A B Salleh ldquoA modeling study by response surfacemethodology and artificial neural network on culture param-eters optimization for thermostable lipase production from anewly isolated thermophilic Geobacillus sp strain ARMrdquo BMCBiotechnology vol 8 p 96 2008

[59] M Sifour T I Zaghloul H M Saeed M M Berekaa and YR Abdel-fattah ldquoEnhanced production of lipase by the ther-mophilic Geobacillus stearothermophilus strain-5 using statisti-cal experimental designsrdquo New Biotechnology vol 27 no 4 pp330ndash336 2010

[60] C Breuil D B Shindler J S Sijher and D J Kushner ldquoStimu-lation of lipase production during bacterial growth on alkanesrdquoJournal of Bacteriology vol 133 no 2 pp 601ndash606 1978

[61] S Takac and B Marul ldquoEffects of lipidic carbon sources onthe extracellular lipolytic activity of a newly isolated strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 35 no 9 pp 1019ndash1025 2008

[62] H A El-Shafei and L A Rezkallah ldquoProduction purifica-tion and characterization of Bacillus lipaserdquo MicrobiologicalResearch vol 152 no 2 pp 199ndash208 1997

[63] M M Berekaa T I Zaghloul Y R Abdel-Fattah H M Saeedand M Sifour ldquoProduction of a novel glycerol-inducible lipasefrom thermophilic Geobacillus stearothermophilus strain-5rdquoWorld Journal of Microbiology and Biotechnology vol 25 no 2pp 287ndash294 2009

[64] N G Edwinoliver K Thirunavukarasu R B Naidu M KGowthaman T N Kambe and N R Kamini ldquoScale up ofa novel tri-substrate fermentation for enhanced productionof Aspergillus niger lipase for tallow hydrolysisrdquo BioresourceTechnology vol 101 no 17 pp 6791ndash6796 2010

[65] J G S Mala N G Edwinoliver N R Kamini andRPuvanakrishnan ldquoMixed substrate solid state fermentationfor production and extraction of lipase from Aspergillus nigerMTCC 2594rdquo Journal of General and Applied Microbiology vol53 no 4 pp 247ndash253 2007

[66] M L Rua C Schmidt-Dannert S Wahl A Sprauer andR D Schmid ldquoThermoalkalophilic lipase of Bacillus thermo-catenulatus large-scale production purification and propertiesaggregation behaviour and its effect on activityrdquo Journal ofBiotechnology vol 56 no 2 pp 89ndash102 1997

[67] S Imamura and S Kitaura ldquoPurification and characterizationof a monoacylglycerol lipase from the moderately thermophilicBacillus sp H-257rdquo Journal of Biochemistry vol 127 no 3 pp419ndash425 2000

[68] A Ebrahimpour R N Abd Rahman M Basri and A BSalleh ldquoHigh level expression and characterization of a novelthermostable organic solvent tolerant 13-regioselective lipasefrom Geobacillus sp strain ARMrdquo Bioresource Technology vol102 no 13 pp 6972ndash6981 2011

[69] M Tayyab N Rashid and M Akhtar ldquoIsolation and identifi-cation of lipase producing thermophilicGeobacillus sp SBS-4Scloning and characterization of the lipaserdquo Journal of Bioscienceand Bioengineering vol 111 no 3 pp 272ndash278 2011

[70] T C Leow R N Abd Rahman M Basri and A B Salleh ldquoAthermoalkaliphilic lipase of Geobacillus sp T1rdquo Extremophilesvol 11 no 3 pp 527ndash535 2007

[71] N A Soliman M Knoll Y R Abdel-Fattah R D Schmidand S Lange ldquoMolecular cloning and characterization of ther-mostable esterase and lipase from Geobacillus thermoleovoransYN isolated fromdesert soil in Egyptrdquo Process Biochemistry vol42 no 7 pp 1090ndash1100 2007

[72] J P Henley and A Sadana ldquoCategorization of enzyme deacti-vations using a series-type mechanismrdquo Enzyme and MicrobialTechnology vol 7 no 2 pp 50ndash60 1985

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


previously been isolated from the volcanic habitat of theSantorini volcano at South Aegean Sea [20] A thoroughbiochemical screening for extracellular lipase activity wasperformed both in solid and liquid media in an attempt toestablish benchmark criteria for such an analysis The mostactive strains were selected and characterized with respect totheir 16S rDNA sequences and the effect of various carbonsources on the production of extracellular lipolytic activityFinally the pH and temperature-activity optima as well as thecorresponding thermal stability kinetics were determinedfor the partially purified and concentrated lipases from allselected isolates

2 Materials and Methods

21 Microbial Strains The 101 thermophilic bacterial strainsused in this study have been previously isolated from theseawater and sediment near the active volcano of Santoriniisland at Aegean Sea Greece [20] All isolates were preservedin 30 (wv) sterile glycerol solution at minus80∘C

22 Growth Media and Conditions All isolates were prelim-inary screened for their lipolytic activity both in solid andliquid cultures A standard inoculum was prepared for eachstrain from glycerol stocks on Nutrient Agar (Nutrient brothplus 3 wv agar) plates Following incubation for 24 h at60∘C bacterial biomass collected in sterile water and dilutedto an OD

600of 03 was served as inoculum

Solid cultures were conducted in sterile 12-well platesfilled with Rhodamine BmdashOlive Oil agar medium containing10 gsdotLminus1 olive oil (ROA) [8] Twenty 120583L of standard inoculumwas placed in the middle of each well (3 wells per strain)and the plates were incubated at 60∘C up to 48 hoursLipase production was evaluated from the diameter of theorange fluorescent halo (reaction of liberated fatty acids withRhodamine B) observed under UV exposure [21]

Liquid cultures were conducted in 100mL Erlenmeyerflasks with 20mL working volume The basal medium usedconsisted of (gsdotLminus1) NaNO

3 3 K2HPO4 1 KCl 05 CaCl

2

01 MgSO4sdot7H2O 05 yeast extract 1 trace elements solu-

tion 1mLsdotLminus1 supplemented with 20 gsdotLminus1 olive oil as solecarbon and energy source Triplicate flasks were inoculatedfor each strain with 05mL standard inoculum and all flaskswere incubated in a thermostated orbital shaker (SANYOBiomedical UK) at 60∘C and 180 rpm Evaporation was dailychecked gravimetrically and compensated with the additionof sterile water Samples were collected every 24 h for thedetermination of biomass concentration and extracellularlipolytic activity The effect of other carbon sources onlipase production was determined in a similar manner bysubstituting olive oil with either glucose mineral oil corncob wheat bran eicosane Tween-80 or glycerol at an initialconcentration of 20 gsdotLminus1 Lipase production was followed byassaying of the lipase activity in the supernatant as describedin enzyme assays All lipolytic activities were determined intriplicate and reported as averages

23 Molecular Characterization of Isolates Bacterial identifi-cation of the nine selected isolates was based on 16S rDNAsequence analysis Genomic DNA extraction from liquidcultures was performed according to Haught et al [22] andthe quantity of the isolated DNA was determined photomet-rically [23]The 16S rDNAwas amplified by polymerase chainreaction (PCR) using two universal primers [24 25]

pA (51015840-AGA GTT TGA TCC TGG CTC AG-31015840) and

R1492 (51015840-TACGGYTACCTTGTTACGACTT-31015840)

Amplification reactions were performed in volumes of50120583L containing 40 ng templateDNA 04 120583Mof each primer1X buffer with Mg2+ 1 U KAPA Taq DNA polymerase(Kapa Biosystems Woburn MA USA) and 02mM dNTPsNucleases free water was used to bring the reaction vol-ume to 50 120583L After initial denaturation at 95∘C for 2minsamples were cycled for 30 PCR cycles using the followingcycle profile 95∘C denaturation for 30 s primer annealingat 53∘C for 30 s and primer extension at 72∘C for 2minplus a final 2min elongation step at 72∘C Amplified PCRproducts were separated by gel electrophoresis on 12 (wv)agarose gel and then purified using NucleoSpin ExtractPCR kit (MACHEREY-NAGEL Germany) The 16S rDNAfragment (gt1400 bp) was fully sequenced in both direc-tions (Macrogen Republic of Korea) and the correspond-ing gene sequences have been submitted to the GenBankdatabases under accession numbers JQ808132 to JQ808140(httpwwwncbinlmnihgov) The 16S sequences wereused for the determination of the evolutionary history (phy-logenetic tree) of the nine isolates using theNeighbor-Joiningmethod [26] in MEGA5 software [27] The percentage ofreplicate trees in which the associated taxa clustered togetherwas calculated by the bootstrap test (1000 replicates) [28]Evolutionary distances were computed using the MaximumComposite Likelihood method [29]

24 Preparation of Partially Purified Lipase Samples Liquidcultures of the nine selected strains were conducted in 2 LErlenmeyer flasks of 400mL working volume (3 flasks perstrain) as described above Biomass was removed by cen-trifugation (6000 rpm 5min) and the resulting supernatantwas passed through Whatman number 1 filter paper inorder to remove the residual olive oil The filtrate was thensubjected to ammonium sulphate precipitation Optimumprecipitation range was determined by employing a gradualammonium sulphate procedure (5 saturation step) andmeasuring specific lipase activity on the resulting super-natants and precipitates The optimum precipitation condi-tions were subsequently applied to each culture supernatantand the collected precipitates were resuspended in 20mMpotassium phosphate buffer (pH 8) and desalted using PD-10 gel filtration columns (GE Healthcare) using the abovebuffer for elution The concentrated and desalted enzymesolutionswere used for the determination of the optimumpHand temperature and thermal stability of the correspondinglipolytic activities



















Table 1 Continued













Table 1 Continued














SP73 (JQ808135)

SP93 (JQ808140)

SP75 (JQ808136)

SP22 (JQ808133)

SP79 (JQ808138)

SP76 (JQ808137)

SP29 (JQ808134)

SP14 (JQ808132)

SP83 (JQ808139)

93100

91

88

42

52

45

41

54

75

85

83

100

100

99

87

74

100

001

























Glycerol

Eicosane

Wheat bran

Tween-80

Mineral oil

Olive oil

Glucose

118 11 143 34

2861872413822

488 113

11562156121128185

165 159 103 348 123 28

158116148461129161205264

178 192 412

141















119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864



2are


1119864

0

and 1205722= 119864

2119864





120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)


1specific activity









40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Table 1 Continued













Table 1 Continued














SP73 (JQ808135)

SP93 (JQ808140)

SP75 (JQ808136)

SP22 (JQ808133)

SP79 (JQ808138)

SP76 (JQ808137)

SP29 (JQ808134)

SP14 (JQ808132)

SP83 (JQ808139)

93100

91

88

42

52

45

41

54

75

85

83

100

100

99

87

74

100

001

























Glycerol

Eicosane

Wheat bran

Tween-80

Mineral oil

Olive oil

Glucose

118 11 143 34

2861872413822

488 113

11562156121128185

165 159 103 348 123 28

158116148461129161205264

178 192 412

141















119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864



2are


1119864

0

and 1205722= 119864

2119864





120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)


1specific activity









40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued














SP73 (JQ808135)

SP93 (JQ808140)

SP75 (JQ808136)

SP22 (JQ808133)

SP79 (JQ808138)

SP76 (JQ808137)

SP29 (JQ808134)

SP14 (JQ808132)

SP83 (JQ808139)

93100

91

88

42

52

45

41

54

75

85

83

100

100

99

87

74

100

001

























Glycerol

Eicosane

Wheat bran

Tween-80

Mineral oil

Olive oil

Glucose

118 11 143 34

2861872413822

488 113

11562156121128185

165 159 103 348 123 28

158116148461129161205264

178 192 412

141















119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864



2are


1119864

0

and 1205722= 119864

2119864





120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)


1specific activity









40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SP73 (JQ808135)

SP93 (JQ808140)

SP75 (JQ808136)

SP22 (JQ808133)

SP79 (JQ808138)

SP76 (JQ808137)

SP29 (JQ808134)

SP14 (JQ808132)

SP83 (JQ808139)

93100

91

88

42

52

45

41

54

75

85

83

100

100

99

87

74

100

001

























Glycerol

Eicosane

Wheat bran

Tween-80

Mineral oil

Olive oil

Glucose

118 11 143 34

2861872413822

488 113

11562156121128185

165 159 103 348 123 28

158116148461129161205264

178 192 412

141















119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864



2are


1119864

0

and 1205722= 119864

2119864





120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)


1specific activity









40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Glycerol

Eicosane

Wheat bran

Tween-80

Mineral oil

Olive oil

Glucose

118 11 143 34

2861872413822

488 113

11562156121128185

165 159 103 348 123 28

158116148461129161205264

178 192 412

141















119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864



2are


1119864

0

and 1205722= 119864

2119864





120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)


1specific activity









40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








119864

0

1198961

997888rarr 119864

1

1198962

997888rarr 119864

2

(1)

where 1198640 1198641 and 119864



2are


1119864

0

and 1205722= 119864

2119864





120572 = [1 +

120572

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198961119905)

minus [

119886

1119896

1

119896

2minus 119896

1

] sdot exp (minus1198962119905)

(2)


1specific activity









40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


40 50 60 70 80 90

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 100

20

40

60

80

100

pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10pHpH pH

5 6 7 8 9 10 5 6 7 8 9 105 6 7 8 9 10

pHpH pH

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)40 50 60 70 80 90

Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

40 50 60 70 80 90Temperature (∘C)

Rela

tive a

ctiv

ity (

)

SP22SP14

SP73

SP83

SP75

SP79 SP93

SP29

SP76

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)

0

20

40

60

80

100

Rela

tive a

ctiv

ity (

)



0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Rela

tive a

ctiv

ity

0

02

04

06

08

1

Resid

ual a

ctiv

ity

0

02

04

06

08

1

SP93

SP14

SP29

SP79

SP75

SP22

SP73

SP76

0 60 120 180 2401200 2400

SP83

Time (min)

Time (min)

0 60 120 180 24012002400

0 60 120 180 24012002400

Rela

tive a

ctiv

ity

0

02

04

06

08

1

0 60 120 180 2401200 2400

Rela

tive a

ctiv

ity0

02

04

06

08

1

0 60 120 180 24012002400Re

lativ

e act

ivity

0

02

04

06

08

1

0 60 120 180 24012002400

0 60 120 180 24012002400

0 60 120 180 24012002400





120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 9372 17158 12524 9356 6096

119896

2

(minminus1)





119864

119886

(kJmole) 12529 18585 8047 8319 16509SP76 SP79 SP83 SP93

120572

1


119896

1

(minminus1)





119864

119886

(kJmole) 6600 6792 18506 14711

119896

2

(minminus1)





119864

119886

(kJmole) 8526 9124 11504 16559








4 Conclusions






References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





References




















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Unraveling the Lipolytic Activity of...

Documents

Transcript of Research Article Unraveling the Lipolytic Activity of...