Bioresource Technology 98 (2007) 1238–1245

Production and partial characterization of dehairing proteasefrom Bacillus cereus MCM B-326

S.S. Nilegaonkar, V.P. Zambare, P.P. Kanekar ¤, P.K. Dhakephalkar, S.S. Sarnaik

Microbial Sciences Division, Agharkar Research Institute, G.G. Agarkar Road, Pune, Maharashtra State 411 004, India

Received 12 January 2006; received in revised form 4 May 2006; accepted 7 May 2006Available online 19 June 2006

Abstract

Bacillus cereus MCM B-326, isolated from buValo hide, produced an extracellular protease. Maximum protease production occurred(126.87§ 1.32 U ml¡1) in starch soybean meal medium of pH 9.0, at 30 °C, under shake culture condition, with 2.8£ 108 cells ml¡1 asinitial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25–65 °C and pH6–12, with maximum activity at 55 °C and pH 9.0. The enzyme required Ca2+ ions for its production but not for activity and/or stability.The partially puriWed enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be eVectively usedto remove hair from buValo hide indicating its potential in leather processing industry.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Bacillus cereus; BuValo hide; Dehairing; Protease; Starch–soybean meal

1. Introduction

In leather processing, the most constrained operationfrom environmental point of view is the dehairing of skin/hide. The conventional method of dehairing involves theuse of lime and sodium sulphide. Presence of these chemi-cals in tannery waste is responsible for tremendous pollu-tion, causing health hazards to the tannery workers. Limeproduces a poisonous sludge while sodium sulphide ishighly toxic and has obnoxious odor. Although enzymeassisted dehairing process reduces the pollution load tosome extent, a technology based on enzyme alone, withoutthe use of sulphide and other chemical inputs, has yet to beexplored (Thanikaivelan et al., 2004).

Proteases Wnd applications at various steps of leatherprocessing, e.g., neutral proteases in soaking (Deshpandeet al., 2004), alkaline proteases in dehairing (Dayanandanet al., 2003), and acid proteases in bating (Padmavathi et al.,1995). Dehairing enzymes from Bacillus sp. have been

* Corresponding author. Tel.: +91 20 25653680; fax: +91 20 25651542.E-mail address: PPKanekar@aripune.org (P.P. Kanekar).

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2006.05.003

reported by many researchers (Huang et al., 2003; RiZeet al., 2003; Alexandre et al., 2005). The concrete mixture ofdehairing enzymes from Bacillus subtilis and Bacillus cereuswith sodium carbonate, caustic soda and thioglycolic acid,is described in a patent (Monsheimer and PXeiderer, 1976).The aim of the present work was to study the production,optimization and properties of extracellular proteolyticenzyme of B. cereus B-326 having application in dehairingof buValo hide.

2. Methods

2.1. Microorganism and taxonomic study

B. cereus BSA-26 producing protease was isolated frombuValo hide obtained from Local Municipal CorporationSlaughterhouse (Pune, India). The strain was identiWedaccording to the methods described in Bergey’s Manual ofSystematic Bacteriology (Sneath, 1984) and on the basis of16 s ribosomal DNA sequence The stock culture was main-tained on nutrient agar at 4 °C and as a glycerol stock at¡20 °C.

S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 1239

2.2. Inoculum preparation and production

Seed inoculum was prepared by growing the isolate onnutrient agar slants/Roux bottle at 30 °C for 24 h. The cellswere suspended in saline and cell density was measuredspectrophotometrically (Shimatzu UV-2501 PC, Japan) at600 nm. Following media were studied for protease produc-tion: synthetic medium casein (SMC: 0.7% K2HPO4, 0.3%KH2PO4, 0.01% MgSO4, 1% casein), nutrient broth supple-mented with 1% casein (NBC), starch soybean meal (SS:2% starch, 1% soybean meal, 0.3% CaCO3) and soybeanmeal-tryptone (ST: 1% soybean meal, 1% tryptone). TheXasks were inoculated with 24 h old inoculum and incu-bated on orbital shaker (150 rpm) at 30 °C for 60 h. Themedium yielding maximum protease activity was selectedfor further investigation.

2.3. Protease assay

The protease activity was determined by caseinolyticassay method of (Kanekar et al., 2002). The cell free super-natant (1 ml) was mixed with 4 ml of casein (0.625% w/v)and incubated at 37 °C for 30 min. The reaction wasstopped by addition of 5 ml of 5% trichloroacetic acid.Enzymatically hydrolyzed casein was measured by modi-Wed Folin Ciocalteu method (Jayaraman, 2003), againstcasein treated with inactive enzyme as blank. A standardgraph was generated using standard tyrosine solutions of5–50 �g ml¡1. One unit of protease activity was deWned asthe amount of enzyme which liberated 1 �g tyrosine per minat 37 °C.

2.4. EVect of diVerent environmental conditions on protease production

For optimization, production of protease by isolateBSA-26 was studied using SS medium, under the followingconditions: shake and static culture, age of inoculum-21 to24 h, initial inoculum density: 0.5–5.0% (v/v) of cell density2.8£ 108 cells ml¡1, temperature: 25–40 °C, with incrementsof 5 °C, pH: 6–12, with increments of one unit. The Xaskswere incubated for 36 h and cells were removed by cold cen-trifugation at 13,000£ g for 10 min. The cell free superna-tant was analyzed for protease activity.

2.5. EVect of diVerent nitrogen and carbon sources

To test the eVect of diVerent nitrogen source on the pro-tease production, the liquid medium of starch (2%) and cal-cium carbonate (0.3%) was supplemented with variouscomplex nitrogen source such as yeast extract, beef extract,casein, tryptone, peptone, soybean meal, corn steep liquorand inorganic nitrogen sources viz. ammonium chloride,ammonium sulphate, ammonium phosphate, ammoniumnitrate and sodium nitrite all at 1% concentration. Likewisethe eVect of diVerent carbon sources on protease produc-tion was studied by supplementing; a liquid medium of soy-

bean meal (1%) and calcium carbonate (0.3%), with starch,glucose, sucrose at 1% concentration. The Xasks were incu-bated for 36 h and cell free supernatant was analyzed forprotease activity.

2.6. EVect of metal ions

To determine the eVect of metal ions on the proteaseproduction, diVerent metal ions viz. CaCO3, CaCl2,K2HPO4, KH2PO4, FeSO4, MgSO4, NaCl and MnSO4 wereindividually added with 0.3% strength in starch (2%) andsoybean meal (1%) liquid medium. The Xasks were incu-bated for 36 h and cell free supernatant was analyzed forprotease activity.

2.7. Time course of protease production

The growth of the organism and production of proteasewas studied under all optimum conditions viz SS medium,pH 9.0, 30 °C, shake culture condition, 1% inoculum size at6 h interval. The cell growth was measured at 600 nm. Cellfree supernatants were analyzed for enzyme activity, pro-tein and residual starch, at each interval, up to 72 h. Proteincontent was measured by Buret method (Jayaraman, 2003).The residual starch was estimated by iodometric method(Jayaraman, 2003).

2.8. Scale-up studies

Production of the enzyme was carried out in a 2 l glassbottle with a working volume of 800 ml of SS medium.Parallel experiment was conducted at Xask level with thesame medium. The culture was inoculated in nutrientbroth. After an incubation period of 21 h, cell growth of2.8£ 108 cells ml¡1 was inoculated in 800 ml SS medium(pH 9.0) and incubated at 30 °C with constant agitationspeed of 150 rpm. Enzyme samples were removed at 12 hintervals for measuring cell growth by recording OD at600 nm. Biomass was separated by centrifugation. The cellfree culture broth was assayed for extracellular proteaseactivity and protein content as described above. Theenzyme was partially puriWed using ammonium sulphate(60% saturation). The precipitated enzyme was used as acrude enzyme for further studies.

2.9. Substrate speciWcity

Protease was produced in SS medium (pH 9.0) at 30 °Cfor 36 h, and cell free supernatant was assayed using diVer-ent substrates such as casein, Bovine serum albumin (BSA),haemoglobin, collagen, gelatin and keratin. The enzymatichydrolysis of the casein and BSA was studied by assay asdescribed above. The proteolytic activity of the enzymewith 2% haemoglobin as substrate was carried out accord-ing to Sarath et al. (1996). The enzymatic hydrolysis of col-lagen, gelatin was studied as described by Woessner (1961).Keratinase activity was determined using keratin azure

1240 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245

substrate (Takami et al., 1990). Enzyme units were deWnedaccording to the respective assay.

2.10. EVect of pH and temperature

The pH stability of precipitated protease was determinedwith casein (0.625% w/v) as a substrate dissolved in diVer-ent buVers [sodium acetate, pH 4–6, sodium phosphate, pH7–8, Tris HCl, pH 9–10.6 and Glycine-NaOH, pH 11–12].The pH stability of the protease was determined by pre-incubating the enzyme in diVerent buVers for 30 min at37 °C. Likewise, thermal stability of the precipitated prote-ase was determined by pre-incubating the enzyme at diVer-ent temperatures from 25–80 °C for 30 min. All experimentswere carried out in duplicate and each analysis was alsoperformed in duplicate.

2.11. EVects of diVerent inhibitors, metals, detergents and oxidant on enzyme activity

To study inhibition of the protease, enzyme was pre-incubated with inhibitors such as phenylmethylsulphonylXuoride (PMSF, 1 mM), ethylenediamine tetraacetic acid(EDTA, 2 & 5 mM), dithiothretol (DTT, 2 & 5 mM), iodo-acetamide (2 mM), trypsin inhibitor (100 �g), metal ionssuch as Hg+ (1 & 5 mM), Na+, Fe2+, Cu2+, Ca2+, Mg2+,Mn2+, Zn2+ (5 mM); detergents such as sodium dodecylsulfate (SDS, 1%), sodium tripolyphosphate (1%), sodiumtetraborate (1%) and oxidizing agent H2O2 (5%, 10% and15%) for 30 min at 37 °C. Subsequently the enzyme assaywas performed as described above. The percent residualenzyme activity was calculated with reference to the activityof the enzyme without these supplements.

2.12. Gel electrophoresis and zymogram

Proteins were analyzed by tube gel electrophoresis usingcomassive brilliant blue R250. The proteins were precipi-tated with ammonium sulphate (60% saturation) followedby membrane dialysis. The protein pattern of crude enzymewas carried out with native PAGE. A zymogram of dia-lyzed enzyme was obtained using polyacrylamide gel elec-trophoresis followed by treatment with 1% (w/v) casein assubstrate in Tris buVer (pH 9.0). The molecular weight ofthe protease enzyme was determined by compairing withmobility of standard molecular weight marker proteins(bovine albumin, 66 kDa, chicken ovalbumin, 45 kDa, gly-ceroldehyde-3-phosphate dehydrogenase 36 kDa, trypsino-gen 24 kDa, cytochrome C 12.4 kDa).

2.13. Dehairing of buValo hide

Ammonium sulfate precipitate of enzyme was applied at1% concentration of to piece buValo hide (2.5 cm£ 2.5 cm),presoaked in tap water from Xesh side, and kept at ambienttemperature (28§ 2 °C) in a dry place. Loosening of hair

and epidermis were observed by mechanical means at anhourly interval.

2.14. Statistical analysis

Analysis of variance with repeated measures was carriedout using GLM command of software SPSS (SPSS version10, Windows 98 version). Activity means and standarddeviations were calculated. Univariate analysis of variance(ANOVA) was employed on the data for protease activityat diVerent % inoculum, pH, temperature, culture condi-tion-incubation period, complex and inorganic nitrogensources, carbon supplement and metal ions supplement andtested for their signiWcance.

3. Results and discussion

The culture B. cereus BSA-26 was deposited in MACScollection of microorganisms (WFCC code 561) and desig-nated as MCM B-326.The isolate was identiWed as on thebasis of morphological and physiological characteristics,biochemical tests and 16 S rRNA sequencing. A compari-son of the DNA sequence with sequences in the NationalCenter for Biotechnology Information (NCBI) databasewith BLAST software (Altschul et al., 1997) showed 98.5%sequence identity with the published 16 s rRNA sequencesof B. cereus. The 16 s rDNA sequence of the isolate hasbeen deposited in GenBank database with accession num-ber DQ479314. Strains of Bacillus species producing pro-tease activity have been widely described in literature(Mehrotra et al., 1999; Kanekar et al., 2002; He et al., 2006).Their applications, especially those of alkaline protease, aremainly directed towards detergent industry (Anwar andSaleemuddin, 1998).

3.1. Optimization of protease production

Maximum production of protease by B. cereus MCM B-326 was obtained in SS medium at 36 h of incubation, andwas signiWcantly (p < 0.001) higher than that observed inother media tested. The protease activity was highest with2% starch, 1% soybean meal and 0.3% CaCO3 with 0.5–1.0% inoculum of cell density 2.8£108 cells/ml of 21 h age.The results showed close resemblance with the alkalineprotease by thermophilic B. licheniformis (Sinha and Satya-narayana, 1991). The results on the eVect of initial pH andtemperature on protease production showed maximumactivity at pH 9.0 (p < 0.001) and 30 °C (p < 0.001)(113.33§ 14.43 and 112§14.83 U ml¡1, respectively, resultsnot shown). Comparable results were obtained for B. alcal-ophilus, isolated from Lonar lake, India (Kanekar et al.,2002).

It was observed that, in presence of sucrose, starchor glucose (1%), the protease activity (116.27§5.72–122.45§20.59 U ml¡1, results not shown) was almost con-stant; however it decreased signiWcantly in the absence ofcarbon sources (29.35§9.36 U ml¡1) at 36 h. The activity

S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 1241

obtained with 1% starch was similar to that with 2% starchin SS medium. The protease activity enhanced 4 fold inpresence of starch. Such enhancement of alkaline proteasehas been reported in alkaliphilic B. licheniformis (Sinha andSatyanarayana, 1991). EVect of diVerent complex and inor-ganic nitrogen sources on protease activity are presented inTable 1. The protease activity was highest with soybean

Table 1EVect of nitrogen sources and metal ions on protease production byB. cereus MCM B-326

Data represent the mean values and standard deviation (n D 9).

Substrates supplied Mean enzyme activity (U ml¡1)

Complex nitrogen sources (1%)Soybean meal 122.24 § 2.06Yeast extract 61.26 § 7.63Peptone 39.97 § 10.03Tryptone 30.37 § 5.40Corn steep liquor 22.31 § 4.43Beef extract 9.04 § 2.53Casein 1.36 § 0.72

Inorganic nitrogen sources (1%)Ammonium sulphate 58.91 § 1.41Ammonium nitrate 58.38 § 0.19Ammonium chloride 100.88 § 8.03Ammonium phosphate 0.42 § 0.80Sodium nitrite 112.13 § 6.99

Metal ions (0.3%)CaCO3 125.99 § 1.91CaCl2 62.63 § 3.65K2HPO4 43.30 § 5.47KH2PO4 41.51 § 2.75FeSO4 30.76 § 1.61MgSO4 21.20 § 1.72NaCl 17.66 § 1.73MnSO4 13.90 § 3.19Without metal ions 17.06 § 0.86

meal (122.24§ 2.06 U ml¡1, p < 0.001). Soybean meal wasused as inducer for protease production from Conidioboluscoronatus (Deshpande et al., 2004). The protease activitywas increased when production medium was supplementedwith potassium, magnesium, iron and calcium ions.Enhancement of 7.4 fold in activity was achieved by supple-mentation of 0.3% CaCO3 (p < 0.001) (Table 1). This indi-cated that the calcium ion was necessary for enzymeinduction (Ghorbel et al., 2005).

Thus, optimization studies resulted in the followingWndings: the most suitable nutrient medium starch (1%),soybean meal (1%) and CaCO3 (0.3%) of initial pH 9.0,temperature 30 °C, 1% inoculum and period of incubation36 h. (Tayler et al., 1987) have reported the best productionof dehairing protease using nutrient medium containingcorn steep liquor, lactose, sodium sulfate and potassiumacid phosphate at initial pH 6.8–7.2 and 37 °C for 96 h.

3.2. Time course of protease production

Under the optimum conditions, the protease activityreached to 126.87§1.32 U ml¡1 within 36 h of the fermenta-tion when the cell growth reached late log phase or earlystationary phase (Fig. 1). An alkaline protease from marineyeast produced protease activity within 30 h when the cellgrowth reached mid-log phase (Chi et al., 2006). The reduc-tion in starch level indicated that B. cereus MCM B-326had strong amylolytic activity in addition to protease activ-ity. The starch level was almost negligible after 6 h. The pHof the medium decreased towards acidic side because ofhydrolysis of starch to acids via glucose (data not shown).The protein content was decreased because of the utiliza-tion of protein by the organism. The decline in proteaseactivity upon prolonged incubation may be due to autolysisof the enzyme.

Fig. 1. Time course of protease production by B. cereus MCM B-326. Standard deviations are represented by error bars.

Time (h)0 10 20 30 40 50 60 70 80

Pro

tein

(m

g m

l-1)

0

1

2

3

4

5

Protein

Enzym

e activity (U m

l -1)

0

20

40

60

80

100

120

140

Enzyme activity

Cel

l den

sity

(x1

09 cells

ml-1

)

0.0

0.5

1.0

1.5

2.0

2.5

Cell density

Residual starch (m

g ml -1)

0

2

4

6

8

10

Residual starch

1242 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245

3.3. Scale-up studies

Protease activity was 150.52§11.24 U ml¡1 in a 2 l glassbottle, (Fig. 2) and 126.87§1.32 U ml¡1 in Xask level at theend of 36 h, showing 1.13 fold increase. (Wang and Shih,1999) reported that keratinase activity from B. licheniformisand a recombinant B. subtilis was increased 1.04 and 1.01fold respectively in 15 l fermentor as compared to Xasklevel.

3.4. Characteristics of protease

The protease from B. cereus MCM B-326 hydrolyzedcasein, haemoglobin, BSA with activity of 128.64 U ml¡1,14.25 U ml¡1 and 50.89 U ml¡1 respectively (data notshown). But it was not able to hydrolyze collagen, gelatin,and keratin. Protease from Alkaligenes faecalis could nothydrolyze the Wbrous proteins such as collagen and kera-tin (Thangam and Rajkumar, 2002). The enzyme wasactive in the pH range of 6–12, with optimum activity atpH 9.0, although, a small peak at pH 10.6 was alsoobserved, suggesting a presence of two alkaline proteases(Fig. 3). The preliminary studies on the extracellulardehairing protease secreted by the Bacillus sp. showed thatit has dual pH maxima at 7.5 and 9.0 (Annapurna et al.,1996).

The enzyme was active in the temperature range of 30–65 °C with maximum activity at 55 °C, although, a smallpeak at 30 °C was also observed, suggesting presence of twoproteases. The activity increased from 134% to 149% withincrease in temperature from 40–55 °C. The enzyme wascompletely inactivated at 80 °C (Fig. 3). In an earlier report,the dehairing enzyme was active between the temperaturerange of 30–60 °C, and showed optimum activity at 55 °C.

After 30 min, activity decreased to 53% and 85% at 50 °Cand 60 °C, respectively and completely inactivated at 70 °C(Huang et al., 2003). (Annapurna et al., 1996) reported thatthe enzyme was active in temperature range of 20–50 °C,with optimum activity at 37 °C. Thus the protease of B.cereus under study is more thermo tolerant than the pro-teases reported above.

The eVect of inhibitors, metal ions, detergents and oxi-dant on the ammonium sulphate precipitated enzyme isdetailed in Table 2. The protease was completely inhibitedby EDTA indicating that it might be a metalloprotease.Similar results for B. cereus KCTC 3674 were observed by(Kim et al., 2001). The protease activity was 97% inhibited

Fig. 3. EVect of pH and temperature on activity of B. cereus MCM B-326.Protease symbols and bars represent the mean values and the standarddeviations. The 100 ml¡1 for pH and 1721.38 U ml¡1 for temperature.

Temperature (ºC)

20 30 40 50 60 70 80 90

% E

nzym

e ac

tivity

0

20

40

60

80

100

120

140

160

Temperature

pH

8642 10 12 14

pH

Fig. 2. Scale-up of protease by B. cereus MCM B-326. Standard deviations are represented by error bars.

Time (h)

0 10 20 30 40 50 60 70 80

Enz

yme

acti

vity

(U

ml-1

)

0

20

40

60

80

100

120

140

160

180

Enzyme activity

Protein (m

g ml -1)

0

1

2

3

4

5

Protein (mg ml-1)

Cell density (x10

9 cells ml -1)

0

5

10

15

20

25

Cell density

S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 1243

by DTT, thus the enzyme might contain S–S bond as apart of its monomeric structure. The eVect was consistentwith its above-described thermal stability, which had beenshown primarily to be the result of disulWde bond (Kara-dzic et al., 2004). Protease activity was inhibited by 15%by PMSF and trypsin inhibitor had no eVect on activity.This suggested that this protease was not of serine type. Inan earlier report, dehairing protease was completely inhib-ited by PMSF and partially inhibited by EDTA (Huanget al., 2003). Similarly, the dehairing protease of Bacillussp. kr10 was completely inhibited by PMSF, whereas 84%inhibited by EDTA (RiZe et al., 2003).Thus, both thereported dehairing proteases of Bacillus sp. were serineproteases.

The protease was markedly inhibited (80–93%) by Cu2+,Mg2+, Zn2+, and sodium tripolyphosphate; while partiallyinhibited (30–60%) by Na+, Fe2+, Mn2+. Sodium tetra-borate and iodoacetamide inhibited the activity by about20%. The protease enzyme was stable and active in the pres-ence of oxidizing agent H2O2 (5% and 10%) and, therefore,could be used in bleach-based detergent formulations. Sim-ilar results were obtained for alkaline protease from alkalitolerant B. patagoniensis (Olivera et al., 2006). Results alsoshowed that (data not shown) Ca2+ was required for theproduction only and not for activity and/or stability of theenzyme. However, protease from B. cereus BG1 requiredcalcium ion for its activity as well as stability (Ghorbelet al., 2005). Thus, the present enzyme diVered from theprevious reports on B. cereus.

The crude enzyme preparation showed Wve proteinbands. After dialysis the ammonium sulphate precipitatedenzyme showed two bands, viz., Cereus 1 and Cereus 2 onzymogram. The non-denaturing PAGE showed two pro-teases with approximate molecular weights 45 kDa and36 kDa of band Cereus 1 and Cereus 2 respectively (Fig. 4).It has been reported that B. cereus has two proteases withmolecular masses of approximately 38 kDa and 36 kDa

(Kim et al., 2001). A calcium dependent protease fromB. cereus BG1 was reported to have molecular weight of34 kDa (Ghorbel et al., 2005). According to (Huang et al.,2003) the molecular weight of the puriWed dehairing prote-ase from B. pumilus was 32 kDa. Thus, molecular weight ofCereus 2 was similar to the molecular weight reportedfor other proteases from B. cereus while molecular weightof Cereus 1 was diVerent. The enzyme showed two optimafor both pH and temperatures and two enzyme bands onPAGE-zymogram, suggesting the extracellular secretion oftwo proteases from B. cereus MCM B-326.

3.5. Dehairing of buValo hide

The enzyme dehaired the buValo hide within 21 h at pH7.0 and ambient temperature 28§2°C, with 1% enzyme

Fig. 4. Enzyme proWle of protease from B. cereus MCM B-326.

Table 2EVect of inhibitors and activators on protease activity of B. cereus MCM B-326

Data represent the mean values and standard deviation (n D 3).The 100% activity correspond to 1393.22 U ml¡1.

Abbreviation: PMSF– phenymethylsulphonyl Xuoride, EDTA– Ethylenediamine tetraaceticacid, DTT– Dithiothreitol, SDS– Sodium dodecyl sulphate.

Compound Concentration % residual activity Compound Concentration % residual activity

Control – 100 § 1.96 CuSO4 5 mM 17.03§ 1.16Inhibitors CaCl2 5 mM 101.03 § 5.07PMSF 1 mM 84.63 § 3.93 MnSO4 5 mM 49.6 3§ 1.86EDTA 2 mM 7.68 § 1.55 MgSO4 5 mM 7.81§ 2.69

5 mM 0.00 § 0.00 ZnSO4 5 mM 17.96§ 1.08DTT 2 mM 11.52 § 1.60 Detergents

5 mM 2.80 § 1.11 Tween 80 1% 79.64§ 1.78Iodoacetamide 2 mM 78.81 § 1.92 SDS 1% 0.72§ 0.92Trypsin inhibitor 100 �g 98.13 § 0.81 Sodium tripolyphosphate 1% 11.00§ 0.77Metal ions Sodium tetraborate 1% 62.30§ 2.94NaCl 5 mM 69.78 § 3.55 OxidantHgCl2 1 mM 15.78 § 2.31 H2O2 5% 121.91 § 1.88

5 mM 0.00 § 0.53 10% 113.60 § 3.04FeSO4 5 mM 36.24 § 2.99 15% 104.77 § 2.80

1244 S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245

used for a 2.5 cm£ 2.5 cm piece of buValo hide (results notshown). The enzyme used in dehairing process should notbe collagenolytic in nature, so that hide matrix remainsintact. Earlier studies with the dehairing protease fromB. subtilis S14 have reported elastase, keratinase and colla-genase activities (Alexandre et al., 2005) while the presentenzyme had non collagenolytic and non keratinolytic prop-erties. In dehairing process, pH tolerance for the enzymeis an important factor. In earlier report, an alkaline prote-ase from Aspergillus tamarri, dehaired the goat skin at pH9–11, temperature 30–37 °C with 1% enzyme concentrationand incubation period of 18–24 h (Dayanandan et al.,2003). Similarly, an enzyme isolated from Bacillus sp. wasused for dehairing of goat skin with 2–3% concentrationand was active in pH 7.5 and 9.0 at 37 °C (Annapurna et al.,1996). In general, the dehairing process required activity ofenzyme under alkaline condition; this criterion was satisWedby protease from B. cereus MCM-B326 and thus suitablefor dehairing.

4. Conclusion

The production of extracellular protease from B. cereusMCM B-326 was optimum in starch- soybean meal-CaCO3medium of pH 9.0 at 30 °C under shake culture conditionwith 2.8£ 108 cells ml¡1 as an initial inoculum density andincubation of 36 h. The protease exhibited important prop-erties such as activity under alkaline pH, at broad tempera-ture range, in presence of oxidizing agent, and dehairingactivity for animal hide without chemical assistance andwithout hydrolyzing Wbrous proteins. Due to these proper-ties, the enzyme could be potentially useful in leather indus-try for dehairing of animal hide without damaging thecollagen layer, resulting in a better quality product andavoiding the pollution problem associated with the use ofchemicals.

Acknowledgements

The work was carried out under NMITLI project on,“Biotechnology for leather towards cleaner processing.”sponsored by CSIR, Govt. of India. The authors also thankMrs. Smita Kale, consultant, State Family PlanningBureau, Pune, for her help in the statistical analysis.

References

Alexandre, J.M., Walter, O., Beys, Da S., Renata, G., David, D., Joao, A.,Pegas, H., Carlos, T., 2005. Novel Keratinase from Bacillus subtilis S14Exhibiting remarkable dehairing capabilities. Appl. Environ. Micro-biol. 71, 594–596.

Altschul, S.F., Madden, T.L., SchaVer, A.A., Zhang, J., Zhang, Z., Miller,W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new gen-eration of protein database search programs. Nucl. Acids Res. 25,3389–3402.

Annapurna, R.A., Chandrababu, N.K., Samivelu, N., Rose, C., Rao,N.M., 1996. Eco-friendly enzymatic dehairing using extracellularprotease from Bacillus species isolate. J. Am. Leath. Chem. Assoc. 91,115–119.

Anwar, A., Saleemuddin, M., 1998. Alkaline proteases: a review. Biores.Technol. 64, 175–183.

Chi, Z., Ma, C., Wang, P., Li, H.F., 2006. Optimization of medium and cul-tivation conditions for alkaline protease production by the marineyeast Aureobasidium pullulans. Biores. Technol. in press, doi:10.1016/j.biortech.2006.02.006.

Dayanandan, A., Kanagraj, J., Sounderraj, L., Govindaraju, R., Rajkumar,G.S., 2003. Application of an alkaline protease in leather processing:an eco-friendly approach. J. Clean. Product. 11, 533–536.

Deshpande, V.V., Laxman, R.S., More, S.V., Rele, M.V., Rao, B.S.R., Jog-dand, V.V., Rao, M.B., Naidu, R.B., Maniknandan, P., Kumar, D.A.,Kanagaraj, J., Samayavaram, R., Samivelu, N., Rengarajulu, P., 2004.Process for preparation of an alkaline protease. US patent No.6777219.

Ghorbel, F.B., Sellami, K.A., Fakhfakh, N., Haddar, A., Manni, L.,Nasri, M., 2005. Production and puriWcation of a calcium-dependentprotease from Bacillus cereus BG1. J. Ind. Microbiol. Biotechnol.32, 186–194.

He, H., Chen, X., sun, C., Zhang, Y., Gao, P., 2006. Preparation and func-tional evaluation of oligopeptide-enriched hydrolysate from shrimp(Acetes chinensis) treated with crude protease from Bacillus sp.SM98011. Biores. Technol. 97, 385–390.

Huang, Q., Peng, Y., Li, X., Wang, H.F., Zhang, Y.Z., 2003. PuriWcationand characterization of an extracellular alkaline serine proteasewith dehairing function from Bacillus pumilus. Curr. Microbiol. 46,169–173.

Jayaraman, J., 2003. Biuret method of protein estimation, Lowry’s methodand quantitative estimations of amylase activity. In: Laboratory Man-ual in Biochemistry. New Age International (P) Limited, New Delhi,pp. 78–79. & 123.

Kanekar, P.P., Nilegaonkar, S.S., Sarnaik, S.S., Kelkar, A.S., 2002. Optimi-zation of protease activity of alkaliphilic bacteria isolated form analkaline lake in India. Biores. Technol. 85, 87–93.

Karadzic, I., Masui, A., Fujiwara, N., 2004. PuriWcation and characteriza-tion of a protease from Pseudomonas aeruginosa grown in cutting oil.J. Biosci. Bioeng. 4, 145–152.

Kim, S.S., Kim, Y.J., Rhee, I.K., 2001. PuriWcation and characterization ofa novel extracellular protease from Bacillus cereus KCTC 3674. Arch.Microbiol. 175, 458–461.

Mehrotra, S., Pandey, P.K., Gaur, S., Dharmwal, N.S., 1999. The produc-tion of alkaline protease by a Bacillus species isolate. Biores. Technol.67, 201–203.

Monsheimer, R., PXeiderer, E., 1976. Method for preparing tannable peltsfrom animal skins and hides US Patent 3966551.

Olivera, N., Sequeiros, C., SiWeriz, F., Breccia, D., 2006. Characterization ofalkaline proteases from a novel alkali- tolerant bacterium Bacillus pat-agoniensis. World J. Microbiol. Biotechnol. (doi:10.1007/s11274-005-9099-8).

Padmavathi, S., Chandrababu, N.K., Chitra, S., Chandrakasan, G., 1995.Application of the extracellular proteases from the culture Wltrate ofStreptomyces spp. G157 in bating. J. Soc. Leath. Technol. Chem 79,88–90.

RiZe, A., Ortolan, S., Brandelli, A., 2003. Dehairing activity of extracellu-lar proteases produced by keratinolytic bacteria. J. Chem. Technol.Biotechnol. 78, 855–859.

Sarath, G., Motte, R.S.D.L., Wagner, F.W., 1996. In: Beynon, R.J., Bond,J.S. (Eds.), Proteolytic Enzymes- A Practical Approach. Oxford IRLPress, New York, p. 7.

Sinha, N., Satyanarayana, T., 1991. Alkaline protease production by ther-mophilic Bacillus licheniformis. Ind. J. Microbiol. 31, 425–430.

Sneath, P.H.A., 1984. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual ofSystematic Bacteriology, vol. 1. Williams and Wilkin, Baltimore, pp.104–1141.

Takami, H., Akiba, T., Horikoshi, K., 1990. Characterization of an alka-line protease from Bacillus sp. No. AH- 101. Appl. Microbiol. Biotech-nol. 33, 519–523.

Tayler, M.M., Bailey, D.G., Feairheller, S.H., 1987. A review of the uses ofenzymes in the tannery. J. Am. Leath. Chem. Assoc. 82, 153–165.

S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 1245

Thangam, E.B., Rajkumar, S.G., 2002. PuriWcation and characterization ofalkaline protease from Alkaligenes faecalis. Biotechnol. Appl. Biochem.35, 149–154.

Thanikaivelan, P., Rao, J.R., Nair, B.U., Ramaswami, T., 2004. Progressand recent trends in biotechnological methods for leather processing.Trends Biotechnol. 22, 181–188.

Wang, J.J., Shih, J.C.H., 1999. Fermentation production of keratinase fromBacillus licheniformis PWD-1 and a recombinant B. subtilis FDB-29.J. Ind. Microbiol. Biotechnol. 22, 608–616.

Woessner, J.F., 1961. The determination of hydroxyproline in tissue andprotein samples containing small portions of this amino acids. Arch.Biochem. Biophys. 93, 440–447.