Vp Zambare-bioresource Technology

Bioresource Technology 98 (2007) 1238–1245

Production and partial characterization of dehairing protease from 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 2006 Available 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 as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25–65 °C and pH 6–12, 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 used to 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 operation from environmental point of view is the dehairing of skin/ hide. The conventional method of dehairing involves the use of lime and sodium sulphide. Presence of these chemicals in tannery waste is responsible for tremendous pollution, causing health hazards to the tannery workers. Lime produces a poisonous sludge while sodium sulphide is highly toxic and has obnoxious odor. Although enzyme assisted dehairing process reduces the pollution load to some extent, a technology based on enzyme alone, without the use of sulphide and other chemical inputs, has yet to be explored (Thanikaivelan et al., 2004). Proteases Wnd applications at various steps of leather processing, e.g., neutral proteases in soaking (Deshpande et al., 2004), alkaline proteases in dehairing (Dayanandan et 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: [email protected] (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; RiZe et al., 2003; Alexandre et al., 2005). The concrete mixture of dehairing enzymes from Bacillus subtilis and Bacillus cereus with 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 proteolytic enzyme of B. cereus B-326 having application in dehairing of buValo hide. 2. Methods 2.1. Microorganism and taxonomic study B. cereus BSA-26 producing protease was isolated from buValo hide obtained from Local Municipal Corporation Slaughterhouse (Pune, India). The strain was identiWed according to the methods described in Bergey’s Manual of Systematic Bacteriology (Sneath, 1984) and on the basis of 16 s ribosomal DNA sequence The stock culture was maintained 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

2.2. Inoculum preparation and production Seed inoculum was prepared by growing the isolate on nutrient agar slants/Roux bottle at 30 °C for 24 h. The cells were suspended in saline and cell density was measured spectrophotometrically (Shimatzu UV-2501 PC, Japan) at 600 nm. Following media were studied for protease production: synthetic medium casein (SMC: 0.7% K2HPO4, 0.3% KH2PO4, 0.01% MgSO4, 1% casein), nutrient broth supplemented with 1% casein (NBC), starch soybean meal (SS: 2% starch, 1% soybean meal, 0.3% CaCO3) and soybean meal-tryptone (ST: 1% soybean meal, 1% tryptone). The Xasks were inoculated with 24 h old inoculum and incubated on orbital shaker (150 rpm) at 30 °C for 60 h. The medium yielding maximum protease activity was selected for further investigation. 2.3. Protease assay The protease activity was determined by caseinolytic assay method of (Kanekar et al., 2002). The cell free supernatant (1 ml) was mixed with 4 ml of casein (0.625% w/v) and incubated at 37 °C for 30 min. The reaction was stopped by addition of 5 ml of 5% trichloroacetic acid. Enzymatically hydrolyzed casein was measured by modiWed Folin Ciocalteu method (Jayaraman, 2003), against casein treated with inactive enzyme as blank. A standard graph was generated using standard tyrosine solutions of 5–50 g ml¡1. One unit of protease activity was deWned as the amount of enzyme which liberated 1 g tyrosine per min at 37 °C.

1239

bean meal (1%) and calcium carbonate (0.3%), with starch, glucose, sucrose at 1% concentration. The Xasks were incubated for 36 h and cell free supernatant was analyzed for protease activity. 2.6. EVect of metal ions To determine the eVect of metal ions on the protease production, diVerent metal ions viz. CaCO3, CaCl2, K2HPO4, KH2PO4, FeSO4, MgSO4, NaCl and MnSO4 were individually added with 0.3% strength in starch (2%) and soybean meal (1%) liquid medium. The Xasks were incubated for 36 h and cell free supernatant was analyzed for protease activity. 2.7. Time course of protease production The growth of the organism and production of protease was studied under all optimum conditions viz SS medium, pH 9.0, 30 °C, shake culture condition, 1% inoculum size at 6 h interval. The cell growth was measured at 600 nm. Cell free supernatants were analyzed for enzyme activity, protein and residual starch, at each interval, up to 72 h. Protein content was measured by Buret method (Jayaraman, 2003). The residual starch was estimated by iodometric method (Jayaraman, 2003). 2.8. Scale-up studies

For optimization, production of protease by isolate BSA-26 was studied using SS medium, under the following conditions: shake and static culture, age of inoculum-21 to 24 h, initial inoculum density: 0.5–5.0% (v/v) of cell density 2.8 £ 108 cells ml¡1, temperature: 25–40 °C, with increments of 5 °C, pH: 6–12, with increments of one unit. The Xasks were incubated for 36 h and cells were removed by cold centrifugation at 13,000 £ g for 10 min. The cell free supernatant was analyzed for protease activity.

Production of the enzyme was carried out in a 2 l glass bottle with a working volume of 800 ml of SS medium. Parallel experiment was conducted at Xask level with the same medium. The culture was inoculated in nutrient broth. After an incubation period of 21 h, cell growth of 2.8 £ 108 cells ml¡1 was inoculated in 800 ml SS medium (pH 9.0) and incubated at 30 °C with constant agitation speed of 150 rpm. Enzyme samples were removed at 12 h intervals for measuring cell growth by recording OD at 600 nm. Biomass was separated by centrifugation. The cell free culture broth was assayed for extracellular protease activity and protein content as described above. The enzyme was partially puriWed using ammonium sulphate (60% saturation). The precipitated enzyme was used as a crude enzyme for further studies.

2.5. EVect of diVerent nitrogen and carbon sources

2.9. Substrate speciWcity

To test the eVect of diVerent nitrogen source on the protease production, the liquid medium of starch (2%) and calcium carbonate (0.3%) was supplemented with various complex nitrogen source such as yeast extract, beef extract, casein, tryptone, peptone, soybean meal, corn steep liquor and inorganic nitrogen sources viz. ammonium chloride, ammonium sulphate, ammonium phosphate, ammonium nitrate and sodium nitrite all at 1% concentration. Likewise the eVect of diVerent carbon sources on protease production was studied by supplementing; a liquid medium of soy-

Protease was produced in SS medium (pH 9.0) at 30 °C for 36 h, and cell free supernatant was assayed using diVerent substrates such as casein, Bovine serum albumin (BSA), haemoglobin, collagen, gelatin and keratin. The enzymatic hydrolysis of the casein and BSA was studied by assay as described above. The proteolytic activity of the enzyme with 2% haemoglobin as substrate was carried out according to Sarath et al. (1996). The enzymatic hydrolysis of collagen, gelatin was studied as described by Woessner (1961). Keratinase activity was determined using keratin azure

2.4. EVect of diVerent environmental conditions on protease production

1240

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

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

and epidermis were observed by mechanical means at an hourly interval.

2.10. EVect of pH and temperature

2.14. Statistical analysis

The pH stability of precipitated protease was determined with casein (0.625% w/v) as a substrate dissolved in diVerent buVers [sodium acetate, pH 4–6, sodium phosphate, pH 7–8, Tris HCl, pH 9–10.6 and Glycine-NaOH, pH 11–12]. The pH stability of the protease was determined by preincubating the enzyme in diVerent buVers for 30 min at 37 °C. Likewise, thermal stability of the precipitated protease was determined by pre-incubating the enzyme at diVerent temperatures from 25–80 °C for 30 min. All experiments were carried out in duplicate and each analysis was also performed in duplicate.

Analysis of variance with repeated measures was carried out using GLM command of software SPSS (SPSS version 10, Windows 98 version). Activity means and standard deviations were calculated. Univariate analysis of variance (ANOVA) was employed on the data for protease activity at diVerent % inoculum, pH, temperature, culture condition-incubation period, complex and inorganic nitrogen sources, carbon supplement and metal ions supplement and tested for their signiWcance.

2.11. EVects of diVerent inhibitors, metals, detergents and oxidant on enzyme activity To study inhibition of the protease, enzyme was preincubated with inhibitors such as phenylmethylsulphonyl Xuoride (PMSF, 1 mM), ethylenediamine tetraacetic acid (EDTA, 2 & 5 mM), dithiothretol (DTT, 2 & 5 mM), iodoacetamide (2 mM), trypsin inhibitor (100 g), metal ions such as Hg+ (1 & 5 mM), Na+, Fe2+, Cu2+, Ca2+, Mg2+, Mn2+, Zn2+ (5 mM); detergents such as sodium dodecyl sulfate (SDS, 1%), sodium tripolyphosphate (1%), sodium tetraborate (1%) and oxidizing agent H2O2 (5%, 10% and 15%) for 30 min at 37 °C. Subsequently the enzyme assay was performed as described above. The percent residual enzyme activity was calculated with reference to the activity of the enzyme without these supplements.

3. Results and discussion The culture B. cereus BSA-26 was deposited in MACS collection of microorganisms (WFCC code 561) and designated as MCM B-326.The isolate was identiWed as on the basis of morphological and physiological characteristics, biochemical tests and 16 S rRNA sequencing. A comparison of the DNA sequence with sequences in the National Center for Biotechnology Information (NCBI) database with BLAST software (Altschul et al., 1997) showed 98.5% sequence identity with the published 16 s rRNA sequences of B. cereus. The 16 s rDNA sequence of the isolate has been deposited in GenBank database with accession number DQ479314. Strains of Bacillus species producing protease 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, are mainly directed towards detergent industry (Anwar and Saleemuddin, 1998).

2.12. Gel electrophoresis and zymogram

3.1. Optimization of protease production

Proteins were analyzed by tube gel electrophoresis using comassive brilliant blue R250. The proteins were precipitated with ammonium sulphate (60% saturation) followed by membrane dialysis. The protein pattern of crude enzyme was carried out with native PAGE. A zymogram of dialyzed enzyme was obtained using polyacrylamide gel electrophoresis followed by treatment with 1% (w/v) casein as substrate in Tris buVer (pH 9.0). The molecular weight of the protease enzyme was determined by compairing with mobility of standard molecular weight marker proteins (bovine albumin, 66 kDa, chicken ovalbumin, 45 kDa, glyceroldehyde-3-phosphate dehydrogenase 36 kDa, trypsinogen 24 kDa, cytochrome C 12.4 kDa).

Maximum production of protease by B. cereus MCM B326 was obtained in SS medium at 36 h of incubation, and was signiWcantly (p < 0.001) higher than that observed in other media tested. The protease activity was highest with 2% 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 alkaline protease by thermophilic B. licheniformis (Sinha and Satyanarayana, 1991). The results on the eVect of initial pH and temperature on protease production showed maximum activity 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, results not shown). Comparable results were obtained for B. alcalophilus, isolated from Lonar lake, India (Kanekar et al., 2002). It was observed that, in presence of sucrose, starch or glucose (1%), the protease activity (116.27 § 5.72– 122.45 § 20.59 U ml¡1, results not shown) was almost constant; however it decreased signiWcantly in the absence of carbon sources (29.35 § 9.36 U ml¡1) at 36 h. The activity

2.13. Dehairing of buValo hide Ammonium sulfate precipitate of enzyme was applied at 1% concentration of to piece buValo hide (2.5 cm £ 2.5 cm), presoaked in tap water from Xesh side, and kept at ambient temperature (28 § 2 °C) in a dry place. Loosening of hair

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

obtained with 1% starch was similar to that with 2% starch in SS medium. The protease activity enhanced 4 fold in presence of starch. Such enhancement of alkaline protease has been reported in alkaliphilic B. licheniformis (Sinha and Satyanarayana, 1991). EVect of diVerent complex and inorganic nitrogen sources on protease activity are presented in Table 1. The protease activity was highest with soybean

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

Mean enzyme activity (U ml¡1)

Complex nitrogen sources (1%) Soybean meal Yeast extract Peptone Tryptone Corn steep liquor Beef extract Casein

122.24 § 2.06 61.26 § 7.63 39.97 § 10.03 30.37 § 5.40 22.31 § 4.43 9.04 § 2.53 1.36 § 0.72

Inorganic nitrogen sources (1%) Ammonium sulphate Ammonium nitrate Ammonium chloride Ammonium phosphate Sodium nitrite

58.91 § 1.41 58.38 § 0.19 100.88 § 8.03 0.42 § 0.80 112.13 § 6.99

Metal ions (0.3%) CaCO3 CaCl2 K2HPO4 KH2PO4 FeSO4 MgSO4 NaCl MnSO4 Without metal ions

125.99 § 1.91 62.63 § 3.65 43.30 § 5.47 41.51 § 2.75 30.76 § 1.61 21.20 § 1.72 17.66 § 1.73 13.90 § 3.19 17.06 § 0.86

5

2.0

4

meal (122.24 § 2.06 U ml¡1, p < 0.001). Soybean meal was used as inducer for protease production from Conidiobolus coronatus (Deshpande et al., 2004). The protease activity was increased when production medium was supplemented with potassium, magnesium, iron and calcium ions. Enhancement of 7.4 fold in activity was achieved by supplementation of 0.3% CaCO3 (p < 0.001) (Table 1). This indicated that the calcium ion was necessary for enzyme induction (Ghorbel et al., 2005). Thus, optimization studies resulted in the following Wndings: 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 incubation 36 h. (Tayler et al., 1987) have reported the best production of dehairing protease using nutrient medium containing corn steep liquor, lactose, sodium sulfate and potassium acid 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 activity reached to 126.87 § 1.32 U ml¡1 within 36 h of the fermentation when the cell growth reached late log phase or early stationary phase (Fig. 1). An alkaline protease from marine yeast produced protease activity within 30 h when the cell growth reached mid-log phase (Chi et al., 2006). The reduction in starch level indicated that B. cereus MCM B-326 had strong amylolytic activity in addition to protease activity. The starch level was almost negligible after 6 h. The pH of the medium decreased towards acidic side because of hydrolysis of starch to acids via glucose (data not shown). The protein content was decreased because of the utilization of protein by the organism. The decline in protease activity upon prolonged incubation may be due to autolysis of the enzyme.

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

2.5

1241

140

10

-1

1.0

-1

Protein (mg ml )

1.5

3

80 60

2

4

-1

40

6

1

-1

)

0.5

Residual starch (mg ml )

9

8 100

Enz yme ac tivity (U m l

Cell density (x10 cells ml )

120

2 20

0.0

0

0

0 Cell density

10

20 Protein

30

40 50 60 Tim e ( h) Enzyme activity

70

80

0

Residual starch

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

1242

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

3.3. Scale-up studies

160 140 % Enzyme activity

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

120

Temperature

100 80 60 pH

40 20

3.4. Characteristics of protease

0

The protease from B. cereus MCM B-326 hydrolyzed casein, haemoglobin, BSA with activity of 128.64 U ml¡1, 14.25 U ml¡1 and 50.89 U ml¡1 respectively (data not shown). But it was not able to hydrolyze collagen, gelatin, and keratin. Protease from Alkaligenes faecalis could not hydrolyze the Wbrous proteins such as collagen and keratin (Thangam and Rajkumar, 2002). The enzyme was active in the pH range of 6–12, with optimum activity at pH 9.0, although, a small peak at pH 10.6 was also observed, suggesting a presence of two alkaline proteases (Fig. 3). The preliminary studies on the extracellular dehairing protease secreted by the Bacillus sp. showed that it 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 small peak at 30 °C was also observed, suggesting presence of two proteases. The activity increased from 134% to 149% with increase in temperature from 40–55 °C. The enzyme was completely inactivated at 80 °C (Fig. 3). In an earlier report, the dehairing enzyme was active between the temperature range of 30–60 °C, and showed optimum activity at 55 °C.

2

4

6

8

10

20

30

40

50

60

70

After 30 min, activity decreased to 53% and 85% at 50 °C and 60 °C, respectively and completely inactivated at 70 °C (Huang et al., 2003). (Annapurna et al., 1996) reported that the 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 proteases reported above. The eVect of inhibitors, metal ions, detergents and oxidant on the ammonium sulphate precipitated enzyme is detailed in Table 2. The protease was completely inhibited by 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

5

25

4

20

Enzyme activity (U ml-1)

3 100 80 2 60 40

15

10

5

1

Cell density (x109 cells ml-1)

Protein (mg ml-1)

120

20 0

0

0

Enzyme activity

30

40

90

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 standard deviations. The 100 ml¡1 for pH and 1721.38 U ml¡1 for temperature.

140

20

80

Temperature (ºC)

160

10

14

pH

180

0

12

50

Time (h) Protein (mg ml-1)

60

70

80

Cell density

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

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

1243

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

Concentration

Control Inhibitors PMSF EDTA

–

DTT Iodoacetamide Trypsin inhibitor Metal ions NaCl HgCl2 FeSO4

% residual activity 100 § 1.96

1 mM 2 mM 5 mM 2 mM 5 mM 2 mM 100 g

84.63 § 3.93 7.68 § 1.55 0.00 § 0.00 11.52 § 1.60 2.80 § 1.11 78.81 § 1.92 98.13 § 0.81

5 mM 1 mM 5 mM 5 mM

69.78 § 3.55 15.78 § 2.31 0.00 § 0.53 36.24 § 2.99

Compound

Concentration

% residual activity

CuSO4 CaCl2 MnSO4 MgSO4 ZnSO4 Detergents Tween 80 SDS Sodium tripolyphosphate Sodium tetraborate Oxidant H 2O 2

5 mM 5 mM 5 mM 5 mM 5 mM

17.03 § 1.16 101.03 § 5.07 49.6 3 § 1.86 7.81 § 2.69 17.96 § 1.08

1% 1% 1% 1%

79.64 § 1.78 0.72 § 0.92 11.00 § 0.77 62.30 § 2.94

5% 10% 15%

121.91 § 1.88 113.60 § 3.04 104.77 § 2.80

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.

by DTT, thus the enzyme might contain S–S bond as a part of its monomeric structure. The eVect was consistent with its above-described thermal stability, which had been shown primarily to be the result of disulWde bond (Karadzic 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. In an earlier report, dehairing protease was completely inhibited by PMSF and partially inhibited by EDTA (Huang et al., 2003). Similarly, the dehairing protease of Bacillus sp. kr10 was completely inhibited by PMSF, whereas 84% inhibited by EDTA (RiZe et al., 2003).Thus, both the reported dehairing proteases of Bacillus sp. were serine proteases. The protease was markedly inhibited (80–93%) by Cu2+, Mg2+, Zn2+, and sodium tripolyphosphate; while partially inhibited (30–60%) by Na+, Fe2+, Mn2+. Sodium tetraborate and iodoacetamide inhibited the activity by about 20%. The protease enzyme was stable and active in the presence of oxidizing agent H2O2 (5% and 10%) and, therefore, could be used in bleach-based detergent formulations. Similar results were obtained for alkaline protease from alkali tolerant B. patagoniensis (Olivera et al., 2006). Results also showed that (data not shown) Ca2+ was required for the production only and not for activity and/or stability of the enzyme. However, protease from B. cereus BG1 required calcium ion for its activity as well as stability (Ghorbel et al., 2005). Thus, the present enzyme diVered from the previous reports on B. cereus. The crude enzyme preparation showed Wve protein bands. After dialysis the ammonium sulphate precipitated enzyme showed two bands, viz., Cereus 1 and Cereus 2 on zymogram. The non-denaturing PAGE showed two proteases with approximate molecular weights 45 kDa and 36 kDa of band Cereus 1 and Cereus 2 respectively (Fig. 4). It has been reported that B. cereus has two proteases with molecular masses of approximately 38 kDa and 36 kDa

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

(Kim et al., 2001). A calcium dependent protease from B. cereus BG1 was reported to have molecular weight of 34 kDa (Ghorbel et al., 2005). According to (Huang et al., 2003) the molecular weight of the puriWed dehairing protease from B. pumilus was 32 kDa. Thus, molecular weight of Cereus 2 was similar to the molecular weight reported for other proteases from B. cereus while molecular weight of Cereus 1 was diVerent. The enzyme showed two optima for both pH and temperatures and two enzyme bands on PAGE-zymogram, suggesting the extracellular secretion of two proteases from B. cereus MCM B-326. 3.5. Dehairing of buValo hide The enzyme dehaired the buValo hide within 21 h at pH 7.0 and ambient temperature 28 § 2°C, with 1% enzyme

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 not shown). The enzyme used in dehairing process should not be collagenolytic in nature, so that hide matrix remains intact. Earlier studies with the dehairing protease from B. subtilis S14 have reported elastase, keratinase and collagenase activities (Alexandre et al., 2005) while the present enzyme had non collagenolytic and non keratinolytic properties. In dehairing process, pH tolerance for the enzyme is an important factor. In earlier report, an alkaline protease from Aspergillus tamarri, dehaired the goat skin at pH 9–11, temperature 30–37 °C with 1% enzyme concentration and incubation period of 18–24 h (Dayanandan et al., 2003). Similarly, an enzyme isolated from Bacillus sp. was used for dehairing of goat skin with 2–3% concentration and was active in pH 7.5 and 9.0 at 37 °C (Annapurna et al., 1996). In general, the dehairing process required activity of enzyme under alkaline condition; this criterion was satisWed by protease from B. cereus MCM-B326 and thus suitable for dehairing. 4. Conclusion The production of extracellular protease from B. cereus MCM B-326 was optimum in starch- soybean meal-CaCO3 medium of pH 9.0 at 30 °C under shake culture condition with 2.8 £ 108 cells ml¡1 as an initial inoculum density and incubation of 36 h. The protease exhibited important properties such as activity under alkaline pH, at broad temperature range, in presence of oxidizing agent, and dehairing activity for animal hide without chemical assistance and without hydrolyzing Wbrous proteins. Due to these properties, the enzyme could be potentially useful in leather industry for dehairing of animal hide without damaging the collagen layer, resulting in a better quality product and avoiding the pollution problem associated with the use of chemicals. 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 thank Mrs. Smita Kale, consultant, State Family Planning Bureau, 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 S14 Exhibiting remarkable dehairing capabilities. Appl. Environ. Microbiol. 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 generation 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 extracellular protease 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 cultivation conditions for alkaline protease production by the marine yeast 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., Jogdand, 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-dependent protease from Bacillus cereus BG1. J. Ind. Microbiol. Biotechnol. 32, 186–194. He, H., Chen, X., sun, C., Zhang, Y., Gao, P., 2006. Preparation and functional 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. PuriWcation and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr. Microbiol. 46, 169–173. Jayaraman, J., 2003. Biuret method of protein estimation, Lowry’s method and quantitative estimations of amylase activity. In: Laboratory Manual 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. Optimization of protease activity of alkaliphilic bacteria isolated form an alkaline lake in India. Biores. Technol. 85, 87–93. Karadzic, I., Masui, A., Fujiwara, N., 2004. PuriWcation and characterization 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 of a 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 production of alkaline protease by a Bacillus species isolate. Biores. Technol. 67, 201–203. Monsheimer, R., PXeiderer, E., 1976. Method for preparing tannable pelts from animal skins and hides US Patent 3966551. Olivera, N., Sequeiros, C., SiWeriz, F., Breccia, D., 2006. Characterization of alkaline proteases from a novel alkali- tolerant bacterium Bacillus patagoniensis. World J. Microbiol. Biotechnol. (doi:10.1007/s11274-0059099-8). Padmavathi, S., Chandrababu, N.K., Chitra, S., Chandrakasan, G., 1995. Application of the extracellular proteases from the culture Wltrate of Streptomyces spp. G157 in bating. J. Soc. Leath. Technol. Chem 79, 88–90. RiZe, A., Ortolan, S., Brandelli, A., 2003. Dehairing activity of extracellular 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 IRL Press, New York, p. 7. Sinha, N., Satyanarayana, T., 1991. Alkaline protease production by thermophilic Bacillus licheniformis. Ind. J. Microbiol. 31, 425–430. Sneath, P.H.A., 1984. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1. Williams and Wilkin, Baltimore, pp. 104–1141. Takami, H., Akiba, T., Horikoshi, K., 1990. Characterization of an alkaline protease from Bacillus sp. No. AH- 101. Appl. Microbiol. Biotechnol. 33, 519–523. Tayler, M.M., Bailey, D.G., Feairheller, S.H., 1987. A review of the uses of enzymes in the tannery. J. Am. Leath. Chem. Assoc. 82, 153–165.

S.S. Nilegaonkar et al. / Bioresource Technology 98 (2007) 1238–1245 Thangam, E.B., Rajkumar, S.G., 2002. PuriWcation and characterization of alkaline protease from Alkaligenes faecalis. Biotechnol. Appl. Biochem. 35, 149–154. Thanikaivelan, P., Rao, J.R., Nair, B.U., Ramaswami, T., 2004. Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol. 22, 181–188.

1245

Wang, J.J., Shih, J.C.H., 1999. Fermentation production of keratinase from Bacillus 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 and protein samples containing small portions of this amino acids. Arch. Biochem. Biophys. 93, 440–447.