Purification and characterization of keratinase from a new Bacillus subtilis strain

Purification and characterization of keratinase from a new Bacillus subtilis strain 1 2 first-author 3 2 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xdzheng@zju.edu.cn †E-mail:xdzheng@zju.edu.cn xdzheng@zju.edu.cn College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China College of Food Science and Biosystem Engineering, Zhejiang University, Hangzhou 310029, China College of Food Science and Biosystem Engineering, Zhejiang University, Hangzhou 310029, China Institute of Bioengineering, Zhejiang Sci-Tech University, Hongzhou 310018, China Institute of Bioengineering, Zhejiang Sci-Tech University, Hongzhou 310018, China The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase. Ammonium sulfatate Bacillus subtilis Characterization Feather Keratin Keratinase Purification Reducing agents INTRODUCTION 1 Feathers are largely produced as a waste byproduct at poultry plants (Williams et al., 1991 1991 ). They are insoluble structural proteins cross-linked by disulfide, hydrogen and hydrophobic bonds but could represent a rich protein resource because they contain over 90% (w/w) keratins. Keratins cannot be degraded by the usual proteolytic enzymes such as pepsin, trypsin and papain. Nevertheless, feathers do not accumulate in nature because keratins could be degraded by keratinases (EC 3.4.21/24/99.11) produced by some microorganisms (Onifade et al., 1998 1998 ). Many keratinases from species of Bacillus (Williams et al., 1990 1990 ; Riffel et al., 2003 2003 ; Lucas et al., 2003 2003 ), fungi (El-Naghy et al., 1998 1998 ; Gradišar et al., 2000 2000 ; Friedrich et al., 2005 2005 ) and Actinomycetes (Ignatova et al., 1999 1999 ; Gushterova et al., 2005 2005 ) had been reported and some of them were purified and characterized (Lin et al., 1992 1992 ; Böckle et al., 1995 1995 ; Nam et al., 2002 2002 ). Different keratinases vary in their characteristics. Until now, only a few keratinases from Bacillus sp. had been purified. Keratinases from microorganisms have many applications in the feed, fertilizer, detergent, leather and pharmaceutical industries (Gupta and Ramnani, 2006 2006 ). For example, the feather hydrolysates of Bacillus licheniformis PWD-1 and Vibrio sp. strain kr2 (Williams et al., 1991 1991 ; Grazziotin et al., 2006 2006 ) can be used as animal food. Indeed, addition of the crude keratinase from B . licheniformis PWD-1 improved the poultry growth (Odetallah et al., 2003 2003 ). Also, the keratinase can degrade the infectious form of prion, PrP sc , in the presence of detergents and heat treatment (Langeveld et al., 2003 2003 ), which could be important for the utilization of animal meal as feed. As for leather industry, the keratinase from Bacillus subtilis S14 exhibits remarkable dehairing capabilities (Macedo et al., 2005 2005 ) without the degradation of collagen and this ecofriendly dehairing approach shows great utilization potential. We had screened a new feather-degrading Bacillus subtilis KD-N2 strain which degraded feathers completely within 30 h in submerged cultivation (Cai et al., 2008 2008 ), and the crude keratinase from the isolate showed capabilities of removing calf hair and sheep wool from the skins. The aim of this study was to purify and characterize the keratinase produced in feathers substrate by this Bacillus spp. isolate. MATERIALS AND METHODS 1 Bacteria and growth conditions 2 Strain KD-N2 was screened from a local poultry plant and kept in our laboratory (Cai et al., 2008 2008 ). The medium (pH 7.2) used for keratinase production contained the following constituents (g/L): NaCl 0.5, KH 2 PO 4 0.7, K 2 HPO 4 1.4, MgSO 4 0.1 and feathers 10. Cultivation was performed using 500 ml Erlenmeyer flasks containing 100 ml medium for 24 h at 28 °C with constant shaking at 200 r/min. As inocula, 5% (v/v) bacteria grew in Luria-Bertani broth [peptone 1% (w/v), yeast extract 0.3% (w/v) and NaCl 0.5% (w/v), pH 7.2] for 20 h. Culture supernatants obtained after centrifugation at 8000× g for 20 min were used for further study. Assay of keratinase activity 2 Keratin azure (Sigma-Aldrich, USA) was used as the substrate. It was first frozen at −20 °C and then ground into a fine powder. The 5 mg keratin azure powder was suspended in 1 ml 50 mmol/L Tris-HCl buffer (pH 8.0). The reaction mixture contained 1 ml keratin azure suspension and 1 ml appropriately diluted enzyme. The reactions were carried out at 50 °C in a water bath with constant agitation of 200 r/min for 30 min. After incubation, the reactions were stopped by adding 2 ml 0.4 mol/L trichloroacetic acid (TCA) and followed by centrifuging at 3000× g for 20 min to remove the substrate. The supernatant was spectrophotometrically measured for release of the azo dye at 595 nm. The 1 ml keratin azure suspension in the same buffer (like that of the sample) was agitated for 30 min at 50 °C, then was added 2 ml 0.4 mol/L TCA and 1 ml enzyme solution as a control. One unit (U) keratinase activity was defined as the amount of enzyme causing 0.01 absorbance increase between the sample and control at 595 nm under the conditions given. Protein determination 2 Protein concentration was measured by the method of Bradford ( 1976 1976 ), using bovine serum albumin (BSA) (Sigma, USA) as standard. The specific activity was expressed as the enzymatic activity (U) per mg of protein. Purification of keratinase 2 All operations were performed at room temperature. After centrifugation at 8000× g for 20 min, solid ammonium sulphate was added to the supernatant to achieve 30% saturation, and then centrifuged to remove the pellet. The enzyme was precipitated from the supernatant by addition of solid ammonium sulphate, with gentle stirring until 80% saturation, and then allowed to stand for 12 h followed by centrifugation at 8000× g . The pellet was dissolved in 20 mmol/L Tris-HCl buffer (pH 8.0) and applied to a Sephadex G-75 column (6.0 cm×60.0 cm), which was processed at a flow rate of 10 ml/h with 20 mmol/L Tris-HCl buffer (pH 8.0) and every 3 ml fraction was collected. Active fractions were collected, concentrated by polyethylene glycol 2000 (PEG 2000) and applied to a DEAE (diethylaminoethyl)-Sepharose Fast Flow (FF) column (1.9 cm×20.0 cm). Samples were eluted at a flow rate of 6 ml/min with different concentrations of sodium chloride solution in 20 mmol/L Tris-HCl buffer (pH 8.0) and every 6 ml fraction was collected. Then the active fractions with PEG 2000 were concentrated and applied to a Sephadex G-75 column (1.0 cm×60.0 cm) at a flow rate of 6 ml/h. The 3 ml fractions were collected. The active fractions were then concentrated by PEG 2000 for further analysis. Molecular mass determination 2 Polyacrylamide gel electrophoresis (PAGE) (12%, w/w) in the presence of sodium dodecyl sulfate (SDS) was carried out by the method of Laemmli ( 1970 1970 ). The electrophoresed protein gels were stained with Coomassie brilliant blue R250 (Sigma, USA). Characterization of the keratinase 2 To determine the optimal temperature for keratinolysis, enzyme reactions were carried out at different temperatures for 30 min. In order to investigate thermostability, the enzyme solution was pre-incubated for 20, 40, 60, 80, 100 and 120 min at 55 °C, and then the residual activity was measured. The optimum pH was determined at 55 °C using the following buffers (50 mmol/L): sodium phosphate buffer (pH 6.0~7.5), Tris-HCl buffer (pH 7.5~9.0), and Glycine/NaOH buffer (pH 9.0~10.0). The effects of metal ions, enzyme inhibitor, detergent and organic solvents on keratinase activity were studied by assaying the enzyme activity as described above after pre-incubation with each chemical for 10 min at room temperature. The concentrations of the chemicals are as follows: 5.0 mmol/L of Ca 2+ , Mg 2+ , Cu 2+ , Mn 2+ , Zn 2+ , Al 3+ , SDS and phenylmethanesulfonyl fluoride (PMSF); 2.5, 5.0 and 10.0 mmol/L ethylene diamine tetraacetic acid (EDTA); 1% (v/v) of methanol, ethanol, dimethyl sulfoxide and isopropyl alcohol. Substrate specificity of keratinase 2 To determine the keratinase specificity, the following substrates were used: casein (Sigma, USA), BSA (Sigma, USA), ovalbumin, type I collagen (Worthington, USA), chicken feather, goat hair, calf hair and human hair. The incubation procedures were the same as described for keratinase activity determination except that every incubation contained 20 mg substrate and 50 μl purified keratinase. The extent of hydrolysis was determined spectrophotometrically at 280 nm by measuring TCA-soluble peptides released from the substrates during incubation. Effects of chemicals on feather meal hydrolysis 2 To determine the effects of chemicals on feather meal hydrolysis, 1 or 5 mmol/L dithiothreitol (DTT), SDS, sodium sulphite, L-cysteine, ammonium sulfamate and 1% (v/v) or 5% (v/v) mercaptoethanol, dimethylsulfoxide (DMSO) were added to 2 ml of 50 mmol/L Tris-HCl (pH 8.5) buffer containing 20 mg feather meal and 50 μl purified enzyme. After incubation for 1 h, the reaction was stopped by adding 2 ml 0.4 mol/L TCA. An incubation of feather meal and TCA for 1 h before enzyme addition was carried out as a control. The extent of feather meal hydrolysis was determined spectrophotometrically at 280 nm by measuring TCA-soluble peptides released from the substrate during incubation. RESULTS AND DISCUSSION 1 Feather degradation and keratinase production 2 B . subtilis KD-N2 strain produced inducible keratinase in feathers substrate and degraded feathers completely in 30 h (Cai et al., 2008 2008 ). Feathers were the mostly utilized substrate for keratinase production. Accompanied by feather degradation and keratinase production, the pH value of the culture increased to about 8.5. B . subtilis strains had been widely utilized for enzyme production, including the keratinases (Lal et al., 1999 1999 ; Suh and Lee, 2001 2001 ; Kim et al., 2001 2001 ; Macedo et al., 2005 2005 ). Other Bacillus species including B . licheniformis , B . pumilis , B . cereus , B . halodurans and B . pseudofirmus (Williams et al., 1990 1990 ; Takami et al., 1999 1999 ; Rozs et al., 2001 2001 ; Kim et al., 2001 2001 ; Gessesse et al., 2003 2003 ; El-Refai et al., 2005 2005 ) had also been reported for their keratinolytic activity. Among these strains, B . subtilis KD-N2 degraded feathers more quickly than any other reported until now, except for B . licheniformis RG1, which degraded feathers completely in 24 h (Ramnani and Gupta, 2004 2004 ). Among the keratinases reported, only two of them had been purified (Suh and Lee, 2001 2001 ; Macedo et al., 2005 2005 ). Enzyme purification 2 The results of the purification procedures are summarized in Table 1 1 . SDS-PAGE analysis of the sample revealed a single band (Fig. 1a 1a ), indicating that the keratinase was purified. The overall purification factor was about 12.7-fold, and the final yield was 4.6%. The final product had a specific activity of about 63.3 U/mg. Step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification Yield (%) Crude enzyme 206.40 1040.0 5.0 1.0-fold 100.0 Sephadex G-75 (6.0 cm×60.0 cm) 10.20 132.6 13.0 2.6-fold 12.8 DEAE-Sepharose FF (1.9 cm×20.0 cm) 1.40 73.0 51.9 10.4-fold 7.0 Sephadex G-75 (1.0 cm×60.0 cm) 0.76 48.0 63.3 12.7-fold 4.6 Lane 1: Purified keratinase; Lane M: Molecular mass of marker proteins (Promega, USA): rabbit muscle phosphorylase b 97.4 kDa, bovine serum albumin 66.2 kDa, chicken egg ovalbumin 42.7 kDa, bovine erythrocytes carbonic anhydrase 31 kDa and chicken egg white lysozyme 14.4 kDa Molecular mass of the keratinase 2 The molecular mass of the keratinase was estimated by comparing the electrophoretic mobility of the enzyme with the electrophoretic mobilities of marker proteins. The apparent molecular mass was 30.5 kDa (Fig. 1b 1b ). Molecular masses of keratinases range from 18 to 200 kDa, except for a unique enzyme from a pathogenic fungi (Gupta and Ramnani, 2006 2006 ). For Bacillus species, they are of medium size, such as 33 kDa ( B . licheniformis ) (Lin et al., 1992 1992 ), 25.4 kDa ( B . subtilis ) (Suh and Lee, 2001 2001 ) and 24 kDa ( B . paeudofirmis ) (Gessesse et al., 2003 2003 ). Effects of temperature and pH on the keratinase activity 2 Fig. 2 2 shows that the keratinase was active over a temperature range of 40~70 °C, with an optimum at 55 °C. Fig. 3 3 shows that the keratinase was active at neutral and alkaline conditions, with optimum at pH 8.5, and the most suitable buffer seemed to be Tris-HCl. Most keratinases possess an activity optimum in the range of 30~80 °C, for example, keratinase from B . pseudofirmus AL-89 is of 60~70 °C (Gessesse et al., 2003 2003 ), Nocardiopsis sp. TOA-1 is of 60 °C (Mitsuiki et al., 2004 2004 ), and a few have exceptionally high temperature optimum of 100 °C (Nam et al., 2002 2002 ). Most keratinases are active in neutral to alkali conditions, from pH 7.0 to pH 9.5. For example, the activity optimum of keratinase from Microbacterium kr10 is pH 7.0 (Thys et al., 2004 2004 ), B . pumilus FH9 of pH 8.0 (El-Refai et al., 2005 2005 ), Fervidobacterium islandicum AW-1 of pH 9.0 (Nam et al., 2002 2002 ), and a few of extreme alkalophilic optima at pH 12~13 (Takami et al., 1999 1999 ) and pH 12.5 (Mitsuiki et al., 2004 2004 ). Assays were performed at different temperatures in 50 mmol/L Tris-HCl buffer (pH 8.0) for 30 min Assays were performed at 55 °C using the following buffers (50 mmol/L): sodium phosphate buffer (pH 6.0~7.5), Tris-HCl buffer (pH 7.5~9.0), and glycine/NaOH buffer (pH 9.0~10.0) Effects of metal ions, enzyme inhibitor, detergent and organic solvents on the keratinase activity 2 The keratinase from B . subtilis KD-N2 was totally inhibited by Cu 2+ , Mn 2+ and partially by Ca 2+ , Mg 2+ , Zn 2+ , Al 3+ , methanol, ethanol, dimethyl sulfoxide, isopropyl alcohol, and was stimulated by SDS and EDTA (Table 2 2 ). Generally, heavy metal ions such as Cu 2+ (Nam et al., 2002 2002 ; Riffel et al., 2003 2003 ; Thys et al., 2004 2004 ), Hg 2+ (Riffel et al., 2003 2003 ; Thys et al., 2004 2004 ) and Zn 2+ (Thys et al., 2004 2004 ) have inhibitory effects on keratinolytic activity. Contrarily, Ca 2+ , Mg 2+ and Mn 2+ stimulate some keratinases (Nam et al., 2002 2002 ; Riffel et al., 2003 2003 ). But for the keratinase from B . subtilis KD-N2 strain, all metal ions have negative effects on its activity. Serine-proteinase inhibitor PMSF partially inhibited the keratinase activity, which is identical to most of the keratinases studied except for a few from Paecilomyces marquandii , Doratomyces microsporus and Xanthomonas maltophilia , which are fully inhibited (de Toni et al., 2002 2002 ; Gradišar et al., 2005 2005 ). EDTA has negative effects on activities of mostly reported keratinases, but we observed in this study that the keratinase from B . subtilis KD-N2 strain was stimulated by 5 mmol/L EDTA, similar to the keratinase from Fervidobacterium islandicum AW-1 (Nam et al., 2002 2002 ). Further results indicate that 2.5 and 10 mmol/L EDTA could also stimulate the keratinase activity (Table 2 2 ). Organic solvents inhibited the keratinase activity to some degree, which is different from keratinases from Chryseobacterium sp. (Riffel et al., 2003 2003 ) and Nocardiopsis sp. TOA-1 (Mitsuiki et al., 2004 2004 ). The detergent SDS has positive effects on keratinase activity, which is similar to the keratinase from Streptomyces albidoflavus (Bressollier et al., 1999 1999 ), but differs from the keratinases from Streptomyces pactum DSM 40530 (Böckle et al., 1995 1995 ), Chryseobacterium sp. (Riffel et al., 2003 2003 ) and Nocardiopsis sp. TOA-1 (Mitsuiki et al., 2004 2004 ). Proteases are widely used in the detergent industry, and it is important for the keratinase to be used in the presence of detergents such as SDS. Compared with previously reported keratinolytic enzymes, the keratinase from B . subtilis KD-N2 showed some novel characterizations and utilization potential. Values are mean of three independent determinations Chemicals Concentration Relative activity * * (%) None 100 Ca 2+ 5 mmol/L 44.8±3.4 Mg 2+ 5 mmol/L 41.4±3.2 Cu 2+ 5 mmol/L 0 Mn 2+ 5 mmol/L 0 Zn 2+ 5 mmol/L 34.5±3.1 Al 3+ 5 mmol/L 75.9±6.9 SDS 5 mmol/L 158.6±13.8 EDTA 2.5 mmol/L 131.8±5.6 EDTA 5 mmol/L 136.4±6.2 EDTA 10 mmol/L 144.8±6.6 Methanol 1%, v/v 79.3±7.9 Ethanol 1%, v/v 62.1 Dimethyl sulfoxide 1%, v/v 51.7±3.5 Isopropyl alcohol 1%, v/v 75.9±6.9 PMSF 5 mmol/L 79.3±3.4 Substrate specificity of the keratinase 2 The keratinase from KD-N2 shows activity on the soluble proteins casein, BSA, ovalbumin and the insoluble substrates feather meal, feather keratin, human hair and sheep wool, but no activity was observed upon collagen, calf hair and silk (Table 3 3 ). Many keratinases are capable of hydrolyzing a broad range of soluble and insoluble proteins, as the enzymes from Streptomyces pactum DSM40530 (Böckle et al., 1995 1995 ), B . subtilis KS-1 (Suh and Lee, 2001 2001 ) and Fervidobacterium islandicum AW-1 (Nam et al., 2002 2002 ). The keratinase from the isolate had the ability to remove hair from calf and sheep skins (data not shown), but could not hydrolyze collagen, which is the main constituent of the dermis. Indeed, similar result had been reported for keratinase from B . subtilis S14 (Macedo et al., 2005 2005 ). Type I collagen could be hydrolyzed by keratinases from Streptomyces albidoflavus (Bressollier et al., 1999 1999 ), B . subtilis KS-1 (Suh and Lee, 2001 2001 ) and Fervidobacterium islandicum AW-1 (Nam et al., 2002 2002 ). And keratinases from Paecilomyces marquandii and Doratomyces microsporus were able to hydrolyze different keratin-containing substrates such as stratum corneum keratin, human nail, porcine nail and bovine keratin (Gradišar et al., 2005 2005 ). Comparing with the substrates listed in Table 3 3 , it seems that insoluble substrates such as human hair and feather meal, which have more disulfide bonds, were more easily hydrolyzed by the purified keratinase than sheep wool, which has less disulfide bonds. Previous studies showed that ball milling could destroy the keratin structure, which makes the substrates easily to be degraded by proteases (Stahl et al., 1949 1949 ; Noval and Nickerson, 1959 1959 ). The keratinase from B . subtilis KD-N2 has a broad range of substrates specificity and could degrade substrates containing both α and β keratins. The enzyme was incubated with 20 mg of substrate in 2 ml of 50 mmol/L Tris-HCl buffer (pH 8.5) for 30 min at 55 °C. Feather keratin was prepared by the method of Wawrzkiewicz et al.( 1987 1987 ) with little modification. After solubilization of native feather keratin in dimethyl sulfoxide and precipitation by cold acetone, the keratin was washed twice with distilled water and dried at 40 °C in a vacuum drier to constant weight. Other insoluble substrates were cut into short fragments Substrates Relative activity (%) Ovalbumin 100 BSA 56±5.2 Casein 68±6.7 Feather keratin 20±2.4 Feather meal 80±5.3 Collagen 0 Human hair 62±4.5 Wool 12±0.83 Calf hair 0 Silk 0 Effects of chemicals on feather meal hydrolysis 2 Fig. 4 4 shows that sulphur-containing chemicals, especially reducing agents, stimulated the keratinase activity towards feather meal. All tested chemicals had positive effects on the feather meal hydrolysis except 1 mmol/L sodium sulfite. Five mmol/L DTT, 5 mmol/L ammonium sulfamate and 5% (v/v) β -mercaptoethanol enhanced keratinolytic activity maximally. Five mmol/L of DTT, β -mercaptoethanol, ammonium sulfamate and sodium sulfite had more positive effects than 1 mmol/L, but for L-cysteine, SDS and DMSO, lower concentrations had more effects. Reducing agents such as DTT, β -mercaptoethanol, cysteine and sodium sulfite had been largely discussed on their positive effects on keratinases (Böckle et al., 1995 1995 ; Bressollier et al., 1999 1999 ; Ignatova et al., 1999 1999 ; Nam et al., 2002 2002 ; Gradišar et al., 2005 2005 ) and some of the enzymes are thiol-activated (Gupta and Ramnani, 2006 2006 ). To test the effects of some sulphur-containing chemicals on the keratinase activity, ammonium sulfamate was firstly utilized in the study, and it had positive effects in both low and high concentrations. DMSO is able to solubilize feather keratin (Wawrzkiewicz et al., 1987 1987 ), and has positive effects on feather meal hydrolysis in this study. Generally, purified keratinases cannot degrade keratin in vitro without the presence of reducing agents (Böckle et al., 1995 1995 ; Bressollier et al., 1999 1999 ; Suh and Lee, 2001 2001 ; Riffel et al., 2003 2003 ). And until now, detailed mechanisms of keratin hydrolysis has not been elucidated. One of the possible mechanisms is the reduction of disulfide bonds or sulfitolysis of the disulfide bonds by secreted sulfite. In this study, the reducing agents, DTT, β -mercaptoethanol, cysteine, sodium sulfite and sulphur-containing chemicals as SDS, ammonium sulfatate and DMSO stimulated the feather meal hydrolysis, which was probably due to the direct breakdown of the disulfide bonds by the reducing agents or due to reactions caused by the sulphur-containing chemicals. Chemicals were added to the incubation mixtures to reach a final concentration as follows: 1: Control; 2: 1 mmol/L DTT; 3: 5 mmol/L DTT; 4: 1% (v/v) β -mercaptoethanol; 5: 5% (v/v) β -mercaptoethanol; 6: 1 mmol/L SDS; 7: 5 mmol/L SDS; 8: 1 mmol/L sodium sulphite; 9: 5 mmol/L sodium sulphite; 10: 1 mmol/L L-cysteine; 11: 5 mmol/L L-cysteine; 12: 1 mmol/L ammonium sulfamate; 13: 5 mmol/L ammonium sulfamate; 14: 1% (v/v) DMSO; 15: 5% (v/v) DMSO. Bar represents standard error ( SE ) of the mean. Means with the different letters are significantly different according to Duncan’s multiple range test at P =0.05 using SAS (SAS Institute, version 6.12, Cary, NC) CONCLUSION 1 The B . subtilis KD-N2 degraded feathers quickly, and the enzyme produced by this isolate seems to be a new keratinase. The enzyme had a molecular mass of 30.5 kDa and was partially inactivated by PMSF. The keratinase is different from another purified B . subtilis keratinase (Suh and Lee, 2001 2001 ) in molecular mass and sensitivity to inhibitors, which has a molecular mass of 25.4 kDa and was completely inactivated by PMSF. The keratinase from B . subtilis KD-N2 was inhibited by metal ions and organic solvents to some degree and stimulated by SDS and EDTA. The enzyme showed activity upon feather meal, human hair as well as sheep wool. SDS and some reducing agents had positive effects on the keratinase activity. The keratinase was able to remove hair from the calf and sheep skins. The hydrolysis of collagen and degradation of the dermis should be further characterized, although the enzyme was not able to hydrolyze pure collagen. All the results present potential utilization of the keratinase industrially in keratin hydrolysis, feed nutrient improvement, and hair removing. References B Böckle , B Galunsky , R Muller . 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Poultry Sci , 1991 . 70 ( 1 ): 85 - 94 . . Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein 10.1631/jzus.B0820128 2008-04-15 2008-08-13 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 09