Degradation of keratin by keratinase and disulfide reductase from Bacillus sp. MTS of Indonesian origin

Biocatalysis and Agricultural Biotechnology 1 (2012) 152–158

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Degradation of keratin by keratinase and disulfide reductase from Bacillus sp. MTS of Indonesian origin Sri Rahayu a,b,1, Dahrul Syah a, Maggy Thenawidjaja Suhartono a,n a b

Department of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agricultural University, PO Box 220, Bogor 16002, Indonesia Alumni, Graduate School (Food Science) Bogor Agricultural University, Bogor 16002, Indonesia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 October 2011 Received in revised form 30 January 2012 Accepted 5 February 2012 Available online 9 February 2012

Bacillus sp. MTS isolated from Tangkuban Perahu crater Indonesia was found capable of degrading whole chicken feather effectively. The bacteria produced extracellular alkaline keratinase and disulfide reductase. When grown in feather media, Bacillus sp. MTS produced multi-fractions of both enzymes. The purified enzymes worked optimally at alkaline pHs, for keratinase at pH 8–12, and for disulfide reductase at pH 8–10. Optimum temperature for the extracellular keratinase was within 40–70 1C, while that for disulfide reductase was 35 1C. When the purified keratinase was mixed with purified disulfide reductase, enzyme activities on the natural keratin substrates (feather and wool) were greatly increased compared to activity of each enzyme alone, activity of proteinase K or activity of purified keratinase in the presence of reducing agents. The mutual action of the two enzymes on feather was examined by Scanning Electron Microscope. & 2012 Elsevier Ltd. All rights reserved.

Keywords: Bacillus Purification Feather Keratinase Disulfide reductase

1. Introduction Keratin, a tough protein substance, is the chief constituent of epidermal layer of skin, hair, feather, nail, hoof, wool and certain shell. As much as 90% of feather is made up of keratin, the fibrous and insoluble structural protein. Mechanical stability of keratin and its resistance to biochemical degradation depend on tight packing of protein chains in the a-helix (a-keratin) or b-sheet (b-keratin) structure and linkage of the structures through disulfide bonds. Digestive enzymes, such as trypsin and pepsin, are not effective for keratin degradation (Bockle and Muller, 1997; Brandelli et al., 2010; Gupta and Ramnani, 2006; Onifade et al., 1998). The majority of reports on keratinase enzymes focussed mainly on their proteolytic action. Nonetheless, reduction of disulfide bonds affects degradation of keratin significantly (Bockle and Muller, 1997; Cai et al., 2008; Prakash et al., 2010; Ramnani et al., 2005; Yamamura et al., 2002). Thiol formation by Vibrio strain kr2 grown in feather keratin media suggested disulfide reduction (Sangali and Brandelli, 2000). Reduction of disulfide bond was observed during the growth of Streptomyces pactum and Bacillus megaterium on feather (Bockle and Muller, 1997; Swerdlow and Setlow, 1983). Stenotrophomonas sp. strain

D-1 produced two types of extracellular proteins, a proteolytic enzyme and a disulfide bond-reducing protein. The protease belongs to a serine enzyme and the disulfide bond-reducing protein was suggested as a disulfide reductase enzyme (Yamamura et al., 2002). We had screened and isolated a feather degrading bacteria from Tangkuban Perahu crater West Java Indonesia and based on its morphology and biochemical reactions, the isolate was grouped as a Bacillus species and tentatively refered to as Bacillus sp. MTS. The aerobic mesophillic bacteria was very effective in degradation of whole chicken feather and this appeared to be related to activity of the extracellular keratinase and disulfide reductase enzymes. We had optimized conditions for enzymes production and characterized the crude enzymes (Rahayu et al., 2010). In this study, we purified the extracellular keratin hydrolyzing enzyme (keratinase) and disulfide reductase from Bacillus sp. MTS grown in feather containing media, characterized the purified enzymes and analyzed the mutual actions of both enzymes in degradation of keratin substances.

2. Materials and methods 2.1. Growth conditions

n

Corresponding author. Tel./fax: þ 62251 8621724. E-mail address: [email protected] (M. Thenawidjaja Suhartono). 1 Present address: Laboratory of Animal Feed and Nutrition, Faculty of Animal Science Jendral Sudirman University, Purwokerto, Indonesia. 1878-8181/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bcab.2012.02.001

The aerobic mesophilic Bacillus sp. MTS screened and isolated from Tangkuban Perahu crater West Java-Indonesia was used in these experiments. The agar medium for culture maintenance

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contained 0.6% crushed dried feather (powder), 0.03% K2HPO4, 0.04% KH2PO4, 0.05% NaCl and 0.05% NH4Cl (Macedo et al., 2005). For enzyme production, 250 ml medium containing 0.03% K2HPO4, 0.01% MgCl2.6H2O, 0.05% NaCl, 0.05% NH4Cl and 1.0% chicken feather powder was used (Lin et al., 2001). pH was adjusted to 7.5 and incubation was carried out in a 1 l flask at 37 1C 100 rpm for 48 h. The culture was filtered and centrifuged at 4000 g 4 1C for 10 min to harvest the extracellular enzymes. 2.2. Protein and enzyme assay Protein content was measured at 595 nm according to Bradford, using bovine serum albumin as the standard protein (Waterborg, 2002). The Bradford reagent consisted of 100 mg Coomassie Brilliant Blue G-250 in 50 ml ethanol 95%. As much as 100 ml of phosphoric acid 85% (b/v) was added and the mixture was made to 1 l by adding aquadest. This reagent was stored at refrigeration temperatures; 0.2 ml of sample was mixed with 4 ml of Bradford reagent, vortexed and kept for 5 min and the absorbance was measured at 595 nm. Keratinase activity was determined according to Walter (Walter, 1984) using 1% feather powder in Tris/HCl (50 mM, pH 8.0) as the substrate. As much as 200 ml of enzyme sample was mixed with 800 ml of 0.5% w/v keratin substrate. Following incubation for 10 min at 37 1C, 500 ml of Trichloracetic acid (5%) was added and the mixture was kept at 37 1C for another 30 min, then centrifuged at 1000 g for 10 min. The supernatant was mixed with 1 ml of Na2CO3 and 200 ml of Follin reagent which had been previously diluted with deionized water (1:2). Incubation was further conducted at 30 min 37 1C to develop the color. After centrifugation at 1000 g for 10 min, the absorbance was read at 660 nm. A tyrosin standard curve was made for quantification. Similar procedure was repeated using varying tyrosin concentration instead of the enzyme samples. One unit of enzyme activity was defined as the amount of enzyme which liberate 1 mmol tyrosine in 1 min. Disulfide reductase activity was measured as described by Serrano et al. Serrano et al. (1984) with a few modifications. As much as 100 ml of enzyme was incubated with 500 ml of Tris/HCl buffer (0.13 mM, pH 9.0) containing 0.05 mM oxidized glutathione (GSSH) and 0.05 mM EDTA at room temperature for 10 min. The reaction mixture was centrifuged at 1000 g 4 1C for 10 min and the reaction product was detected by addition of 100 ml 20 mM DTNB (dithiobis-nitro benzoic acids) and 1.35 ml Tris/HCl buffer (0.13 mM, pH 9.0) to the 50 mL of supernatant. Absorbance was measured at 405 nm after 2 min of stable color development. 2.3. SDS-PAGE and zymogram SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was carried out using 10% of separating polyacrylamide gel according to Walker (Walker, 2002). Electrophoresis was conducted at 100 V and 50 A for 1.5 h and the gel was stained in silver nitrate solution (Dunn, 1994; Walker, 2002). Gelatin (0.5%, w/v) in Tris–HCl buffer (50 mM, pH 8.0) was mixed into the separating gels for zymogram analysis. After electrophoresis, the gel was washed with 2.5% (v/v) Triton X-100 for 60 min followed by overnight incubation at 55 1C in Tris–HCl buffer (50 mM, pH 8.5). The gel was stained with Coomassie Brilliant Blue R-250 for 30 min and destained in acetate ethanol solution. Low molecular weight (LMW) proteins containing b-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), restriction endonuclease (25 kDa), b-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa) were used as the protein standard.

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2.4. Enzyme purification At the end of incubation, the bacterial culture was filtered and centrifuged at 4000 g 4 1C for 10 min to separate the cell and harvest the extracellular enzymes. The supernatant was precipitated by addition of 50% (w/v) ammonium sulphate and centrifuged at 4000 g 4 1C for 30 min. The enzyme precipitate was resuspended in 3 ml of Tris/HCl buffer (50 mM, pH 8.0) and further dialyzed for 5 h in the same buffer using 12 kDa cut off membrane. Enzyme solution was then applied onto Butyl Sepharose FF coloumn (10/20 mm) which was previously equilibrated with 30% ammonium sulphate in Tris/HCl buffer (50 mM, pH 8.0). The same buffer with 30%, 15% and 0% ammonium sulphate was used to wash the gel and elution was performed at 0.5 ml/min. Fractions of 3 ml were collected and assayed for keratinase and disulfide reductase and the fractions with highest activity was further applied to the Sephacryl S-200HR column (10/40 mm). A Tris/HCl buffer (50 mM, pH 8.0) solution was used to elute the column at 0.2 ml/min. The purified enzyme fractions were used for analysis of optimum pHs and temperatures and for further experiments. 2.5. Degradation of keratin by mutual activities of keratinase and disulfide reductase In this experiment, we used purified keratinase and disulfide reductase fractions which showed highest stabilities and purification fold. The commercial enzyme Proteinase K which is known to have keratin degrading ability was used to compare keratinase activities of this enzyme with that of Bacillus sp. MTS. The protein content of enzymes used was 0.2 mg/ml. Three kinds of natural keratin were used as enzyme substrates, namely native chicken feather, prehydrolyzed chicken feather and natural wool. 100 ml of enzyme was mixed with 1 ml of substrate at 1% in 50 mM buffer Tris–HCl pH 8.0. In this case, the volume ratio of keratinase and disulfide reductase applied was 4:1. Incubation was conducted for 60 min at the optimum enzyme temperature: 35 1C for Disulfide reductase reaction or 50 1C for keratinase reaction or 37 1C for proteinase K. The product was measured according to Walter (Walter, 1984). We also tested the effect of reducing agents on keratinase ability to degrade the keratine substrates. Dithiothreitol (DTT), b-mercaptoetanol (BMT) and urea were used at concentrations of 0.1 mM (DTT), 0.2 mM (BMT) and 0.3 mM (urea). Initially, we incubated keratin with disulfide reductase or reducing agents (DTT and BMT) or urea in 50 mM Tris/HCl buffer (pH 10.0) at 35 1C for 10 min, then keratinase or proteinase K was added and the mixtures were further incubated at 50 1C for 60 min. 2.6. Scanning electron microscopy For scanning electron microscope analysis, we used whole native feather, which was incubated with 50 mL of keratinase and 50 mL of disulfide reductase, or 0.1 mM dithiothreitol at 50 1C in Tris/HCl buffer (50 mM, pH 9.0) for 90 min. The reaction was stopped by addition of trichloroacetic acid and the feather was washed several times with the same buffer. Fixation was carried out with 2.5% (v/v) glutaraldehyde and 2% (v/v) formaldehyde for 48 h. The specimens were dehydrated several times with 70–100% acetone and dried at 50 1C for 10 min. The gold treated specimens were analyzed in JSM-5310LV SEM JEOL Japan. 3. Results Results of purification of keratinase and disulfide reductase from Bacillus sp. MTS are summarized in Tables 1 and 2 and

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Figs. 1 and 2. The specific activities of keratinase and disulfide reductase were increased after purification through Butyl Sepharose FF. Keratinase was purified to about 76 folds with specific activity of 6.6 U/mg and purity of disulfide reductase was increased 38 folds to a specific activity of 17.4 U/mg. Further purification of F52 with Sephacryl S-200 h resulted in several keratinase fractions and the purification factor of F61 was increased to approximately 460 folds with specific activity of 40 U/mg (Fig. 1). We analyzed optimum temperatures and pHs of the crude (ammonium sulphate fraction) and the purified enzymes. We selected F 4, 15, 32 and 61 for keratinase not only due to their high specific activities, but also their stability after purification. The result is shown in Fig. 3 and Table 3. Keratinases and disulfide reductases excreted by Bacillus sp. MTS appeared as alkaline enzymes. Optimum pHs of the crude keratinase were within 8–10. Optimum pHs of the purified keratinase were shifted to more alkaline pHs, namely 8–12. Except for F 15 keratinase which showed two optimum pHs (9 and 11), the other fractions showed broad range of optimum working pHs which reflects the insensitivity of enzyme conformation to the hydrogen ions in the environment. The F15 enzyme fractions may consist of more than 1 subunit and the present purification method apparently released at least 2 forms (isozyme) with different pH sensitivities. The activities of F4 and F32 fractions at pH 12 were still high. We assume that the alkaline and pH insensitive enzymes might contain a larger percentage of hydrophobic and basic amino acids. The optimum temperature of keratinase enzymes were within the

range of 40–70 1C, while that of disulfide reductase was lower, i.e. 35 1C. The F4 keratinase showed optimum temperature of 40 1C, but the activity was still about 25% at temperature up to 70 1C and a little bit elevated at 80 1C and higher temperature. The optimum temperature of F 61 was 50 1C and at higher temperature up to 90 1C, the activity still remained at about 20% which reflects the heat stability of this fraction. Zymogram analysis in the previous work indicated the presence of six keratinase bands of approximately 122, 96, 53, 32, 25 and 17 kDa (Rahayu et al., 2010). SDS PAGE analysis for the purified keratinase indicated 3 protein bands of approximately 16, 32 and 50 kDa (Fig. 4). Purification through Sephacryl S-200 h resulted in three fractions of disulfide reductase and the highest specific activity was increased to 58.57 U/mg (F65). The three disulfide reductase fractions (F32, 65 and 86) were used for optimum pH and temperature analysis but only fraction with

Table 1 Purification of keratinase from Bacillus sp. MTS. Sp. activity (U/mg protein)

Yield (%)

Purification (fold)

Steps

Total protein (ug)

Enzyme activity (mU/ml)

Culture supernatant Ammonium sulphate precipitate Dialysis

40,000

3487

510

118

0.23

3.38

2.66

560

115

0.21

3.29

2.36

5

33

6.6

0.95

75.86

0.6 2.1 1.5 1.5 0.6 0.6

12 24 36 15 24 18

20 11.43 24 10 40 30

0.34 0.69 1.03 0.43 0.69 0.52

229.88 131.36 275.86 114.94 459.77 344.83

Butyl sepharose FF F52 Sephacryl S-200 HR F4 F12 F15 F32 F61 F66

0.087

100

Fig. 1. Elution profile of keratinase of Bacillus sp. MTS from Sephacryl S-200HR column. Enzyme activity (K) and protein (J).

1

Purification was conducted using ammonium sulphate precipitated enzyme through Butyl sepharose FF followed by Sephacryl S-200HR coloumn. Proteins and enzyme activity were analyzed as written in the methods.

Fig. 2. Elution profile of Sephacryl S-200HR from disulfide reductase Bacillus sp. MTS Enzyme activity (K) and protein (J).

Table 2 Purification of disulfide reductase from Bacillus sp. MTS. Steps

Total protein (ug)

Culture supernatant 40,000 Ammonium sulphate precipitate 510 Dialysis 560 Butyl sepharose FF F28 5 Sephacryl S-200 HR F32 1 F65 0.7 F86 2

Enzymeactivity (mU/ml)

Specific activity (U/mg of protein)

Yield (%)

Purification (fold)

18,400 170 130

0.46 0.33 0.23

100 0.92 0.71

1 0.72 0.50

87

17.4

0.47

37.83

52 41 67

52 58.57 33.50

0.28 0.22 0.36

113.04 127.33 72.83

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Fig. 3. Optimum pH and temperature of purified keratinase and disulfide reductase. (A) The effect of temperature on activities of purified keratinase fractions F 4 (-m-), F 15 (-D-), F 32 (-J-) and F 61 (-K-), (B) The effect of pH on activities of purified keratinase fractions F 4 (-m-), F 15 (-D-), F 32 (-J-) and F 61 (-K-), (C) The effect of temperature on activities of purified disulfide reductase fractions F32 (-J-), F 65 (-’-) and F 86 (-m-), (D) The effect of pH on activities of purified disulfide reductase fractions F32 (-J-), F 65 (-’-) and F 86 (-m-).

Table 3 Optimal temperature and pH keratinase and disulfide reductase from ammonium sulphate (50% w/v) and the purified fractions eluted from Sephacryl S-200HR column. Fraction

Keratinase

Fraction

116 kDa 66.2 kDa

Disulfide reductase

45 kDa pH

Ammonium 8 and 10 sulphate F4 9–12 F15 9 and 11 F32 8–12 (Still active at pH 412) F61 10

Temp. (1C) 55 40 70 50–70

pH

Ammonium sulphate F32 F65 F86

Temp. (1C)

9

28

9 10 10–12

35 35 35

50

highest stability and purification fold was used for synergistic experiment with keratinase. Fig. 4 indicated that the purified disulfide reductase contained 2 protein bands of approximately 13 and 35 kDa. In further experiments, we mixed the crude and purified keratinase with purified disulfide reductase. In this case, we used only fractions F 61 (for keratinase) and F 65 (for disulfide reductase) due to their highest specific activities and stability. The substrates

35 kDa 25 kDa 18.4 kDa 14.4 kDa 1

2

3

4

5

Fig. 4. SDS-PAGE of keratinase and disulfide reductase from Bacillus sp. MTS. (1) Marker protein, (2) crude extract, (3) ammonium sulphate fraction, (4) purified keratinase, (5) purified disulfide reductase Low molecular weight (LMW) proteins containing b-galactosidase (116 kDa), bovine serum albumine (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), endonuclease restriction (25 kDa), b-lactoglobulin (18.4 kDa) and lisozyme (14.4 kDa) were used as the standard proteins.

used for this experiments were chicken feather and wool. In one of the experiment, the feather was prehydrolysed with NaOH 0.02% for 15 min. The treatment was expected to physically modify the

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Table 4 Comparative keratinolytic activities (U/mg) of keratinase Bacillus sp. MTS and Proteinase-K on 1% prehydrolyzed feather with and without addition of disulfide reductase (DR) or reducing agents and urea. Source of enzyme Crude enzyme DR Purified enzyme (keratinase or proteinase K) Ker Bacillus sp. MTS Proteinase-K

0.4

0.9 5.0 1.6

þDR

þ DTT

þ BMT

þ Urea

10.9 1.2

9.8 1.5

2.6 0.7

2.0 0.5

Table 5 Comparative keratinolytic activities (U/mg) of keratinase Bacillus sp. MTS and Proteinase K on 1% natural feather with and without addition of disulfide reductase (DR) or reducing agents and urea. Source of enzyme Crude enzyme DR

Purified keratinase or Proteinase K Ker

Bacillus sp. MTS Proteinase K

0.1

1.3 4.5 0.5

þ DR

þ DTT

þBMT

þUrea.

27.5 0.6

12.8 0.7

10.8 0.5

4.6 0.0

Table 6 Comparative keratinolytic activities (U/mg) of keratinase Bacillus sp. MTS and Proteinase K on 1% natural wool with and without addition of disulfide reductase (DR) or reducing agents and urea. Source of enzyme

Bacillus sp. MTS Proteinase K

Crude enzyme

0.09

DR

0.5

Purified keratinase or proteinase K Ker

þ DR

þ DTT

þ BMT

þ Urea

8.7 0.5

24.3 0.5

11.0 0.5

8.5 0.3

7.9 0.0

substrate and improved enzyme substrate interaction. Our experimental data proved that the NaOH prehydrolysed treatment increased activity of the crude keratinase alone. However, total effect of the mixture of keratinase and disulfide reductase on this subtrate were lower than on the native feather and wool. Addition of disulfide reductase on three keratin substrates increased the activity of Bacillus MTS keratinase by 2–10 fold, but addition of disulfide reductase did not affect the activity of proteinase K (Tables 4–6). The synergestic activities of keratinase and disulfide reductase were clearly observed and proved to be more effective for keratin degradation in comparison to the treatment with keratinase plus reducing agent. The activity of purified keratinase on 1% prehydrolized feather was 5 U/mg and addition of reducing agent dithiothreitol still increased the activity to 9.8 U/mg but addition of beta-mercaptoethanol and urea reduced 50% of the activity down to 2.6 and 2 U/mg (Table 4). Activity of proteinase K was lower on all substrates tested, and addition of disulfide reductase or reducing agents did not affect the activity, while urea inhibited the enzyme activity. Degradation of native chicken feather was observed by scanning electron microscope (Fig. 5) which showed that there were no changes in the feather epidermis layer without enzyme addition (A) but this structure was degraded (stripped off) after incubation with keratinolytic enzymes (B–D). A combination treatment of keratinase with disulfide reductase and keratinase with reducing agent dithiothreitol showed better degradation effect, while addition of Proteinase K was less effective (E).

4. Discussion The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Reduction of disulfide bonds plays significant role in degradation of the whole keratin structure. Therefore, addition of reducing agents or enzyme capable of breaking/reducing S–S bridge will accelerate activity of the protein/peptide hydrolyzing keratinase to complete degradation of the whole keratin substrate (Gupta and Ramnani, 2006; Prakash et al., 2010). We had purified and characterized extracellular keratinase (EC 3.4.4.25), the protein hydrolyzing enzyme and disulfide reductase (EC 1.6.4.2), the disulfide reducing enzymes from local Bacillus sp. MTS. In our study, the effect of disulfide reductase addition was much more significant to total degradation of keratin compared with the effect of adding reducing agent such as dithiothreitol. The presence of disulfide reductase in several bacteria and the synergistic actions of both keratinase and disulfide reductase enzymes have been reported earlier. Stenotrophomonas sp. produced serine protease and disulfide bond-reducing protein capable of degradation of native keratin. After addition of the two enzymes, the total keratinolytic activity was increased tremendously over that of the single keratinase alone (Yamamura et al., 2002). Extracellular keratinolytic activity of B. licheniformis RG1 was synergistically enhanced by addition of intracellular disulfide reductase (Ramnani et al., 2005). Reduction of disulfide bonds was observed when Streptomyces pactum and Chryseobacterium sp. were grown in feather media (Bockle and Muller, 1997; Riffel et al., 2003). Disulfide reductase has not been detected in several eucaryotic sources and is present at most in a low level in a number of gram-negative bacteria. However, the presence of bacilithiol disulfide reductase in Bacillus subtilis has been suggested (Newton et al., 2009). Feather degradation by Bacillus subtilis S 8 resulted in free SH group, soluble protein and amino acid production (Jeong et al., 2010). An NADH linked disulfide reductase was present and purified from the spore of Bacillus megaterium (Swerdlow and Setlow, 1983). The presence of thiol-disulphide reductases is apparently beneficial not only for catalysis of S–S bond reduction but also for secretion of disulphide-bond containing proteins. In this research, we purified the two enzymes important for degradation of the whole/natice chicken feather, analyzed their optimum pHs and temperatures and found their synergistic activities for total degradation of keratinous substances. Bacillus sp. MTS produce multiple keratinases with various molecular weight (16, 32 and 50 kDa). Researchers have reported keratinase enzymes of diversed molecular weight. Keratinase from Thermoanaerobacter keratinophilus was reported as a 135 kDa molecule (Riessen and Antranikian, 2001), Lysobacter 148 kDa (Allpress et al., 2002) and Bacillus sp. 134 kDa (Lee et al., 2002). Fervidobacterium islandicum AW-1 produced homomultimer keratinase of molecular weight higher than 200 kDa (Nam et al., 2002). Some bacteria produce smaller keratinase: Xanthomonas maltophilia 36 kDa (Toni et al., 2002) and Bacillus pumilus 32 kDa (Huang et al., 2003), Bacillus subtilis 30 kDa (Cai et al., 2008), Bacillus halodurans PPKS-2: 30 and 66 kDa (Prakash et al., 2010). Streptomyces sp. strain 16 produced four keratinases of 19–50 kDa (Xie et al., 2010). Chryseobacterium indologenes also produced three keratinases with molecular weight of 56, 40 and 40 kDa (Wang et al., 2008). The fractions eluted from Sephacryl column in our study, showed several individual/separated keratinase activity indicating that this Bacillus produces diversed keratinase enzymes. Multiforms of keratinases might be important to the microorganism producers for their survival in nature, that is enable them to utilize vast array of protein (keratine) structures found in their

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Fig. 5. Scanning electron micrographs of chicken whole feather after incubation with enzyme produced by Bacillus sp. MTS. (A) Control feather. (B) feather þ crude enzyme. (C) feather þ keratinase þDisulfide Reductase. (D) featherþ keratinase. þ 0.1 mM Dithiothreitol. (E) feather þ Proteinase K.

environment. In addition to the multiform of keratinase, Bacillus sp. MTS may produce enzymes with different subunits which could possibly create several isozymes with slightly different characteristics. This may explain the presence of fraction (F15) which has two optimum pHs (9 and 11). In general, the extracellular enzymes in our study are stable at alkaline pHs, and some fractions are quite insensitive towards hydrogen ion concentrations in their microenvirenment. It is possible that our enzymes contain larger percentage of hydrophobic and basic amino acids. The F4 fraction exhibited quite unique characteristic: the optimum temperature for this fraction was 40 1C, but at 80 1C and higher temperature, the activity was elevated. This might be due to the possible heat inactivation or heat induced liberation of several contaminants (impurities) previously bound to the enzyme molecule which occured at higher temperature. In the absence of these unwanted molecules, the free enzyme regained its optimum folding and exhibited higher activity Report on disulfide reductase from Bacillus sp. has not been as frequently found as those for keratinase, especially the extracellular disulfide reductase. The result of our research is important in enzyme application originated from Bacillus group, as Bacillus species continue to be dominant bacterial workhorses among the microbial enzyme producers, and for many industrial productions, extracellular enzymes are preferable due to the production efficiency. Several types of gel have been reported as effective for disulfide reductase purification. Octyl sepharose was used to purify disulfide reductase from Bacillus megaterium (Swerdlow and Setlow, 1983), while NADPH-dependent disulfide reductase from Anabaena sp. Strain 7119 was purified by Sephacryl S-300 (Serrano et al., 1984). Our data indicated that two steps purification using hydrophobic interaction (with Buthyl Sepharose gel) and gel filtration (with Sephacryl gel) chromatography is quite effective for disulfide purification. Generally, intracellular disulfide reductase produced by bacteria exhibit small molecular weight. The molecular weight of reductase produced by Streptomyces clavuligerus is 15 kDa (Aharonowitz et al., 1983), thioredoxin reductase B. acidocaldarius 11.5 kDa (Bartolucci et al., 1997) and Clostridium sticklandii 36 kDa (Harms et al., 1998). Only few reports of extracellular bacterial disulfide reductase were found. Molecular weight of extracellular disulfide reductase produced by Stenotrophomonas sp. D-1 is 15 kDa (Yamamura et al., 2002), Bacillus megaterium 122 kDa (Swerdlow and Setlow 1983) and Lactobacillus sanfranciscensis produced

extracellular glutathione reductase with molecular weight of 48 kDa (Jansch et al., 2007). In our study, SDS PAGE analysis indicated that Bacillus sp. MTS excreted small size disulfide reductase of approximately 13 and 36 kDa. Proteinase K activity remained non-responsive to addition of disulfide reductase or reducing agent DTT. Proteinase K of Tritirachium album consists of a single peptide chain with 277 amino acids which also harbor two disulfide bonds (34–124, 179– 248) and a free cysteine (Branden and Tooze, 1999; Jany et al., 1986). Reduction of the S–S bond in keratine substrates promoted by addition of disulfide reductase or reducing agent which is expected to increase the activity of proteinase K is contradicted by negative effect of disulfide reductase or reducing agent to S–S bond within the proteinase K enzyme. Thus, the total effect was shown as unchanged activity of proteinase K by addition of disulfide reductase or reducing agent DTT. Synergistic effect of keratinase and disulfide reductase appeared more significant on native feather compared to the prehydrolyzed feather which was prepared by boiling the feather in NaOH solution, continued by HCl treatment to neutralize the pH. In this case the salt (NaCl) formed by this treatment might be unfavorable to the enzyme conformation and enzyme substrate interaction. Addition of urea did not affect the keratinolytic action of Bacillus sp. MTS but inhibit proteinase K. Some denaturants such as urea, sodium dodecyl sulphate (SDS) and guanidium hydrochloride disrupt the noncovalent interactions within the enzyme structure and thus alter (reduce) enzyme activity. Denaturants may perturb microenvironment of the aromatic amino acids residues such as tryptophan accompanied by reduction or loss of catalytic activity (Branden and Tooze, 1999; Sumathi and Dasgupta, 2006). In this case, keratin degrading enzymes secreted by Bacillus sp. MTS was more resistant than proteinase K to this effect. This probably reflects a more compact tertiary structure of our enzyme. Microbial keratinases and disulfide reductase have become biotechnologically important since they can be used for hydrolysis of highly rigid, strongly cross-linked structured proteins such as keratin. Their applications include uses as detergent component, in wool and silk cleaning and in leather industry which lead to development of greener technology. Furthermore, with their capability of hard protein degradation, the enzymes are potential for prion degradation and thus useful for healthy and safe meat preparation.

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Acknowledgments This research was supported by Indonesian Ministry of Education (Research grant 2010) and Atma Jaya Catholic University Jakarta (Research grant 2009). We acknowledge and appreciate Biotechnology Laboratory Marine Research Experiment Station under Dr. Ekowati Chasanah for some of the enzyme purification work.

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