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75-81. 8 Yoshida E & Noda H, Isolation and characterization of collagenase I and II from Clostridium histolyticum, Biochim. Biophys Acta, 105 (1965) 562-574.
Journal of Scientific & Industrial Research Vol. 64, December 2005, pp. 978-983

An extracellular protease with depilation activity from Streptomyces nogalator P Mitra and P K Chakrabartty* Department of Microbiology, Bose Institute, P1/12, CIT Scheme VII M, Kolkata 700 054 Received 29 April 2005; revised 11 August 2005; accepted 03 October 2005 An extracellular protease-producing actinomycete, Streptomyces nogalator strain Ac 80, was isolated from soil. Mutants of the strain designated as Mt C producing protease of higher (3.0 mg tyrosine liberated/min/mg protein) and Mt L of lower (0.9 mg tyrosine liberated/min/mg protein) specific activity as compared to that of the wild type (1.8 mg tyrosine liberated/min/mg protein) were selected. Proteolytic activity of the wild type and the mutants correlated with their depilation capacities. Proteolytic enzyme produced by Mt C in solid state fermentation was purified to homogeneity, and partially characterized (molecular mass, 66 kDa by SDS- PAGE). It was found to be efficient in depilation of goatskin. Keywords: Depilation, Enzyme purification, Proteolytic enzyme, Streptomyces nogalator IPC Code: C12N11/02

Introduction Depilation or dehairing of hides and skins, in leather industry, is traditionally done by chemical methods using lime, sodium sulphide, etc, which create serious pollution hazards. An environment-friendly enzymatic method of dehairing may provide an alternative to chemical method1. Dehairing enzymes, in general, are protease in nature2-4. Actinomycetes, particularly streptomycetes, are known to secrete multiple proteases in culture medium5. Some proteases have been characterized5,6 and are involved mainly in hydrolysis of large peptide molecules. Proteases may conveniently be produced by fermentation using cheap substrate such as wheat bran3,7. In this study, an actinomycete with proteolytic activity was isolated and mutagenised. A hyperproducing mutant with increased level of the proteolytic activity was then selected. It was grown in solid state fermentation (SSF) using wheat bran as the substrate and the culture extract was observed to be more efficient in dehairing of goatskin as compared to that of the wild type. The extracellular protease of the mutant was purified and partially characterized. Materials and Methods Media

Three media were used. Gelatin agar medium __________ *Author for correspondence E-mail: [email protected] 1 Present address: Mahodari High School, PO Mahodari, Distt Birbhum (W B)

contained: peptone, 5; beef extract, 3.0; gelatin, 4.0; and agar 20.0 g/l; pH 7.0. YEM medium contained: yeast extract, 4.0; malt extract, 10.0; and dextrose, 4.0 g/l; pH 7.3. Basal medium contained: (NH4)2SO4, 2.64; KH2PO4, 2.38; K2HPO4.3H2O, 5.65; and MgSO4.7H2O, 1.00 g/l. Isolation of Protease-producing Bacteria

Soil samples were collected from leather and hair dumping areas, serially diluted on Gelatin agar medium and incubated at 28oC for 4 days to allow the colonies to grow. The medium was then flooded with mercuric chloride solution (HgCl3, 15g; HCl, 20 ml; H2O, 80 ml) for screening of colonies producing proteolytic enzyme. The diam of the zone of clearance around colonies furnished a measure of their proteolytic activity. An actinomycete colony with a large zone of clearance was isolated and designated as Ac 80. Organism and Growth Condition

The isolate Ac 80 was identified as Streptomyces nogalator (MTCC 2505) under the family Streptomycetaceae by Microbial type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh. For enzyme production by SSF, wheat bran (100 g/flask) was moistened with different volumes (0-80 ml) of basal medium. The flasks were inoculated with spore suspension of the organism and incubated at 28oC for 3 days. The enzyme was extracted by the addition of 200 ml of 0.1 M

MITRA & CHAKRABARTTY: PROTEASE WITH DEPILATION ACTIVITY FROM STREPTOMYCES NOGALATOR

phosphate buffer, pH 7.5, to each flask and gently stirred for 5 min. This was followed by centrifugation of the preparation at 10,000×g for 15 min. The supernatant thus obtained served as the enzyme extract. For enzyme purification, the cultures (40% substrate moisture) were used. Protease Assay

Proteolytic activity was assayed using casein as the substrate8. A 0.2 ml aliquot of the enzyme extract was incubated with 4 ml of 0.6% casein solution in 0.1 M Tris HCl buffer, pH 7.8 at 37oC for 2 h. The reaction was stopped by the addition of an equal volume of cold 20% trichloroacetic acid. The mixture was filtered. The amount of acid soluble tyrosine released into the filtrate was measured from its absorbance at 273 nm. Protein was estimated9 using bovine serum albumin (BSA) as the standard. One unit of caseinase activity is defined as the amount of enzyme that liberates 1 µg of tyrosine per min under the above conditions.

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(DEAE)-cellulose column (1.1 × 1.0 cm) equilibrated with 20 mM Tris HCl, pH 7.8. The column was washed with the same buffer and elution was performed with a step gradient of NaCl (0.1-1 M) with an increment of 0.1 M at each step. All the fractions were dialyzed overnight at 4oC against 0.01 M Tris HCl, pH 7.8. The fractions showing high enzyme activity were concentrated. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE of protein was performed according to Laemmli10 with 10% polyacrylamide gel slab. The protein bands were visualized by Coomassie Brilliant Blue R-250 staining. The molecular mass of the purified enzyme was estimated from its position relative to those of standard proteins run in a parallel lane. The molecular mass standards used were β-galactosidase (116 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa) and glyceraldehyde3-phosphate dehydrogenase (36 kDa). Characterization of the Purified Enzyme

Induction of Mutation and Selection of Mutants

Conidial suspension was irradiated with ultraviolet (UV) light ranging from 0 to 10 min and plated. The plates were incubated for 4 days in the dark. The colonies developed were counted and percent survivability of the conidia in respect of the control was estimated. The colonies developed at 1% survival were transferred to Gelatin agar plates. Each colony was also spot inoculated on a duplicate plate and incubated at 28oC for one day. After flooding, HgCl3 mutants were scored on a set of plates by measuring the zone of clearance around their colonies. Enzyme Purification

The enzyme extract was first subjected to 80% (NH4)2SO4 at 4°C for overnight to remove nonproteinaceous material. The precipitate formed was dissolved in 0.1 M Tris HCl buffer, pH 7.8 and dialyzed against the same at 4oC with several changes. The dialysate was then subjected to fractional precipitation with increasing saturation of (NH4)2SO4. The protein precipitate obtained with 30-60% (NH4)2SO4 was dissolved in 0.1 M Tris HCl buffer, pH 7.8 and applied on to a column of Sephadex G-150 (1.5 × 24 cm) equilibrated with the same buffer. The flow rate was adjusted to 24 ml per h and 2 ml fractions were collected. The fractions showing the enzyme activity were pooled and loaded on to a

To determine pH optimum of the purified enzyme, it was assayed at 28oC in 0.1M Tris maleate (pH 7.0-7.8) or 0.1 M Tris HCl (pH 7.4-8.8) buffer adjusted to various pH values. Protease activity was also assayed at various temperatures in 0.1 M Tris HCl, pH 7.8, to determine its temperature optimum. Thermal stability of the purified protease was determined by incubating the enzyme at various temperatures (28-60oC) for 0 to 30 min and the residual activity was determined. The effect of phenylmethylsulfonyl fluoride (PMSF, 0.5-2 mM) as well as of EDTA (1-10 mM) on the activity of the protease was examined. The effect of metal ions to a final concentration of 1 mM on the protease activity was also measured. Michaelis Menten constant (Km) of the protease was determined11 using casein (2-10 mg/ml) as the substrate. An enzyme paste was made mixing 10 g of kaolin and 100 mg streptomycin sulphate with 20 ml of enzyme. A portion of the paste was applied to the flesh side of paired goatskin pieces (6′′×6′′ each) and dehairing of skin was ascertained at every 2 h starting from 12 h. In control, water was used in place of the enzyme. Results and Discussion From a total of 270 colonies, an actinomycete, Ac 80 [Streptomyces nogalator (MTCC 2506)], was selected on the basis of a large zone of clearance

J SCI IND RES VOL. 64 DECEMBER 2005

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Table 1Purification of protease of Streptomyces nogalator Mt C Enzyme

Ammonium sulphate (0-80%) ppt. Ammonium sulphate (30-60%) Sephadex G-150 DEAE cellulose

Fold Total Total Caseinase Yield % purification protein activity specific mg activity (µg tyrosine/mg protein/min) 129.74 252.97

1.95

100

1

21.67

101.19

4.67

40

2.4

1.87

31.92

17.07

12.6

8.8

0.035

2.75

78.65

1.1

40.3

further studies were made using the mutant strain Mt C. Effect of Substrate Moisture

Basal medium was added at different percentages to wheat bran. At 20% substrate moisture content, the production of the enzyme was low; further increase in moisture content (up to 40%) allowed linearly increased production of the enzyme and thereafter the level reached a plateau (Fig. 1). The mutant Mt C was grown on wheat bran (40% moisture) for 3 days and the culture was extracted. The culture extract contained non-proteinaceous substances, which interfered with protein estimation. Similar observation was also made in the solid-state culture of Phanerochaete chrysosporum12. The interfering substances were eliminated by precipitation of the protein with 80% (NH4)2SO4. Enzyme Purification

Fig. 1Effect of substrate moisture content on the production of proteolytic activity by Streptomyces nogalator Mt C

around its colony on Gelatin agar medium. For isolation of mutants, conidia of Ac 80 were subjected to UV radiation. At 6 min of UV exposure, only 1% of the conidia survived as ascertained from the growth of irradiated conidia. Screening the resulting colonies, a protease hyperproducing mutant, Mt C, and a protease deficient mutant, Mt L, were selected. Mt C exhibited considerably higher (16 mm) and Mt L exhibited reduced (3 mm) zone of clearance than the wild type (7 mm) on gelatin agar plate. This indicated that the proteolytic activity of Mt C, wild type and of Mt L was in the descending order. The strains were allowed to grow on YME medium for 48 h and activity of the extracellular protease was assayed. The strain Mt C produced the enzyme with highest specific activity (3.0 mg tyrosine liberated/min/mg protein) followed by Ac 80 (1.8 mg tyrosine liberated/min/mg protein) and then the strain Mt L (0.9 mg tyrosine liberated/min/mg protein). As such,

The extracted protease was purified to homogeneity (Table 1). The 30-60% (NH4)2SO4 precipitate was subjected to Sephadex G-150 gel filtration chromatography, which furnished an elution profile with several peaks. The fractions with enzyme activity, corresponding to one of the peaks, were pooled. At this step, the protease was purified 9-fold over the culture extract. Upon DEAE-cellulose chromatography, the protease was eluted at 0.6 M NaCl. Total purification of the protease was achieved in this step with 40-fold purification. The eluate was dialyzed and then lyophilized for further studies The enzyme was subjected to SDS-PAGE on a 10% polyacrylamide slab gel where the purified protein exhibited a single band (Fig. 2) indicating homogeneity of the preparation. Molecular weight of the protein was determined to be 66 kDa corresponding to the BSA marker protein. Miyoshi et al13 also reported a protease from Streptomyces sp. having a similar molecular weight of 65 kDa as estimated from gel filtration. Proteases from several actinomycetes were, however, reported to fall in the molecular mass range of 18 kDa to 36 kDa5,14,15. Enzyme Activity at Different pH and Temperature

Enzyme had a broad pH optimum (7.5-8.5) similar to proteases of many actinomycetes5,15,16. Enzyme from Mt C had an optimum protease activity at 28oC. Extracellular proteases from actinomycetes generally have high temperature optimum of 40oC or above5,17-19 requiring a heated environment for optimum activity.

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Table 2Effect of metal ions on the activity of purified protease of Streptomyces nogalator. Mt C Metal ions 1 mM Control MgSO4.7H2O CaCl2 Pb(NO3)2 AgNO3 MnCl2 CuSO4 ZnSO4 HgCl2

Relative proteolytic activity % 100 98 98 36 33 106 54 64 32

up to 20 min was noted, after which it remained essentially constant. At 20 min of incubation, proteolytic activity was reduced (35%) at 50oC and (40%) at 60oC (Fig. 3). As such, the enzyme may be considered as moderately thermostable. At 28oC, enzyme had a shelf life of more than two weeks. Effect of Inhibitors on Protease Activity Fig. 2SDS-PAGE of purified proteolytic enzyme Streptomyces nogalator Mt C. Lane 1, molecular mass standards; Lane 2, purified enzyme

of

Fig. 3Thermal stability of purified proteolytic enzyme at (--) 28oC, (--).37oC, (-∆-), 50oC and at (-○-) 60oC

However, the enzyme produced by Mt C acts optimally at around ambient temperature of the tropics. Thermal stability of the protease was determined by incubating the enzyme at different temperatures (28-60oC) for different periods. At 28o and 37oC, the enzyme was quite stable. At 50o and 60oC, a progressive decrease in the activity of the enzyme

At 1 mM PMSF, enzyme activity was reduced (20%) and it remained unchanged when the PMSF concentration was increased. Incubating the enzyme with varying concentrations of EDTA, the activity decreased (25%) when the concentration of EDTA was 1 mM. Increased concentrations of EDTA did not cause any further change in the activity of the enzyme. MgSO4, MnCl2 as well as CaCl2 had no significant effect on the activity of the enzyme. ZnSO4 and CuSO4 reduced the proteolytic activity by 36 percent and 46 percent respectively. However, the protease activity was significantly inhibited by Pb(NO3)2, Ag(NO3)2 and HgCl2 (Table 2). A large number of proteases isolated so far for the purpose of dehairing are serine proteases5,6,16,20. Serine proteases are sensitive to PMSF and metalloptoteases are sensitive to EDTA14,16. It seems unlikely that the enzyme produced by Mt C is a serine protease or metalloprotease, but they suggest that enzyme may belong to the class of cysteine protease. Using casein as substrate, Km of the protease was found to be 50 mg/ml. suggesting a low affinity of the enzyme towards casein. Dehairing capacity of culture extract of the wild type Ac 80 as well as of the mutants containing the same amount of protein was ascertained. Enzyme extract (3 ml) in the form of a paste was applied per square inch of goatskin. Enzyme extracts from the cultures of Mt C (7.5 units) and the wild type

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Conclusions The isolated strain, Streptomyces nogalator Ac 80, and its overproducing mutant, Mt C, produces extracellular protease in solid-state fermentation. The activity of the protease appeared to have a relationship with dehairing activity. The enzyme (molecular weight 66 kDa) had optimum activity at a broad pH (7.5-8.5) and at 28°C. References 1

Fig. 4Dehairing of goatskin by enzyme extracts of Streptomyces nogalator. 1, Strain Ac 80 (wild type); 2, strain Mt C (hyperproducing mutant); 3, strain Mt L (deficient mutant); and 4, control

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Fig. 5Dehairing of goatskin by purified proteolytic enzyme of Streptomyces nogalator strain Mt C

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(4.5 units) depilated goatskin at 12 and 22 h respectively. The depilation capacity of the respective enzyme extracts, thus, followed their specific activities and the depilation capacity of the hyperproducing Mt C was highest, followed by that of Ac 80 and then of Mt L (Fig. 4). Thus, it was apparent that dehairing capacities and proteolytic activities were correlated. Estimation of the dehairing capacity of an enzyme by depilation trials on hides or skins, although direct, is a complicated process. However, assay of proteolytic activity as a measure of dehairing capacity appears to be a relatively simple and easy method to adopt. This is especially advantageous where yield of the purified enzyme is low. The purified protease was observed to be highly effective in depilation of goatskin (Fig. 5). It appears that the strain would have potential biotechnological use in process involving hair removal from skins and hides.

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18 Sampath P, Subramanian S & Chandrakasan G, Extracellular proteases from Streptomyces spp.G157: Purification and characterization, Biotechnol Appl Biochem, 26 (1997) 85-89. 19 Lee J K, Kim Y O, Kim H K, Park Y S & Oh T K, Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp.E79 and the DNA sequence of the encoding gene, Biosci Biotechnol Biochem, 60 (1996) 840-846. 20 Tsuchiya K, Nakamura Y, Sakashita H & Kimura T, Purification and characterization of a thermostable alkaline proteases from alkalophilic Thermoactinomyces sp. HS682, Biosci Biotech Biochem, 56 (1992) 246-250.