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of district Sagar, India, was used as test organism in the present study12. It was maintained at 28°C on. ISP-2 agar slants throughout the present study.
Indian Journal of Experimental Biology Vol. 48, February 2010, pp. 174-178

Production and partial characterization of collagenase of Streptomyces exfoliatus CFS 1068 using poultry feather Richa Jain & P C Jain* Department of Applied Microbiology and Biotechnology, Dr. Hari Singh Gour University, Sagar 470 003, India Received 2 April 2009; Revised 30 September 2009 Streptomyces exfoliatus CFS 1068, an isolate of cultivated field soil, produced maximum collagenase activity (58.19 ± 0.83 U ml-1min-1) in 5 days when soybean meal and starch were used as nitrogen and carbon sources, respectively at pH 7 and 30°C in shake cultures (150 rpm). Production of collagenase was higher (40.43± 0.63 U ml-1min-1) when poultry feathers were used as nitrogen source. Thus, the strain was found to be of biotechnological importance. The purified enzyme showed 30.34 fold increase in collagenase activity and was stable at 70°C for 1h. The enzyme was found to be of serine type. Keywords: Azocoll, Collagenase, Poultry feathers, Protease, Streptomyces exfoliatus CFS 1068

Collagen is a major fibrous element of skin, bones, tendons, cartilage, blood vessels and teeth found in all multi-cellular animals. Collagenases are collagen hydrolyzing enzymes that play an important role in connective tissue metabolism1. In animal body they are produced by specific cells and are involved in repair and remodeling processes2. The microorganisms producing collagenase are mostly pathogenic in nature and presumably contribute to their pathogenicity by allowing the organisms to penetrate connective tissue barrier3-5 or involved in degradation of collagen under natural conditions6-8. Microbial collagenases are of wider applications because they cleave collagen helix at multiple sites. Purified collagenases are used in quantitation of collagen, isolation of specific cell types from attendant connective tissue and pulmonary mast cells from bovine tissue9-10. In medicine, collagenases are used in experimental transplantation of pancreatic islet cells to alleviate diabetic symptoms11, enzymic debridment, nonsurgical removal of debris from wounds and removal of dandruff etc2. Keeping importance of collagenase in view, present investigation was planned to partially characterize collagenase produced by Streptomyces exfoliatus CFS 1068, an isolate of cultivated field soil of India. _____________ *Correspondent author Telephone: 91(7582)264475 Ext.120 (O), 265517 (R) E-mail:[email protected]

Materials and Methods Microorganism ⎯ A proteolytic Streptomyces exfoliatus CFS 1068, an isolate of cultivated field soil of district Sagar, India, was used as test organism in the present study12. It was maintained at 28°C on ISP-2 agar slants throughout the present study. Production medium ⎯ A medium containing glucose (10g); soybean meal (10g); MgSO4.7H2O (0.5g); NaCl (0.5g); FeSO4.7H2O (0.1g); K2HPO4 (1.0g) was used as basal medium for production of collagenase. Packed cell volume of the biomass produced was measured by centrifugation of the content of each flask at 5000 rpm for 10 min. The protein content of the culture filtrate was determined following the method of Lowry et al.13. Determination of collagenase activity ⎯ Collagenase activity was measured by using azocoll (Sigma Chemical Co., USA) as substrate following the method of Peterkofsky14. One unit of enzyme activity was defined as decomposition of 1 µg azocoll ml-1 min-1. Optimum culture conditions for Streptomyces exfoliatus CFS 1068 ⎯ To study optimum culture conditions the cultivation of S. exfoliates CFS 1068 was subjected to 25°-45ºC temperatures (Table 1), 1-8 days incubation period and pH 5-10 (Table 2). For pH studies buffers used were phosphate buffer (pH 5-6), Tris- HCl buffer (pH 7-8) and glycine- NaOH buffer (pH 9-10). Effect of different nitrogen sources was studied by replacing soybean meal with an equal amount of test nitrogen source (Table 3) in the production medium. Effect of carbon sources on

JAIN & JAIN: COLLAGENASE OF STREPTOMYCES EXFOLIATUS USING POULTRY FEATHER

enzyme production was tested using 1% (W/V) carbon source in the medium. In all sets of experiments the culture conditions were kept optimum as determined in previous experiments. Purification and determination of molecular weight of collagenase ⎯ Precipitation of proteins from the cell free culture filtrate was done using (NH4)2SO4 Table 4. The precipitated protein sample was dialyzed in 50 mM Tris-HCl buffer (pH 8) and was concentrated overnight at 4°C on sucrose pad. Collagenase was purified by gel permeation chromatography using Sephadex G-75 column (2.5×55 cm2) and proteins were eluted with 50mM of Tris-HCl buffer (pH 8). Fractions containing collagenase were pooled and the purity of enzyme was checked by SDS-PAGE (12% gel). The molecular weight was determined using low molecular weight marker (Sigma Chemical Co.). Characterization of enzyme⎯ Thermotolerance of the purified collagenase was studied by treating enzyme samples at eight different temperatures (30° -100°C) for 1 h and then the collagenase activity was determined at 40°C. Dependence of collagenase on pH was determined using different buffers (0.1M citrate buffer for pH 4-5, 0.1M phosphate buffer for pH 6-8, 0.1M Tris -HCl buffer for pH 9 and glycine - NaOH buffer for pH 10). The effect of metal ions, protease inhibitors and detergents (Table 5) on collagenase activity was studied using standard protocols15-16. The Collagenase activity in each treatment was determined and expressed as residual activity as compared to control.

also reported 30°C as the most suitable temperature for maximum production of protease by a Bacillus sp. B21. Maximum yield of collagenase (43.5 U ml-1min-1) was observed in 5days old cultures when grown at 30°C. On further increase in incubation both biomass yield and enzyme activity titers gradually decreased. Results of present study are supported by the observations on protease production made by the workers who have used Streptomyces species as test organisms16-17. Nascimento et al.18 have studied the production of proteases by Streptomyces malaysiensis. They have also obtained maximum enzyme titers on day 5 of incubation at 30°C. Collagenase production by Streptomyces sp. 1349 has been found to be maximum during 3-4 days of incubation19. The present strain, Streptomyces exfoliatus CFS 1068 was found to grow at a wide range of pH 5-10, but showed maximum collagenase activity (42.83 U ml-1min-1) in cultures having initial pH 7.0 (Table 2). The organism was found to be pH sensitive for production of collagenase. The enzyme yield was found in the range of 12-14 U ml-1min-1 in cultures having initial pH 6.0 and 8.0. Maximum production of collagenase by Streptomyces sp. 21 E has been reported at pH 7.5 (ref. 20). The strain was found to produce collagenases in presence of sodium nitrate, indicating constitutive Table 2⎯ Effect of incubation periods and pH on growth and production of collagenase by S. exfoliatus CFS 1068 in shake cultures (150 rpm) at 30°C [Values are mean ± SD of 2 independent determinations] Parameters

Results and Discussion Collagenase production by Streptomyces exfoliatus CFS 1068 was noted maximum when its cultures were grown at 30°C for 5 days in medium having initial pH 7 (Table 1). Fujiwara and Yamamoto15 have Table 1⎯Effect of temperature on growth and production of collagenase by S. exfoliatus CFS 1068 in 7 days at pH 7.0 [Values are mean ± SD of 2 independent determinations] Temperature (°C) 25 30 35 40 45

Packed cell volume (cm3) 0.60 ± 0.1 2.50 ± 0.1 2.45 ± 0.05 1.20 ± 0.2 0.65 ± 0.05

Collagenase activity (U ml-1min-1) 7.38 ± 0.38 19.91 ± 0.28 18.71 ± 0.56 9.83 ± 0.72 6.95 ± 0.24

Protein content (μg ml-1) 512.96 ± 18.5 871.80 ± 44.92 870.56 ± 20.06 652.38 ± 48.48 226.10 ± 21.84

Cultures were grown at pH 7.0 for 7 days in stationary conditions.

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Packed cell volume (cm3)

Collagenase activity (U ml-1min-1)

Protein content (μg ml-1)

Incubation [Day(s)] 1 2 3 4 5 6 7 8 pH* 5 6 7 8 9 10

1.30 ± 0.1 2.25 ± 0.05 3.75 ± 0.0 4.70 ± 0.1 5.60 ± 0.05 5.25 ± 0.05 4.80 ± 0.0 3.15 ± 0.05

3.83 ± 0.48 14.03 ± 0.12 23.63 ± 0.21 43.50 ± 0.04 39.94 ± 0.12 38.51 ± 0.83 35.27 ± 0.71

2425.04 ± 36.04 2272.28 ± 28.94 1943.68 ± 60.65 2096.44 ± 23.62 2488.60 ± 24.32 2183.16 ± 21.84 2245.12 ± 50.15 2351.21 ± 32.14

2.4 ± 0.0 3.6 0.2 5.5 ± 0.1 4.7 ± 0.1 3.75 ± 0.05 3.85 ± 0.05

9.23 ± 0.12 12.35 ± 0.60 42.84 ± 1.00 13.43 ± 0.95 9.11 ± 0.95 9.83 ± 0.41

362.86 ± 43.16 1092.88 ± 52.66 2318.46 ± 62.43 1817.58 ± 37.82 1488.98 ± 60.65 1012.96 ± 43.16

*Culture grown for 5 days

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secretion of enzyme by S. exfoliatus CFS 1068 (Table 3). Petrova et al.8 have reported constitutive secretion of collagenase by Streptomyces sp. strain 3B during its post-exponential growth phase. Ammonium salts completely inhibited growth of S. exfoliatus. Earlier studies carried out on other proteases have also reported suppression of enzyme production in presence of inorganic nitrogen as compared to organic nitrogen sources21-22. Soybean meal supported both production of collagenase and biomass yield. Earlier reports have also indicated lesser enzyme production in presence of inorganic nitrogen as compared to organic nitrogen sources. Soybean meal supported maximum yield of collagenase (43.55 U ml-1min-1) by the present test strain. Feather

meal as a nitrogen source was found next to soybean meal, it supported 40.43 U ml-1min-1collagenase production by this strain. Casein has not been found as a good nitrogen source for production of collagenase (Table 3). A perusal of the data given in Table 3 indicates that among the carbon sources soluble starch supported the growth as well as collagenase production by S. exfoliatus CFS 1068. Production of collagenase reached to its maximum value (i.e., 58.19 U ml-1min-1) when soluble starch and soybean meal were used as a carbon and nitrogen sources. A summary of purification steps of collagenase is given in Table 4. Yield of purification was 67.21 using ammonium sulphate precipitation with 30.34

Table 3⎯Effect of different carbon and nitrogen sources on production of collagenase by S. exfoliatus CFS 1068 at 30°C and pH 7.0 for 5 days [Values are mean ± SD of 2 independent determinations] Packed cell volume (cm3)

Collagenase Activity (U ml-1min-1)

Protein content (μg ml-1)

1.45 ± 0.05 1.65 ± 0.05 3.80 ± 0.0 3.90 ± 0.1 2.70 ± 0.1 2.55 ± 0.05 2.40 ± 0.0 6.20 ± 0.2 5.70 ± 0.1 3.15 ± 0.15

1.79 ± 0.36 3.71 ± 0.59 4.43 ± 0.36 4.19 ± 0.36 24.35 ± 0.60 4.43 ± 0.12 21.11 ± 0.48 4.31 ± 0.48 43.55 ± 0.36 40.43 ± 0.36

41.11 ± 12.69 49.11 ± 10.03 952.56 ± 28.94 1084.00 ± 66.24 2163.94 ± 43.16 2190.58 ± 89.34 2831.60 ± 44.92 653.22 ± 37.82 1902.50 ± 32.20 3705.68 ± 43.78 2483.64 ± 28.94 3464.12 ± 63.12

5.65 ± 0.05 4.20 ± 0.0 4.25 ± 0.05 5.40 ± 0.0 3.65 ± 0.05 3.35 ± 0.05

43.79 ± 0.35 39.95 ± 0.36 36.95 ± 0.95 58.19 ± 0.83 35.39 ± 1.07 30.35 ± 0.00

2573.00 ± 21.84 2133.74 ± 36.22 2146.18 ± 32.25 3393.06 ± 65.98 1941.90 ± 75.12 1483.64 ± 52.37

Nitrogen sources Ammonium sulphate Ammonium nitrate Sodium nitrate Urea Gelatin Gelatin(Commercial) Peptone Carragenan Yeast extract Casein Soybean meal Feather meal Carbon sources* Glucose Maltose Sucrose Starch soluble Wheat bran Cellulose *Soybean meal was used as nitrogen source Table 4 ⎯ Purification of extracellular collagenase of Streptomyces exfoliatus CFS 1068. Step

Cell free filtrate Protein precipitate by (NH4)2SO4 Purified enzyme by Sephadex G-75

Volume (ml)

Enzyme activity

100 ml 30 ml

Uml-1min-1 54.85 121.41

25 ml

429.53

Total units 5485 12141

Protein content (mg/ml)

Specific activity U mg-1

3.502 2.354

15.66 51.57

0.904

475.14

Yield (%)

67.21

Purification fold(s)

3.29 30.34

JAIN & JAIN: COLLAGENASE OF STREPTOMYCES EXFOLIATUS USING POULTRY FEATHER

purification fold. The molecular mass of purified collagenase enzyme produced by S. exfoliatus CFS 1068 as determined by SDS-PAGE was found to be 14.5 kDa. Park et al.23 have reported that purified collagenase from Scomber japonicus have a molecular weight of 14.8 kDa. Croux et al.24 have reported production of an acidic metalloprotease by Clostridium acetobutylicum having a molecular weight of 44,000 with highest specific activity against azocoll. Collagenases from other microbial sources have been reported to have different molecular masses such as 66±4 kDa for collagenase produced by Rhizoctonia solani25, 20 and 82 kDa for collagenases produced by Trichophyton schoenleinii and Aspergillus fumigatus, respectively26,27. The enzyme was found stable at a broad range of temperature (30° -80°C) and showed its maximum activity at 70°C

Fig.1⎯ Effect of temperature on activity of purified collagenase: Enzyme activity ( • ), relative enzyme activity (--S--).

Fig.2⎯ Effect of pH on activity of purified collagenase: Enzyme activity ( • ) , relative enzyme activity (--S--).

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Table 5⎯ Effect of metal ions and enzyme inhibitors on relative activity of the purified collagenase Treatment Concentration (mM)

Collagenase activity Relative activity Unit (%) (ml-1min-1)

Metal ions None Mg2+ Ca2+ Fe3+ Cu2+ Zn2+ Ba2+

10 10 10 10 10 10

203 219.08 180.68 102.20 122.13 168.93 167.96

100 107.92 89.00 50.35 60.16 83.21 82.74

203.4 0.00 193.16 134.13 71.02 132.22 122.86 183.57 128.14 148.77

100 94.92 65.91 34.90 64.97 60.37 90.21 62.97 80.42

Enzyme inhibitor/detergent None PMSF EGTA EDT A pCMB L-cystine SDS Tween-20 Tween-80 Triton X-100

10 10 10 10 10 10 20% (V/V) 20% (V/V) 20% (V/V)

for 1h (Fig.1). Thermostability of the collagenase of test strain was found to be higher than that of alkaline thermostable collagenase produced by Bacillus subtilis FS-228, Bacillus sp.Strain MO-129, Streptomyces sp.YSA-13030 and S. albidoflavus31. The enzyme showed activity at different pH (4-10) and was found to retain its 65% residual activity at pH 10 as compared to its optimum activity at pH 7(Fig. 2). Enzyme lost its complete activity in presence of PMSF and retained its 65.91 and 94% activity in presence of EDTA and EGTA , respectively (Table 5). Thus, the collagenase of S. exfoliates CFS 1068 was of serine type. Production of collagenase by S. exfoliatus CFS 1068 using cheap nitrogen sources such as soybean meal and feather meal was significant because their use as nitrogen source could significantly reduce the cost of production of this enzyme. Utilization of feathers by the test strain was also important from the point of view of poultry based management. Acknowledgement One of the authors (RJ) is thankful to the Head, Department of Applied Microbiology and Biotechnology, Dr. H. S. Gour. Vishwavidyalaya, Sagar, M.P., India for providing laboratory facilities during the course of the present study.

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