Isolation, production and characterization of protease from Bacillus Sp isolated from soil sample

Transcription

1 Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2012, 2 (1): ( ISSN : CODEN (USA) : JMBRB4 Isolation, production and characterization of protease from Bacillus Sp isolated from soil sample F Soundra Josephine 1, Ramya V S 1, Neelam Devi 1, Suresh Babu Ganapa 2, Siddalingeshwara K. G 1. Venugopal. N 1 and Vishwanatha T 1 1 Dept of Microbiology Maharani Science College for Women College, Bangalore 2 Dr. Swamy s Lab, University of Agricultural Sciences Campus, Dharwad, Karnataka ABSTRACT Bacillus sp being industrially important organisms produces a wide variety of extra-cellular enzymes including proteases. Bacillus sp isolated from local soil samples collected from Maharani Science College Campus, Bangalore produced protease enzyme. The optimum conditions for cell growth and protease production were 37 C in 24 h, agitation speed of 200 rpm and medium ph 7.0. The specific activity of partially purified protease obtained from ammonium sulphate fractionation is found to be and the fractionation is 1.32 fold purified from the crude enzyme preparation yielding 75.75% from the crude protein. The optimum temperature and ph of the partially purified protease from Bacillus Sp is found to be 40 C and ph 7.0 respectively. Key words: Bacillus Sp, Proteases, Partial Purification, Industrial Applications. INTRODUCTION Proteases execute a large variety of functions and have important biotechnological applications. Proteases represent one of the three largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes [1]. Proteases are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth, the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for their various applications [2] Bacillus produces a wide variety of extra-cellular enzymes, including proteases. Several Bacillus species involved in protease production are e.g. B. cereus, B. sterothermophilus, B. mojavensis, B. megaterium and B. Subtilis [3-8] 163

2 Probably the largest application of proteases is in laundry detergents, where they help removing protein based stains from clothing [9 10]. In textile industry, proteases may also be used to remove the stiff and dull gum layer of sericine from the raw silk fiber to achieve improved luster and softness. Protease treatments can modify the surface of wool and silk fibers to provide new and unique finishes. Proteases have been used in the hide dehairing process, where dehairing is carried out at ph values between 8 and 10 [11]. Proteases are also useful and important components in biopharmaceutical products such as contact-lens enzyme cleaners and enzymatic deriders [12]. The proteolytic enzymes also offer a gentle and selective debridement, supporting the natural healing process in the successful local management of skin ulcerations by the efficient removal of the necrotic material [13]. With respect to the factors affecting productivity and properties of the proteases, it was considered of interest to isolate new sources of protease, purify proteases and investigate the factors affecting their activity. In this paper we aimed to isolate newer source of protease producing bacteria from the local soil sample and partially purify protease and to study the factors affecting the activity to present potential application of the proteases for industrial applications. MATERIALS AND METHODS Isolation and Screening of Microorganisms producing Proteases The soil samples were collected from Maharani Science College Campus Bangalore, were diluted in sterile saline solution. The diluted samples were plated onto Gelatin agar plates containing peptone (0.1% wt/vol), NaCl (0.5% wt/vol), agar (2.0% wt/vol), and gelatin (10% vol/vol) ph-7.0. Plates were incubated at 37 C for 24 hours. A clear zone of gelatin hydrolysis gave an indication of protease producing organisms. Depending upon the zone of clearance, the strain SNR01 was selected for further experimental studies. The isolated proteolytic strain was a spore-forming gram-positive rod, identified as Bacilllus sp by slandered biochemical tests[14,15] Production of protease from Bacillus Sp: Production of protease from Bacilllus, Sp was carried out in a medium containing the following: glucose, 0.5% (wt/vol); peptone, 0.75% (wt/vol); and salt solution, 5% (vol/vol) (MgSO 4.7H 2 O, 0.5% [wt/vol]; KH 2 PO 4, 0.5% [wt/vol]; and FeSO 4.7H 2 O, 0.01% [wt/vol]), ph-7.0 and maintained at 37 C for 24 hours in a shaker incubator (200 rpm). The ph of the medium was adjusted with 1N NaOH or 1N HCl. After the completion of fermentation, the whole fermentation broth was centrifuged at 10,000 rpm at 4 C, and the clear supernatant was recovered. The crude enzyme supernatant was subjected to further studies. Partial Purification of Enzyme Ammonium Sulphate Precipitation The organism was grown for 24 hours as described previously. The cells were separated by centrifugation (10000 rpm, 10 minutes), and the supernatant was fractionated by precipitation with ammonium sulfate between 50% and 70% of saturation. All subsequent steps were carried out at 4 C. The protein was resuspended in 0.1M Phosphate buffer, ph 7.0, and dialyzed against the same buffer. 164

3 Analytical Methods Determination of Protease Activity Protease activity was determined by a modified procedure based on the method of[16]. One protease unit is defined as the amount of enzyme that releases 1 µg of tyrosine per ml per minute under the standard conditions. Protein Assay Protein was measured by the method of [17] with Bovine Serum Albumin (BSA) as the standard. The concentration of protein during purification studies was calculated from the absorbance at 280 nm. Factors affecting activity: The following factors were studied to obtain the optimal condition for activity of partially purified proteases. These factors include incubation period, incubation temperature, and ph. Effect of Temperature and ph on Protease from Bacillus Sp: Effect of temperature was studied from 10 to 80ºC. effect of ph was studied from ph 2.0 to 11.9(HCl/KCl buffer for ph2.0; Glycine/HCl buffer for ph 2.5 to 3.5; Acetate buffer for ph 4.0 to 5.5; Phosphate buffer for ph 6.0 to 7.5; Tris/HCL buffer for ph 8.0 to 9.0; Glycine/NaOH buffer for ph 9.5 to 10.5 and NaHPO 4 /NaOH buffer for ph 11.0 to 11.9) RESULTS AND DISCUSSION Isolation and screening of microorganisms producing Protease Fig. 1 shows the organism producing protease enzyme a clear zone around the colony as the protein near the colony is utilized. Depending upon the zone of clearance, the strain of Bacilllus SNR01 was selected for further experimental studies. Fig. 1: The zone of hydrolysis of Bacillus SNR 01 on gelatin agar Production and purification of protease from Bacillus SNR01: The isolated source of Bacillus SNR01 maximum enzyme production was observed at 24 hours in showed in Fig. 2. Other investors, reported that both Bacillus anthracis, S-44 and Bacillus cereus var. mycoides, S-98 exhibited their maximum ability to biosynthesize proteases within

4 h incubation period since the productivity reached up to units/ml-1 for Bacillus anthracis, S-44 corresponding to units/ml - 1 for Bacillus cereus var. mycoides, S-98 respectively. Moreover, Johnvesly et al., [18] found that a high level of extracellular thermostable protease activity was observed after 24 h incubation and hence our results are in complete accordance with earlier reports. Fig.2: Effect of Incubation Period on Protease Activity The protease enzyme from Bacillus SNR01 was partially purified by ammonium sulphate fractionation as described previously. One liter of the bacterial broth was centrifuged at 8000 rpm for 5 min at 4 C. The specific activity of crude protease enzyme was 7.71U/mg of protein. The specific activity of ammonium sulphate fractionation is found to be and the fractionation is 1.32 fold purified from the crude enzyme preparation yielding 75.75% from the crude protein [Table 1]. Purification Step Total Enzyme Activity (U) Total Protein (mg) Specific Activity (U/mg) Purification Fold % Recovery Crude enzyme (NH 4 ) 2 SO 4 precipitation, dialyzed Effect of Temperature and ph on Protease from Bacillus Sp: Effect of temperature was studied from 10 to 80ºC. Effect of ph was studied from ph 2.0 to 11.9 The optimum temperature of protease produced from Bacillus SNR01 is found to be 40 ºC(Fig.3). The earlier reports reveals different optimum temperature were reported as El- Kastawy [19], reported 30 ºC, where as different optimum incubation temperature were reported by other investigators including 35 o C [7], 50 o C [2,3], 70 o C [8]. The optimum ph of protease produced from Bacillus SNR01 shown at the ph 7.0 (Fig.4). Other investigators recorded optimum proteolytic activity at different ph values such as ph 7.8 [21], 8.0 [2], [22], 10.5[2] and [23]. 166

5 Fig.3: Effect of Temperature on Protease Activity Fig. 4: Effect of ph on Protease Activity CONCLUSION Although many potent strains are on market for enzyme production, scientists prefer studying on new isolates because they could be alternative for commercial use. Many studies such as those showed that researches will continue to isolate alternative strains for production of enzymes as 167

6 well as proteases. The isolated new source of protease producing bacteria from the local soil sample and partially purified protease may be alternative source also for the potential industrial applications. Acknowledgements Authors owe the deepest gratitude to Prof. Lalithamma. B. Principal of Maharanis Science College for Women, Bangalore who made us available the support in a number of ways for completing this research project successfully. REFERENCES [1] Gupta, R.; Beg, Q.K. And Lorenz, P. Appl. Microbiol. Biotech :15-32 [2] Anwar, A. and Saleemuddin, M. Biotech. App. Biochem : [3] Ammar, M.S.; El-Louboudy, S.S. and Abdulraouf, U.M. Az. J. Microbiol (13): [4] Soares, V.F.; Castilho, L.R.; Bon, E.P. and Freire, D.M. Appl Biochem Biotechnol., : [5] Beg, Q.K. and Gupta, R. Enzy. Microbial Techn., : [6] Banik, R.M. and Prakash, M., Microbial Research (2): [7] Gerze, A.; Omay, D. and Guvenilir, Y. Appl Biochem Biotechnol., : [8] Sookkheo, B.; Sinchaikul, S.; Phutrakul, S. and Chen, S.T., Prot. Exp. Purification : [9] Vishwanatha T, Spoorthi N.Jain, Reena V, Divyashree B.C, Siddalingeshwara K.G, Karthic j and Sudipta K.M. Intern J of Eng Sci and Technol (11), [10] Banerjee, U.C.; Sani, R.K.; Azmi, W. and Soni, R. Proc. Biochem., : [11] Ishikawa, H.; Ishimi, K.; Sugiura, M.; Sowa, A. and Fujiwara, N., J. Ferme. Bioeng. 1993, 76: [12] Anwar, A. and Saleemuddin, M. Biotech. App. Biochem. 2000, 31: [13] Sjodahl, J.; Emmer, A.; Vincent, J. and Roeraade J. Protein Expression and Purification, : [14] Ellaiah P, Adinarayana K, Pardasaradhi SV, Srinivasulu B. Indian J Microbiol. 2002, 42: [15] Sneath HAP, Halt GJ, eds. Bergey s Manual of Systematic Bac-teriology Vol 2. Baltimore, MD: Williams and Wilkins [16] Tsuchida O, Yamagota Y, Ishizuka J, Arai J, Yamada J, Ta-keuchi M, Ichishima E. Current Microbiol :7-12. [17] Lowry OH, Rosebrough NJ, Farr AL, Randall RL.. J Biol Chem. 1951; 193: [18] Johnvesly B.; Manjunath B. R., and Naik G. R.. Bioresour Technol (1):61-4. [19] El- Kastawy, S.F.M. Egypt. J. Biomed. Sci (2): [20] Ali, O.A. Az. J. Microbiol (11): [21] Tsujibo, H.; Miyamoto, K.; Hasegawa, T. and Inamori, Y., J. App. Bacteriol. 1990, 69: [22] Kumar, C.G.; Tiwari, M.P. and Jany, K. D. Proc. Biochem. 1999, 34: [23] Takami, H.; Akiba, T. and Hprikoshi, K. Appl. Microbiol. Biotechnol. 1989, 30: