SCREENING OF SUBSTRATES FOR PROTEASE PRODUCTION FROM BACILLUS LICHENIFORMIS

Size: px

Start display at page:

Download "SCREENING OF SUBSTRATES FOR PROTEASE PRODUCTION FROM BACILLUS LICHENIFORMIS"

Juliet Cleopatra Horn
6 years ago
Views:

1 SCREENING OF SUBSTRATES FOR PROTEASE PRODUCTION FROM BACILLUS LICHENIFORMIS Vishwanatha T 1, Spoorthi N.Jain 1, Reena V 1, Divyashree B.C 1, Siddalingeshwara K.G 1, Karthic J 2 and Sudipta K.M 3 1. Department of Microbiology, Maharani science college for women, Bangalore Research and Development centre, Bharathiar University, Coimbatore. 3. Department of Biotechnology, Padmashree Institute of information Science Nagarabhavi, Bangalore-72 ABSTRACT Proteases are the key enzyme in industrial application. Microbial protease play role in biotechnological process. Protease production by Bacillus licheniformis were isolated from different soil samples around Maharani s College, Bangalore and used to screen for the protease producing strains and also screened for the substrates for by using casein and skim milk agar plate assay. The agro wastes are also used to screened produce protease. The agro wastes are rice bran, paddy straw and pigeon pea waste, among these substrate rice bran showed maximum synthesis of protease. At 48 hr it showed maximum IU protease production in rice bran medium Key words- casein-skim milk agar, plate assay, solid state fermentation, rice bran, inhibitors and metal ions. INTRODUCTION Proteases are the most important industrial enzymes that execute a wide variety of functions and have various important biotechnological applications (Mohen et al., 2005). They constitute two thirds of the total enzymes used in various industries and it account for at least a quarter of the total global enzyme production (Kumar et al., 2002). These enzymes occupy a pivotal position due to their wide application in food processing (Pastor et al., 2001), pharmaceutical industries (Anwar and Saleemuddin, 1998; Gupta et al., 2002), meat tenderization process (Takagi et al., 1992; Wilson et al., 1992), peptide synthesis (Kumar and Hiroshi, 1999), infant formula preparation (American Academy of Pediatrics Committee on Nutrition, 1989), leather processing (George et al., 1995) and in weaving processing (Helmann, 1995). Microorganisms are the most important sources for enzyme production. Selection of the right organism plays a key role in high yield of desirable enzymes. For production of enzymes for industrial use, isolation and characterization of new promising strains using cheap carbon and nitrogen source is a continuous process. Habitats that contain protein are the best sources to isolate the proteolytic microorganism. Waste products of meat, poultry and fish processing industries can supply a large amount of protein rich material for bioconversion to recoverable products (Dalev, 1994; Gaustevora et al., 2005). A number of bacteria (Shalinisen and Satyanarayana, 1993), fungi (Banerjee and Battacharyya, 1992) and yeast (Ogryziak and Yamada, 1983) have been reported for the protease production. Many of the organisms produce more than one kind of protease. The type of proteolytic enzyme formed may depend on the composition of the medium. Culture conditions play significant role on growth and production of protease by bacteria (Lee et al., 1992). The genus Bacillus contains a number of industrially important species and approximately half of the present commercial production of bulk enzymes derives from the strains of Bacillus sp. (Beg and Gupta, 2003; Priest 1977). These strains are specific producers of extracellular proteases (Singh,et al.,2001) and can be cultivated under extreme temperature and ph conditions to give rise to products that are, in turn, stable in a wide range of harsh environments (Han and Damodaran 1997). ISSN:

The aim of the present study was to isolate the Bacillus licheniformis from the soil, identification of the culture, screen the protease producing culture as well as substrate for the production of

2 The aim of the present study was to isolate the Bacillus licheniformis from the soil, identification of the culture, screen the protease producing culture as well as substrate for the production of protease. MATERIALS AND METHODS Microorganism and Growth The bacterial strain used in this study was alkalophilic Bacillus lichenoformis isolated from garden soil samples from different regions of Maharanis science college campus, Bangalore. Screening of protease production by plate assay Bacillus sp. was also screened for its proteolytic activity as per siddalingeshwara et al (2010). This was done by inoculating the organisms on the agar plates containing casein (1% w/v) and milk powder (1% w/v), incubated for 48 h. The plates were flooded with 25% TCA (trichloro acetic acid) solution and incubated for 15 min at 45 0 C. (Plate. 1). Plate-1. Screening of protease by casein hydrolysis Screening of substrate for extracellular protease production by Solid State Fermentation Production of protease from Bacillus lichenoformis were carried out in Erlenmeyer s flask containing 20 g of ground nut cake, ground nut pod, rice bran and hay. (Figure-1). The agro waste was moistened with 20 ml of mineral salt media containing K2HPO4, NaCl, CaCl2, MgSO4, Peptone, Yeast extract, Na2CO3. The Na2CO3 was autoclaved separately and added into flasks while inoculating. The flasks were inoculated with 1 ml of 18 h culture and incubated at 37 0 C for 72 h. ISSN:

Figure-1 Screening of different substrates for Protease production After the completion of fermentation, the whole fermentation mix was flooded with 25 Mm Glycine-NaOH buffer (ph-10.0) and filtered.

3 Figure-1 Screening of different substrates for Protease production After the completion of fermentation, the whole fermentation mix was flooded with 25 Mm Glycine-NaOH buffer (ph-10.0) and filtered. The filtrate was further centrifuged at 10,000 rpm for 10 min and the clear supernatant was recovered. The crude enzyme supernatant was subjected for further studies. Partial purification by Precipitation and Dialysis The partial purification of enzyme has been carried out as per the method described by Sadashivam and Manikam (1998). The cell free supernatant was precipitated with different concentrations of ammonium sulphate i.e., from 10 80%. The precipitate was dissolved in small amount of 25 Mm glycine-naoh buffer (ph-10) and dialyzed over night against the same buffer. Assay of Protease The protease activity was determined by the method proposed by Keay et al., (1970). 0.5 ml of suitably diluted enzyme was added to 1.0 ml of 1% casein and 0.5 ml of glycine-naoh buffer (25 Mm, Ph 10.0) whole mixture was incubated at 75 0 C for 10 min. The reaction was terminated by the addition of 3 ml of 10% TCA solution. The solution was allowed to stand for 10 min in cool and was filtered. To the clear filtrate, 5 ml 0.4 M Na2CO3 and 0.5 ml of Folin Ciocalteau reagent (FCR) was added, mixed thoroughly and incubated at 37 0 C for 30 min, in dark. The absorbance was measured at 660 nm. International units (IU) One protease unit was defined as the amount of enzyme that released 1 μg of tyrosine per ml per minute under the above assay conditions. ISSN:

4 RESULTS AND DISCUSSION Ten isolates of bacterial strains were identified as Bacillus licheniformis by staining and biochemical analysis. The isolated strains were used to screen for protease by using casein and milk powder plate assay. By cleared zone around colony (Plate-1) were used as protease producers. Out of ten isolates Bacillus licheniformis VSDR 03 strain showed as a potential protease producer. The Bacillus licheniformis VSDR 03 further used to screen the substrates for the production of protease through solid state fermentation. Among the agro wastes used rice bran showed best substrate for the maximum protease production. It showed IU at 48 hr. The minimum enzyme production was observed is IU at 48 hr in ground nut cake (Table-1). The perusal of data indicated that the ammonium sulphate precipitation method showed IU/mg.This clearly indicated that there is an increase in the protein purification by employing ammonium sulphate method. Siddalingeshwara et al (2010) were reported on screening of the substrate for production of protease and also suggested that rice bran was the suitable substrate. Purification by salt precipitation also employed. Our results are close agreement with siddalingeshwara et al., (2010). TABLE- 1.Screening of different substrates for protease production Substrate Used Enzyme Activity(IU) Ground Nut Cake Ground nut pod Paddy straw Rice Bran REFERENCE [1] F. N Mohen, D. Dileep and D. Deepthi. Potential application of protease isolated from Pseudomonas auriginosa PD100. Biotechnol Ind : [2] A. Kumar, A. Sachdev S.D. Balasubramanyam, A.K. Saxena and A. Lata. Optimization of conditions for production of neutral and alkaline protease from species of Bacillus and Pseudomonas. Ind. J. Microbiol : [3] M.D. Pastor, G.S. Lorda and A. Baltti. Protease obtention using Bacillus substills 3411 and amaranth seed meal medium at different aeration ratio. Braz. J. Microbiol : 1-8. [4] A. Anwar and M. Saleemuddin. Alkaline proteases: A review. Bioresour. Technol : [5] R.Gupta, Q.K. Beg and P.Lorenz. Bacterial alkaline proteases: Molecular approaches and industrial application. Appl. Microbiol. Biotechnol : [6] H.Takagi, M. Kondou, T. Hisatsuka, K.Nakamuris, Y.C.Tsai and M.Yamasaki. Effect of an alkaline elastase from an alkalophillic Bacillus stain on the tenderization of beef meat. J. Agric. Food. Chem : [7] S.A.Wilson, O.A. Young, T. Coolbear and R.M. Daniel. The use of protease from extreme thermophilic for meat tenderization. Meat Sci : [8] Kumar CG, Hiroshi T (1999). Microbial alkaline protease from a bioindustrial vino point. Biotechnol. Adv. 17: [9] American Academy of Pediatrics Committee on Nutrition (1989).Pediatries. 83: [10] S, George, V, Raju, M.R.V. Krishnan, T.V.Subramanian and K. Jayaraman (1995). Production of protease by Bacillus amyloliquefaciens in solid state fermentation and its application in the unlairing of hides and skins. Process Biochem. 30: [11] J.D Helmann Compilation and analysis of Bacillus subtilis SAdependent promotes sequences: evidence for extended contact between RNA polymerase and upstream promotes DNA. Nucl. Acids Res : [12] P.G. Dalev. Utilization of waste feather from poultry slaughter for production of protein concentrate. Bioresour. Technol : [13] A. Gaustevora, D. Braikova P. Christov, K. Tishinov, E. Vasileva Tonkova, T.Haertle, P Nedkov. Degradation of keratin and collagen containing wastes by newly isolated. Thermnoactivomycetes or by alkaline hydrolysis. Lett. Appl. Microbiol : [14] Shalinisen and T. Satyanarayana. Optimization of alkaline protease production by thermophilic Bacillus licheniformis S- 40.Indi.J.Microbiol : [15] R. Banerjee and B.C. Battacharyya. Optimization of multiple inducers effect on protease biosynthesis by rhizopus Oryzae.Bioprocess Eng : [16] D.M. Ogrydziak and T.Yamada.Extracellular acid protease produced by saccharomycopsis lipolytica. J.Bacteriology : [17] W.Lee, Young-Jecho, Gyu-Mok Son and Cheong Choi. Characteristic and action pattern alkaline protease produced from Bacillus sp.cw Korean Biochem.J. 24: ISSN:

5 [18] Q.K. Beg and R.Gupta. Purification and characterization of an oxidationstable, thiol- dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microb. Technol : [19] F.G. Priest. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev : , [20] J. Singh, N.Batra and C.R. Sobti,. Serine alkaline protease from a newly isolated Bacillus sp. SSR1. Proc. Biochem : [21] X.Q. Han and S. Damodaran. Isolation, identification and fermentation of Bacillus species producing a detergent-stable endopeptidase. J.Agric. Food Chem , [22] L. Keay, P.W. Moser and B.S. Wildi. Proteases of the genus Bacillus I alkaline proteases, Biotechnol.Bieng : 213. [23] K.G. Siddalingeshwar, J. Uday, C.H, Huchesh H.P. Puttaraju, J. Karthic, K.M. Sudipta, T. Pramod and T.Vishwanatha. Screening and characterization of protease from bacillus sp. International Journal of Applied Biology and Pharmaceutical Technology2010. Volume: I: Issue-2: ISSN:

Volume: I: Issue-2: Aug-Oct ISSN SCREENING AND CHARACTERIZATION OF PROTEASE FROM BACILLUS SP

Volume: I: Issue-2: Aug-Oct ISSN SCREENING AND CHARACTERIZATION OF PROTEASE FROM BACILLUS SP Volume: I: Issue-2: Aug-Oct -2010 ISSN 0976-4550 SCREENING AND CHARACTERIZATION OF PROTEASE FROM BACILLUS SP Siddalingeshwara K.G 1, Uday.J 2, Huchesh C.H 2, Puttaraju H.P 2, Karthic J 3, Sudipta K.M 1,