Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2012, 2 (4):612-618 (http://scholarsresearchlibrary.com/archive.html) Purification and characterization of neutral protease enzyme from Bacillus Subtilis Muthu Padmapriya and *B. Christudhas Williams ISSN : 2231 3168 CODEN (USA) : JMBRB4 Department of Botany & Research Centre, Scott Christian College (Autonomous), Nagercoil ABSTRACT Isolation and partial purification of neutral protease from Bacillus subtilis was the main aim of this study. The production medium for Bacillus subtilis was optimized by using different ph, Temperature, Carbon, Nitrogen sources and Agitation Speed for 48 hours fermentation culture. The protease enzyme was highly active and stable from ph 6.0 to 9.0 with an optimum at ph 7. Its optimum temperature was 37 C and, it proved stable up to 30-60 C.The best carbon and nitrogen sources were starch and whey protein. The best buffer for production of protease enzyme was phosphate buffer. Protease was partially purified by ammonium sulphate precipitation, dialysis. Protease activity was very high in 180 rpm agitation speed. The proteolytic activity was also detected by casein zymography. The molecular weight of purified enzyme was characterized by SDS- PAGE as 50 kda. This result showed that Bacillus subtilis under study is a good producer of extra cellular protease, which can be beneficial for industries. Keywords: Agitation, Bacillus subtilis, Carbon, Nitrogen, Protease, Zymography. INTRODUCTION Microorganisms are the most important sources for enzyme production and serve as a preferred source because of their rapid growth, the limited space required for their cultivation. Selection of the right organism plays a key role in high yield of desirable enzymes. Many of the organisms produce more than one kind of protease enzyme. It is mostly produced from various bacteria, and it was reported that about 35% of the total microbial enzymes used in detergent industry [1]. Proteases (EC 3.4.21-24) are the most important industrial enzymes and widespread in nature. Microbial proteases belong to acid, neutral or alkaline based on their ph optimum for activity, active sites and may depend on the composition of the medium. Culture conditions play significant role on growth and production of protease by bacteria. Bacillus subtilis is one of the most widely used bacteria for the production of industrially important protease enzymes [2]. These strains are specific producers of extracellular proteases 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 [3]. In the present study we report the isolation of protease producer Bacillus subtilis collected from soil sample of Tirunelveli (kumbikulam), and then focus on optimizing the production of extracellular protease by testing various environmental and nutritional factors. MATERIALS AND METHODS Microorganism Soil samples were taken from Tirunelveli (kumbikulam), and used for the isolation of bacterial cultures on nutrient agar. A total of 12 bacterial cultures were isolated. The isolates were purified by streaking on agar plates and picking single colonies. The isolates were examined for various morphological and biochemical characteristics as per Bergey s Manual of determinative Bacteriology. The Culture was maintained on nutrient agar medium at 4 C. 612
Protease production and assay The isolated bacteria were inoculated on casein agar medium containing casein 2.0%; peptone, 0.5% and agar 1.5% and then incubated at 37 C for 24 hours. The clear zone of casein hydrolysis was an indication of protease secretion [4]. Then, the protease producers were subcultured on nutrient agar plates in order to obtain pure isolates of bacteria species. The protease activity was determined by the casein digestion method and concentration was determined by Lowry s method with BSA as a standard. Effect of ph on protease production The ph values investigated were 5.5, 6.0, 7.0, 8.0, and 9.0. In these analyses ph was adjusted using the following buffer systems: sodium acetate (ph 5.5-6.0), sodium phosphate (ph 6.0-7.0), phosphate buffer (ph 7.0), sodium carbonate (ph 8-9), and glycine NaOH (ph 9 11) for 8 hours at 45 C. At the end of incubation period the cell free culture filtrate was obtained and used as enzyme source [5] Effect of Temperature on protease production The optimum temperature of the protease was determined by incubation of the reaction mixture at different temperatures of 30, 35, 45, 55, and 65 ºC for 8 hours. At the end of incubation period the cell free culture filtrate was obtained and used as enzyme source. Effect of Agitation rate on protease production The effect of agitation rate on enzyme production was investigated by incubating culture flasks at different agitation speed of 110, 130, 150,180, 190and 200 rpm [6]. Effect of Carbon and Nitrogen sources on protease production 0.1% of different carbon (Glucose, Fructose, Lactose, Sucrose, Galactose, Starch and Maltose) and 0.5% of different nitrogen (Beef extract, Peptone, Soy bean meal, Whey protein, Tryptone and Glycine) [7] sources and salt solutions (MgSO 4, CaCl 2 ) were supplemented individually to the culture medium with different ph buffer. 10% of culture was inoculated to the media and incubated in shaker (180 rpm). Samples were drawn periodically. Purification and characterization of protease The protease was partially purified by ammonium sulphate (75%) precipitation followed by dialysis. The precipitate was collected by centrifugation at 12,500 rpm for 20 min at 4 C, and dissolved in 0.01 M phosphat buffer (ph 7.0). The protein concentration was measured by Lowry s methods using BSA as standard protein. After partial purification, the enzyme was characterized with its optimal values of ph (5.5-9.0), temperature (30-65 C), and their stabilities were presented. The molecular weight of partially purified enzyme was determined by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). RESULT AND DISCUSSION In the present study forty bacterial isolates were obtained from soil samples, of which 12 isolates were identified as proteolytic Bacillus species based on gram staining, cellular morphology, and some biochemical tests such as lecithin, gelatin, and casein hydrolysis in which all the species were positive in these tests and only one isolated bacterial strain (T5) was identified as Bacillus subtilis [8] (Table. 1). The protease activity was observed as clear zone of inhibition around the casein agar plate well. T5 zone size was 29.8mm and standard culture zone size: 30 mm. and it was found more active at high temperature 35 C and ph 7-8 range (Fig. 1). Effect of ph and Temperature The optimum ph of enzyme was determined as ph 7.0 (Fig. 2). 100% of enzyme activity was still detectable at ph 7.0 after 4 h at 37 C [5]. This might be the neutral protease. (Basu et al. 2007;. The optimum temperature of enzyme was 35 C (Fig. 3). Enzyme activity was stable with temperature within the range of 30ºC to 60ºC. Effect of Agitation rate on protease production Bacillus subtilis grown in culture media containing starch and whey protein showed maximum protease activity at 180 rpm agitation speed after 24 h incubation (Fig. 4). At this speed, aeration of the culture medium was increased which could lead to sufficient supply of dissolved oxygen in the media. Nutrient uptake by bacteria also will be increased resulting in increased protease production. It was also revealed that a decrease in agitation rate drastically lowered the total protease yield [6] 613
Figure 1 Clear zone of Proteolytic Bacillus sp on casein agar medium This figure showed the protease production by casein medium by the isolated B.subtilis. T1-12 strains were used to screen for cleared zone around colony and T5 zone size was 29.8mm around the well with the Standard culture zone size: 30mm. Figure 2 Effect of different ph Concentration on the production of B. subtilis protease The optimum ph for the production of protease enzyme was found at ph 7 614
Figure 3 Effect of different Temperature on the production of B. subtilis protease The optimum temperature for the production of protease enzyme was found at 37 C Figure 4 Effect of different Agitation on the production of B. subtilis protease The optimum agitation speed for the production of protease enzyme was found at 180 rpm 615
Figure 5 Effect of different Carbon sources on the production of B. subtilis protease The protease production was high in 0.1% starch containing medium Figure 6 Effect of different Nitrogen sources on the production of B. subtilis protease The protease production was high in 0.5% Whey protein containing medium 616
Figure 7 SDS-PAGE of the purified protease from Bacillus subtilis Carbohydrates of Biochemical tests Lane A Protease enzyme (50kDa), Lane B Protein markers Table 1 Morphological and Biochemical characteristics of Bacillus subtilis S.No Identification Test Bacterial Isolates C T 1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 1. Morphology Rods Cocci Rods Cocci Rods Cocci Rods Cocci Cocci Cocci Cocci Rods Rods 2. Gram staining + + + + + + + + + + - - 3. Motility Test + + + + + + + + + + + 4. Glucose + + + + - + + + + + + + - 5. Fructose + + + + - + + + + + + + + 6. Sucrose + + + + - + + + + + + + + 7. Lactose - - - - - - - + - - + + + 8. Maltose + + + + - + + + + + + - + 9. Galactose - - - - - - - - - - - + + 10. Arabinaose + + + + + + + + + + + - - 11. Xylose - - - - - - - - - - - + + 12. Raffinose - - - - - - - + - - + - - 13. Mannitol + + + + + + + - + + - - + 14. Salicin + + + + + + + - + + - + - 15. Citrate + + + + + + + + + + + + - Utilization 16. Urease - - - - + - - + - - + + + Activity 17. Starch + + + + + + + + + + + - + 18. Gelatin + + + + + + + + + + - + + 19. Casein + + + + + + + + + + + + + 20. Starch + + + + + + + + + + + + + 21. Catalase + + + + + + + + + + + + + 22. Oxidase Test + + - + + + + + + + + + + 23. Indole Test. - - - - - - - + - - + + + 24. Methyl Red - - - - + - - + - - + - + Test 25. Voge s + + + + - + + - + + - - + Proskaver Test 26. NO 3 + + + - - + + - - + - + - Reduction 27. omacconkey + + + - + + + + - + + - - 28. Spore test + + + - + + + + - + + - + + Indicate Positive result -Indicate Negative result Gr 617
Based on this table T 5 was similar to control and it was conformed as Bacillus subtilis. Effect of Carbon and Nitrogen sources on protease production Among various media tested, Starch and Whey protein medium was found most suitable. When glucose in basal medium was replaced by five various sugars (maltose, sucrose, fructose, glycerol, starch), Starch (339.632 U/ml) was the best source for protease production (Fig.5). The protease production was affected by carbon sources in the order: starch>glucose> fructose> sucrose>lactose> maltose> galactose. Results obtained showed that the best nitrogen source for protease production was Whey protein (241.970 U/ml) (Fig 6). Maximum enzyme production were whey protein> soyabean meal>beef extract > tryptone >glycine > peptone [7]. Purification and characterization of protease The molecular mass of the partially purified enzyme (T5) was found 50 kda by using SDS-PAGE (Fig 7). The protease enzyme was partially purified from B.subtilis by using 75% ammonium sulfate precipitation and their purification fold was 3.12-fold purity after dialysis purification fold was 5.14 fold purity. CONCLUSION In the present study, we could isolate Bacillus subtilis and protease enzyme. The molecular weight was found to be ~50 kda. The protease was partially purified and the activity was analyzed by zymography. The Protease had an optimum ph of around 7, which is a typical characteristic of neutral proteases. The optimum temperature was 37 C and proved stable up to 60 C.The best protease producing Carbon and Nitrogen substrates were starch and whey protein and the activity was maximum at 180 rpm agitation speed. The present study achieved by reducing the production cost, increasing the products quantity and overall profit. Protease applications ranged from industrial, bioremediation process to high quality in case of products involved pharmaceutical medicines. Acknowledgements The authors are thankful to Scott Christian College, Affiliated to Manonmaniam Sundaranar University, Tami Nadu, India for providing the infrastructure facilities for this study. REFERENCES [1] M. A. Ferrero, G. R. Castro, C. M. Abate, M. D. Baigori and F. Sineriz, Applied Microbiology and Biotechnology,1996, 45(3) 327 332. [2] Q. K. Beg, V. Sahai and R. Gupta, Process Biochemistry. (2003), 38 1-7. [3] X. Q. Han and S. Damodaran, Journal of Agricultural and Food Chemistry. 1998, 45(11) 4191 4195. [4] F. M. Olajuyigbe and J. O. Ajele, Afr. J. Biotechnol., (2005), 4(8) 776-779. [5] N. Sevinc and E. Demirkan, J. Biol. Environ. Sci. 2011, 5(14) 95-103. [6] S. Saurabh, I. Jasmine, G. Pritesh and S. Rajendra Kumar, Malaysian Journal of Microbiology., 2007, 3 (1) 1 6. [7] F. Abidi, F. Limam and M. M. NejibProces Biochem. 2008, 43 1202 1208. [8] Shereen Gul, M. U. R. Rahman, A. K. K. Achazai and K. Kamin, J.Chem.Soc.Pak., 2008, 30 900-906. 618