Optimization of Process Parameters for Alkaline Protease Production by Bacillus licheniformis N-2 and Kinetics Studies in Batch Fermentation
Production of alkaline protease from B. licheniformis N-2 was studied in shake flask experiments in growth medium comprised of (g/l): glucose, 10; soybean meal, 10; K2HPO4, 5; MgSO4.7H2O, 0.5; NaCl, 0.5; CaCl2.2H2O, 0.5. Different process parameters, such as fermentation period, initial pH, incubation temperature, agitation speed, and size and age of inoculum, were optimized for the maximum yield of the alkaline protease. Maximum enzyme production (991.04 PU/ml) was obtained after 24 h of incubation at 37 °C in growth medium of pH 10. The optimum inoculum size and age were determined as 2.0% (v/v) and 24 h, respectively. Kinetics of growth and protease production by this microorganism were also analyzed at all optimum conditions. Biomass (Yx/s) and product yield (Yp/s) coefficients were determined as 0.375 g of cells/g of glucose and 99.3 PU/g of glucose, with a growth rate of (0.215 h-1) after 24 h of incubation. Thereafter, a continuous decrease in biomass and product yield was observed. The results indicate that enzyme production by B. licheniformis N-2 was entirely growth-associated and that a major portion of the enzyme was secreted in the post exponential phase.
Optimization of Process Parameters for Alkaline Protease Production by Bacillus licheniformis N-2 and Kinetics Studies in Batch Fermentation
Production of alkaline protease from B. licheniformis N-2 was studied in shake flask experiments in growth medium comprised of (g/l): glucose, 10; soybean meal, 10; K2HPO4, 5; MgSO4.7H2O, 0.5; NaCl, 0.5; CaCl2.2H2O, 0.5. Different process parameters, such as fermentation period, initial pH, incubation temperature, agitation speed, and size and age of inoculum, were optimized for the maximum yield of the alkaline protease. Maximum enzyme production (991.04 PU/ml) was obtained after 24 h of incubation at 37 °C in growth medium of pH 10. The optimum inoculum size and age were determined as 2.0% (v/v) and 24 h, respectively. Kinetics of growth and protease production by this microorganism were also analyzed at all optimum conditions. Biomass (Yx/s) and product yield (Yp/s) coefficients were determined as 0.375 g of cells/g of glucose and 99.3 PU/g of glucose, with a growth rate of (0.215 h-1) after 24 h of incubation. Thereafter, a continuous decrease in biomass and product yield was observed. The results indicate that enzyme production by B. licheniformis N-2 was entirely growth-associated and that a major portion of the enzyme was secreted in the post exponential phase.
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- Sandhya C, Nampoothiri KM, Pandey A. Microbial proteases, In: Barredo JL, ed. Microbial Enzymes and Bio-information. Human Press Inc., Totowa, New Jersey; 2005: pp. 165-180.
- Nadeem M, Shahjahan B, Syed QA et al. Microbial production of alkaline proteases by locally isolated Bacillus subtilis PCSIR-5. Pak J Zool 38: 109-118, 2006. 9. Li Y, Lin J, Meng DJ et al. Effect of pH, cultivation time and substrate concentrations on the endoxylanase production by A. awamori ZH-26 under submerged fermentation using central composite rotary design. Food Technol Biotechnol 44: 473-477, 2006.
- Nadeem M, Qazi JI, Baig S et al. Studies on commercially important alkaline protease from Bacillus licheniformis N-2 isolated from decaying organic soil. Turk J Biochem 32: 171-177, 2007.
- Pirt SJ. Principles of Microbes and cell cultivation. In: Parameters of growth and analysis of growth data. Blackwell Scientific Publications, London, 1975: pp. 4-14.
- Okpokwasili GC, Nweke CO. Microbial growth and substrate utilization kinetics. African J Biotechnol 5: 305-317, 2006.
- Lowry OH, Roserbrough NJ, Farr AL et al. Protein measurement with folin phenol reagent, J Biological Chem 193: 265-275, 1951.
- Dubois M, Gilles KA, Hamilton JK et al. Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356, 1956.
- Yang SS, Huang CI. Proteases production by Amylolytic fungi in solid state fermentation. J Chinese Agric Chem Soc 32: 589-601, 1994.
- Denizci AA, Kazan D, Abeln ECA et al. Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under high alkaline conditions. J Appl Microbiol 96: 320-327, 2004.
- Wellingta CAN, Martins MLL. Production and properties of an extracellular protease from thermophilic Bacillus sp. Brazilian J Microbiol 35: 91-96, 2004.
- Chi Z, Zhao S. Optimization of medium and cultivation conditions for pullulan production by new pullulan-producing yeast. Enz Microb Technol 33: 206-221, 2003.
- Joo HS, Kumar CG, Park GC et al. Oxidant and SDS stable alkaline protease from Bacillus clausii 1-52: production and some properties. J Appl Microbiol 95: 267-72, 2003.
- Joo HS, Kumar CG, Park GC et al. Optimization of the production of an extra cellular alkaline protease from Bacillus horikoshii. Process Biochem 38: 155-159, 2002.
- Gupta R, Beg QK, Lorenz P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59: 15-32, 2002.
- Kanekar PP, Nilegaonkar SS, Sarnaik SS et al. Optimization of protease activity of alkalophilic bacteria isolated from an alkaline lake in India. Biores Technol 85: 87-93, 2002. He G, Chen Q, Ju X et al. Improved elastase production by Bacillus sp. EL31410 - further optimization and kinetics studies of culture medium for batch fermentation. J. Zhejiang Univ Sci 5: 149-156, 2004.