Medium factors affecting extracellular protease activity by Bacillus sp. HTS 102—A novel wild strain isolated from Portuguese merino wool

Ana Catarina  Queiroga; Manuela Estevez  Pintado; Francisco Xavier  Malcata

doi:10.4236/ns.2013.56A007

Ana Catarina Queiroga, Manuela Estevez Pintado, Francisco Xavier Malcata

Abstract: The synthesis of an extracellular protease by Bacillus sp. HTS102—a wild strain recently isolated from the wool of Portuguese Merino ewes, was optimized. This protease is thermostable and particularly resistant to harsh environmental conditions—and appears to bear a unique ability to hydrolyze keratin-rich solid materials. Following a preliminary screening for the most relevant medium factors involved in processing, a fractional factorial design (2VI6-1) was applied to ascertain the effects of six relevant parameters—viz. yeast extract concentration, peptone level, inoculum size, stirring rate, temperature and pH. The concentrations of yeast extract and peptone, as well as the incubation temperature and pH were found to play significant roles; and the 2-factor interaction between yeast extract level and pH was also significant. A 2.2-fold increase in the overall level of protease synthesis was eventually attained, with the improved medium relative to the basal medium—which is noteworthy when compared with competing proteases and previous optimization efforts.

Keywords: Fractional Factorial Design; Thermotolerant Enzyme; Proteolytic Activity; Bacillus sp.; Ewe’s Wool

Cite this paper: Queiroga, A. , Pintado, M. and Malcata, F. (2013) Medium factors affecting extracellular protease activity by Bacillus sp. HTS 102—A novel wild strain isolated from Portuguese merino wool. Natural Science, 5, 44-53. doi: 10.4236/ns.2013.56A007.

References

[1] Ibrahim, A.S.S. and Al-Salamah, A.A. (2009) Optimization of media and cultivation conditions for alkaline pro tease production by alkaliphilic Bacillus halodurans. Research Journal of Microbiology, 4, 251-259. doi:10.3923/jm.2009.251.259

[2] Gupta, R., Beg, Q. and Lorenz, P. (2002) Bacterial alka-line proteases: Molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59, 15-32. doi:10.1007/s00253-002-0975-y

[3] Navaneeth, S., et al. (2009) Optimization of medium for the production of subtilisin from Bacillus subtilis MTCC 441. African Journal of Biotechnology, 8, 6327-6331.

[4] Shafee, N., et al. (2005) Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium Bacillus cereus strain 146. Journal of Applied Sciences Research, 1, 1-8.

[5] Huang, G.R., et al. (2008) Optimization of medium com position for thermostable protease production by Bacillus sp. HS08 with a statistical method. African Journal of Biotechnology, 7, 1115-1122.

[6] Queiroga, A.C., Pintado, M.E. and Malcata, F.X. (2012) Potential use of wool-associated Bacillus species for bio degradation of keratinous materials. International Biodeterioration and Biodegradation, 70, 60-65. doi:10.1016/j.ibiod.2011.12.013

[7] Joo, H.-S. and Chang, C.-S. (2005) Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: Optimization and some properties. Process Biochemistry, 40, 1263-1270. doi:10.1016/j.procbio.2004.05.010

[8] Reddy, L.V.A., et al. (2008) Optimization of alkaline pro tease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches. Bioresource Technology, 99, 2242-2249. doi:10.1016/j.biortech.2007.05.006

[9] Rao, Y.K., et al. (2007) Medium optimization of carbon and nitrogen sources for the production of spores from Bacillus amyloliquefaciens B128 using response surface methodology. Process Biochemistry, 42, 535-541. doi:10.1016/j.procbio.2006.10.007

[10] Puri, S., Beg, Q.K. and Gupta, R. (2002) Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Current Microbiology, 44, 286-290. doi:10.1007/s00284-001-0006-8

[11] Myers, R.H. and Montgomery, D.C. (2002) Response surface methodology: Process and product optimization using designed experiments. Wiley Series in Probability and Statistics, Wiley Interscience, New York, 824.

[12] Queiroga, A.C., Pintado, M.M. and Malcata, F.X. (2007) Novel microbial-mediated modifications of wool. Enzyme and Microbial Technology, 40, 1491-1495. doi:10.1016/j.enzmictec.2006.10.037

[13] Gu, X.B., et al. (2005) Optimization of medium constituents for a novel lipopeptide production by Bacillus subtilis MO-01 by a response surface method. Process Biochemistry, 40, 3196-3201. doi:10.1016/j.procbio.2005.02.011

[14] Kumar, C.G. and Takagi, H. (1999) Microbial alkaline proteases: From a bioindustrial viewpoint. Biotechnology Advances, 17, 561-594. doi:10.1016/S0734-9750(99)00027-0

[15] Chi, Z., et al. (2007) Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresource Technology, 98, 534-538. doi:10.1016/j.biortech.2006.02.006

[16] Joo, H.-S. and Chang, C.-S. (2006) Production of an oxidant and SDS-stable alkaline protease from an alkaophilic Bacillus clausii I-52 by submerged fermentation: Feasibility as a laundry detergent additive. Enzyme and Microbial Technology, 38, 176-183. doi:10.1016/j.enzmictec.2005.05.008

[17] Liu, J., et al. (2005) Optimization of nutritional conditions for nattokinase production by Bacillus natto NLSSE using statistical experimental methods. Process Biochemistry, 40, 2757-2762. doi:10.1016/j.procbio.2004.12.025