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KMID : 1025520090510060527
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Journal of Animal Science and Technology
2009 Volume.51 No. 6 p.527 ~ p.536
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Isolation, Production, and Characterization of Protease from Bacillus subtilis IB No. 11.
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Lee Min-Hyang
Lee Kang-Moon
Choi Yong-Jin
Baek Yeon-Soo
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Abstract
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A potent protein degrading bacterium was isolated from soil samples of different environments. Polyphasic taxonomic studies and phylogenetic 16S rRNA sequence analyses led to identify the isolate IB No. 11 as a strain of Bacillus subtilis. The isolated strain was recognized to produce protease constitutively, and the maximum production(1.64 units/ml) was attained in a shake flask culture when the isolate was grown at 40℃, for 32 h in basal medium supplemented with starch(0.25%) and gelatin(1.25%) as sole carbon and nitrogen source, respectively. The optimum pH and temperature for the protease activity were determined to be pH 7.0 and 50℃, respectively. Ca2+ and Mn2+ enhanced remarkably the protease activity but neither showed positive effect on the protease’s thermal stability. In addition, it was observed that the protease was fairly stable in the pH range of 6.5-8.0 and at temperatures below 50℃, and it could be a good candidate for an animal feed additive. The inhibition profile of the protease by various inhibitors indicated that the enzyme is a member of serine-proteases. A combination of UV irradiation and NTG mutagenesis allowed to develop a protease hyper-producing mutant strain coded as IB No. 11-4. This mutant strain produced approximately 3.23-fold higher protease activity(6.74 units/mg) than the parent strain IB No. 11 when grown at 40℃ for 32h in the production medium. The protease production profile of the selected mutants was also confirmed by the zymography analysis.
Proteases(EC.3.4.21-24 & 99) are a complex group of enzymes collectively known as peptidyl-peptide hydrolases. They are responsible for the hydrolysis of peptide bonds in a protein molecule by the process of proteolysis, and are ubiquitous in occurrence, being found in all living organisms (1,10).
They constitute one of the most important groups of industrial enzymes, accounting for 40~60% of the world total enzyme sales with more than two thirds of the proteases commercially produced from microbial origin(18).
Recently, proteases have attracted renewed interest, mainly due to the recognition that they not only play an important role in the cellular metabolic processes but have also gained considerable attention in the industrial community (1).
Today, the enzymes have become widely used in various industrial sectors, such as detergent, food, pharmaceutical, leather, diagnostics, waste management, and silver recovery (19).
The potential uses of proteases and the need for development of economical processes for the highly active enzyme production still continue to stimulate the search for new microbial strains capable of producing higher levels of proteases with novel catalytic properties.
The data presented here describe the isolation and identification of a bacterial strain producing a high level of protease having the potential for using as an animal feed additive and/or an efficient proteolytic enzyme for the hydrolysis of soy protein. Also described in this report are factors that affect the production of the enzyme by the isolate, some properties of the enzyme activity, and the isolation of protease hyper-producing mutants of the B. subtilis IB No. 11strain.
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KEYWORD
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Protease, Bacillus subtilis IB No. 11, Isolation, Characterization
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