Studies On Cold-adapted Protease Produced By Psychrophlic Bacillus Cereus SYP A2-3

The cold-adapted protease has been widely studied for its high activity in lower temperature as well as potential high values for application on food, chemical and pharmaceutical industry and so on. This paper introduced a psychrophilic strain isolated from the No.1 Glacier of China located in Xinjiang province, which could produce cold-adapted protease. The protease fermentation, enzymatical isolation, purification and the characterization of enzyme properties were intensively investigated. The results could serve as a background for the research and development of cold-adapted microorganism resource. Furthermore, some new valuable viewpoints on the mechanism of adapting cold temperature were proposed in this paper.SYP A2-3 was a gram-postive bacillus and could grow in 0-38℃. Its optimum growing temperature was 25℃. No growth occured in the presence of more than 8.0% NaCl. Many kinds of carbon sources and the nitrogen sources could be used in the medium. Physiological and biochemical test showed that the V-P test, catalase test, liquefication of gelatin and M-R test were all positive. The 16s rDNA of SYP A2-3 was determined and the phylogenetic tree was constructed which indicated that it was closer to Bacillus cereus and Bacillus thuringiensis in kinship. Then the strain was identificated as Bacillus cereus according to its alignment of 16s rDNA and phenotypic classification.The fermentation condition optimization for protease production indicated that the optimum temperatur and initial pH were 15℃and 6.5 to 7.0. Casein with the concentration of 10-20 g/L was the best nitrogen source, while glucose and starch were suitable carbon sources for the protease production in the concentration about 10g/L, and higher concentration could repress the protease producing.The potential inducing ability of the enzyme substrate and repressing action from its degrading products were studied systematically in the course of fermentaion. The peptide with chain length of 35-55 amino acids had the strongest ability to induce protease producing, while the small molecular nitrogen source (small peptide or amino acid) had no ability for the induction but repressing the protease producing. So the dual adjustment function in the protease synthesis by induction and repression from nitrogen source was related to the length of the peptide. To coarse nitrogen source, such as bean flour and peanut flour, prehydrolysis may enhanceα-amino nitrogen level in the culture medium which could promote cell growing in early period and protease producing in later period. For example, prehydrolysising bean dregs could increase yield 1,100 U/mL. This principle may promote the protease production regulation for other strains.