Screening Of Protease Producing Strain From Deep-sea And Studies On Enzymatic Properties

Protease is one of the main enzymes in industry, widely used in the food, cosmetics and pharmaceutical industry and so on. Cold-adapted protease has irreplaceable advantage compare to mesophilic enzyme in the food processing and cosmetics applications, the most notable characteristics of the protease is:the optimum temperature is generally at 10℃-45℃, higher catalytic efficiency in the range of 0-30℃ and lower thermal stability compared to mesophilic enzyme. This paper investigated a protease producing strain isolated and identified from deep-sea sediment. The conditions of producing cold-adapted protease were optimized and the properties of enzyme were investigated, in order to provide basis research for cold-adapted protease application. The results were as follows:By means of the morphological, physiological and biochemical text, the strain was identified as a gram-positive bacillus and many kinds of carbon sources could be used in the medium. The catalase test and methyl red test was negative, nitrate, arginine double enzyme test and VP test were all positive. Further the 16S rDNA of strain was analyzed and the strain was closer to Bacillus subtilis strain Acj 115 in kinship. Then the strain was identified as Bacillus subtilis and named Bacillus subtilis.N-9.Medium composition and fermentation conditions were optimized by single factor and orthogonal experiments. The optimal culture medium components were determined(g/L): xylose 25, peptone 35, yeast extract 16, NaCl 10, KH2PO4 3, K2HPO4 7. The optimal culture conditions were pH 5.5, liquid volume 80 ml/500 ml, inoculation time 15 h, inoculation volume 3.5%. Under the optimal conditions, the enzyme activity was 306.93 U/ml and it had increased by 223.56% than before.Using ammonium sulfate precipitation step by step for purification, the enzymatic properties of protease was analyzed. As a cold-adaptedenzymes, the optimal temperature of the protease is 40℃ and the protease has higher low-temperature calytic activity, at 0℃ the protease activity still remains 7.05%. The enzyme has a typical thermal instablity, after 60℃ and 15 min treatment; the relative enzyme activity only retained 39%. The optimal pH of the protease is 7.0 and the test of pH stability indicated that the enzyme was more stable within pH 6.0-8.0. The protease activity was strongly inhibited by PMSF, but not by metal ions, which indicated that the protease belong to serine protease. Effect of surfactants SDS on the protease is more obvious than Tween-80. The protease was tolerable to some denaturing agents such as methanol, ethanol and glycerin.