The Selection Of A Yield Alkaline Protease Strain Bacillus Pumilus Zk202 And Analysis Of Enzymetic Properties

A strain of bacterium capable of producing saline-alkali endurance protease was isolated and identified.The yield of protease was enhanced by UV and DES treatment. The conditions for production of protease were optimized. The crude extract was purified and characterized. The results showed as below:1. One haloalkaloresistant Bacillus was isolated from the salt-soil. It could grow in broth containing 5%～10% NaCl. It could grow in broth with pH 13 too. Based on morphological, cultural, physiobiochemical characteristics and phylogenetic analysis of 16S rDNA of the strain, it was suggested that strain was the closest relative of Bacillus sp. therefore this strain was named Bacillus haloalkaloresistant sp Zk202.2. A mutant of which protease activity was improved from 81U/ml to 321U/ml was obtained using ultraviolet radiation and DES treatments.3. Based on uniform and orthogonal tests of fermentation condition, the optimized conditions were established as follow: pH 9.0, glucose 1.5 %, peptone 1.5 %, Ca2+ 0.01%, the optimum temperature was 36℃. The protease activity was improved to 383U/ml by optimization.with 45h shaking flask.4. The specific activity of crude protease increased up to 954U/mg, with purifying fold of 6.13 and recovery rate of 27.6% by filtration on Sephadex G-75.5. The molecular weight of the enzyme was determined to be 25000 Da by SDS-PAGE, The enzyme activity was optimal at pH 11 and 50℃.