Identification Of Strain Halomonas Sp.SD-02 Producing Cold-adapted Protease And Purification, Characterization Of The Protease

A strain of bacterium capable of producing cold-adapted protease was isolated and identified from saline soda lake in Inner Mongolia.The conditions for production of protease were optimized. The crude extract was purified and characterized. The results were reported as follows:1. Halomonas sp.SD-02 producing cold-adapted protease was isolated from saline soda lake and identified. It was suggested that strain Halomonas sp.SD-02 was the closest relative of Halomonas sp. Based on morphological, cultural, physiobiochemical characteristics and phylogenetic analysis of 16S rDNA of the strain.2. Under varies fermentation condition, extracellular protease characteristic of Halomonas sp.SD-02 was researched. Based on uniform and orthogonal tests, the optimized conditions were established as follow(g/L): glucose 30, peptone 15, Mn²⁺ 0.4,Tween80 0.1,K2HPO4 1,MgSO4·7H2O 0.2, pH 10,The optimum temperature was 18℃.The protease activity was improved to 305U/mL by optimization.3. After the purification and isolation of crude cold-adapted protease. The specific activity of crude protease increased up to7037.50U/mg with purifying fold of 131.39 and recovery rate of 6.16% by filtration on DEAE-SephadexA25 and Sepharose 6B.4. After the research on the characterization of pure cold-adapted protease. It reached that the molecular weight of the enzyme was determined to be 35KD by DS-PAGE, and its isoelectric point pI 8.5 was determined by PAGE-IEF. The enzyme activity was optimal at pH10 and 30℃,the enzyme was stable over the range of below 45℃.The enzyme was incomplete inhibited by EDTA and of low-dose tolerance of 5% H2O2 and 5mmol/LSDS.The protease activity was obviously stimulated by Mn²⁺,Cu²⁺ ,and was inhibited by by Ag⁺,Al³⁺,Hg²⁺.The Km of the enzyme can reach 3.4mg/ml with Casein.