Identification Of Strain HL211 Producing Cold-adapted Protease And Purification, Characterization Of The Protease

A strain of bacterium capable of producing cold-adapted protease was isolated and identified. The yield of cold-adapted protease was enhanced by UV treatment. The conditions for production of protease were optimized. The crude extract was purified and characterized. The results were reported as follows:1. The isolation and identification of strains producing cold-adapted protease:Strain HL221 producing cold-adapted protease was isolated from soil and identified. It was suggested that strain HL221 was the closest relative of Chryseobacterium sp. based on morphological, cultural, physiobiochemical characteristics and phylogenetic analysis of 16S rDNA of the strain.2. Enhancement of cold-adapted protease yield produced by strain HL221 using UV treatment:A mutant of which protease activity was improved from 110U/mL to 230U/mL was obtained using ultraviolet radiation.3. The optimization of conditions for cold-adapted protease production:Based on uniform and orthogonal tests of fermentation condition, the optimized conditions were established as follow(g · L-1): glucose 30, peptone 15, Mn2+ 0.4, Tween80 0.1, K2HPO4 7, KH2PO4 3, pH 7. The optimum temperature was 25℃. The protease activity was improved to 305U/mL by optimization.4. The purification of crude cold-adapted protease:The specific activity of crude protease was increased up to 6076.9U/mgwith purifying fold of 21.2 and recovery rate of 16.0% by filtration on Sephadex G-75and DEAE Sepharose Fast Flow.5. The characterization of cold-adapted protease:The molecular weight of the enzyme was determined to be 35 OOODa by SDS-PAGE, and its isoelectric point pI 4.9 was determined by PAGE-IEF. The enzyme activity was optimal at pH7 and 25℃. The enzyme was stable over the range of pH 7-10 and below 25 ℃.