【作者】 刘静；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 微生物学， 2005， 硕士

【摘要】 本论文从土壤中分离筛选出一株产低温蛋白酶的菌株HL221,并进行了菌株鉴定、诱变提高酶活、发酵条件优化、粗酶的提纯和酶学特质的研究。结果如下:1.菌株的分离筛选及鉴定 经过富集培养和分离纯化,获得了产低温蛋白酶的菌株HL221。经形态学观察、生理生化特征研究及16S rDNA序列比对结果表明该菌属于金黄杆菌属（Chryseobacterium sp.）菌株。2.紫外线诱变提高酶活 通过不同时间紫外照射进行菌种诱变,结果表明30s照射时间下,菌株产酶效果最佳,酶活由原出发菌株的110U/mL提高到230U/mL。3.发酵条件优化 通过单因子试验和正交试验,在摇瓶条件下对菌株的碳源、氮源、金属离子、培养温度、pH、接种量等产酶条件进行了优化。得到了菌株的最适产酶条件（g.L^-1）为:葡萄糖30,蛋白胨15,Mn²+0.4,Tween80 0.1,K₂HPO₄ 7,KH₂PO₄3,培养温度25℃,培养基初始pH 7,接种量为2%-4%。在此优化条件下酶活可达到305.9U/mL。4.低温蛋白酶粗酶的提纯 粗酶液通过硫酸铵沉淀、G-75和DEAE Sepharose F.F.柱层析从发酵液分离纯化得到纯酶,经过纯化后的蛋白酶比活力达到6076.9U/mg,提纯倍数为27.2,得率为16.0%。5.低温蛋白酶纯酶特性 测得低温蛋白酶纯酶分子量为35 000Da,等电点为4.9。酶促反应最适温度25℃、最适pH为7。酶活在低于25℃、pH7～10范围内稳定,蛋白酶对酪蛋白的Km为3.1mg/mL。更多还原

【Abstract】 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 ℃.更多还原