淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质  

Purification and Characterization of an ε-Poly-L-lysine-degrading Enzyme Isolated from Streptomyces diastatochromogenes TUST2

作　　者：谭之磊[1] 贾士儒[1] 赵颖[1] 袁国栋[1] 曹伟锋[1] 

机构地区：[1]天津科技大学生物工程学院,教育部工业微生物重点实验室,天津300457

出　　处：《高等学校化学学报》2009年第12期2404-2408,共5页Chemical Journal of Chinese Universities

基　　金：国家“八六三”计划项目(批准号:2006AA10Z347);国家“九七三”计划项目(批准号:2007CB714305)资助

摘　　要：分离得到产抗菌聚氨基酸ε-聚赖氨酸菌株淀粉酶产色链霉菌TUST2,从中纯化了ε-聚赖氨酸降解酶,并对其性质进行了研究.结果表明,该酶为膜结合蛋白.为提取该降解酶,先收集菌体细胞并用超声波破碎,细胞膜部分用1.0mol/LNaSCN溶液溶解.将粗酶液进行SephadexG100凝胶柱层析分离.用100mmol/L磷酸缓冲液洗脱,收集活性部分.纯化后的样品用SDS-PAGE检测,酶亚基分子量约为54700.酶活力在pH=6.0～9.0间稳定,最适宜pH=7.0.酶的最适温度为30℃,在10～50℃水浴30min酶活力未见明显下降.研究了不同金属离子对酶活力的影响,结果表明,Zn2+,Cu2+和Fe3+可分别提高酶活力29.72%,15.85%和15.08%;但Ag+,Hg2+,Co2+和Mn2+对酶活力有强烈的抑制作用.Ca2+,K+和Ba2+对酶活力没有影响.添加4%Tween-80能提高酶活力10%,但EDTA能强烈抑制酶活力.研究结果表明,此降解酶的性质与白色链霉菌产生的ε-聚赖氨酸降解酶的性质相似.Streptomyces diastatochromogenes TUST2, which was isolated from Hainan province, produced the antimicrobial poly（amino acid）, ε-poly-L-lysine. In this study, the ε-poly-L-lysine-degrading enzyme was purified from this strain and its properties of this enzyme were determined. Preliminary data suggested that the ε-poly-L-lysine-degrading enzyme was a cell membrane associated protein. To extract this enzyme, bacterial cells were collected and disrupted with an ultrasonic oscillator, the membrane fraction were solubilized with 1.0 mol/L NaSCN solution. The coarse enzyme extraction was subjected to Sephadex G100 column for purification. With 100 mmol/L phosphate buffer as elution solution, the fractions with the enzyme activity were collected. The purified sample was analyzed with SDS-PAGE and the subunit molecular mass of the enzyme was estimated to be about 54700. The enzyme was stable between pH 6.0 and 9.0, with a maximum at pH=7.0. The optimum temperature was 30 ℃, and no significant activity loss was observed when the enzyme was incubated at 10—50 ℃ for 30 min. The effect of different metal ions on the activity of the enzyme were also investigated, some metal ions, including Zn2＋, Fe3＋, and Cu2＋, could increase the enzyme activities by 29.72%, 15.85%, and 15.08%, respectively; while some other metal ions, including Ag＋, Hg2＋, Co2＋ and Mn2＋, strongly inhibited the enzyme activity. The enzyme activity was not affected by Ca2＋, K＋ and Ba2＋. Experiment results also showed that the enzyme activity increased 10% by addition of 4%Tween-80, while EDTA strongly inhibited the enzyme activity. These results suggested that the specificities of this enzyme are similar to that of the ε-poly-L-lysine-degrading enzyme from streptomyces albulus.

关 键 词：淀粉酶产色链霉菌 ε-聚赖氨酸降解酶 分离纯化 

分 类 号：O621.4[理学—有机化学] Q55[理学—化学]