嗜热脂肪芽孢杆菌耐热β-半乳糖苷酶功能位点的累积进化研究  

Coevolutionary study on the functionary amino acid residues of Geobacillus stearothermophilus thermostable β-Galactosidase Bga B

作　　者：董艺凝[1] 陈海琴[2] 张灏[2] 陈卫[2] 

机构地区：[1]滁州学院生物与食品工程学院,安徽滁州239000 [2]江南大学食品学院,食品科学与技术国家重点实验室,江苏无锡214122

出　　处：《食品工业科技》2015年第7期148-153,共6页Science and Technology of Food Industry

基　　金：国家青年科学基金项目(31301523);国家自然科学基金项目(31171636);十二五国家“863”计划(2011AA100905);安徽高校省级科学研究项目(KJ2013B187);滁州学院科研启动基金资助项目(2012qd14)

摘　　要：针对嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)来源耐热β-半乳糖苷酶Bga B底物结合位点构建突变体,研究底物结合位点累积突变的功能进化及水解活性的变化规律。实验结果表明:Y272A与E351R的累积突变体比酶活为野生型酶的3.67倍,为单点突变体Y272A的2倍;Y272A/E351R突变体的Km值增大,其对乳糖的亲和力下降,但由于Kcat值增大,使累积突变体Y272A/E351R催化效率提高为野生型酶催化效率的7.8倍。本研究结果表明底物结合位点间的累积突变可改变底物亲和性,并对水解催化活性进化起到正向促进作用。To study the coevolutionary of the functional residues and the variation of hydrolysis activity, the substrates binding residues simultaneous mutations of Geobacillus stearothermophilus thermostable β-galactosidase were constructed.The results showed that the Y272A/E351R simultaneous mutant let a 3.67-fold increase in specific activity over the wild type enzyme and give a 2-fold increase over the single site mutation Y2？2A.Increased Km value of Y272A/E351 R mutant occurred with decreased affinity of lactose binding,while the mutant increased the catalytic efficiency of the wild type enzyme by 7.8- fold. This study revealed that the simultaneous evolution of the substrate binding residues had its advantage on substrate affinity,and was favorable to the evolution of catalytic activity.

关 键 词：Β-半乳糖苷酶 嗜热脂肪芽孢杆菌 累积进化 功能位点 

分 类 号：TS201.3[轻工技术与工程—食品科学]