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

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。

Abstract: To study the coevolutionary of the functional residues and the variation of hydrolysis activity,the substrates binding residues simultaneous mutations of Geobacillus stearothermophilus thermostableβ-galactosidase were constructed.The results showed that the Y272 A/E351 R 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 Y272 A.Increased Km value of Y272 A/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.