点突变H125L和E145A对L-天冬酰胺酶酶活和热稳定性的改善

摘要

L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia.At present,it has been widely used in food and pharmaceutical industry.However,the application of this enzyme is limited by low enzyme activity and poor stability.In this study,in order to improve enzyme activity and thermal stability of L-asparaginase from Bacillus subtilis B11-06 (BSAII),seven amino acid sites were selected and subjected to mutagenesis according to rational design strategy.The results from enzyme activity assay showed that the specific activity of enzyme H125Lansz E145A and H125L-E145Aansz were increased by 36％,48％ and 64％,respectively,compared with BsAII.The specific activity of E74G was 35.73％ of BSAII and that of H139 was almost 0,and the specific activity of other mutant enzymes were lower than BSAII.The half-inactivation time of H125L at 40 ℃ was 3.9 h longer than that of BSAII.This study showed that residues at position 125 and 145 in amino acid sequence of BsAII had a great influence on its catalytic action and thermal stability,which provided a basis for the directed mutagenesis of L-asparaginase from other bacteria and extended its potential application in food and pharmaceutical industry.%L-天冬酰胺酶可以催化L-天冬酰胺转化为L-天冬氨酸和氨.目前,已被广泛应用于食品和医药行业,但是由于低酶活、稳定性差等缺陷限制了该酶的应用.研究通过理性设计选择了7个位点进行点突变,以提高来源于Bacillus subtilis B11-06的L-天冬酰胺酶(BsAII)的酶活和热稳定性.酶活测定结果表明,突变体酶H125Lansz、E145Aansz、H125L-E145Aansz的酶活较BsAII分别提高36％、48％和64％,突变体酶E74Gansz酶活为BSAII的35.73％,H139ansz酶活几乎为0,其余突变体酶酶活较BSAII均有所降低.其中H125Lansz在40℃的半衰时间较BSAII延长3.9h.研究表明,第125和145位氨基酸残基对酶的催化作用和蛋白结构的稳定性有较大影响,为其他来源L-天冬酰胺酶的改造提供了理论依据,并提高了该酶的工业应用潜力.