食品科学2017，Vol.38Issue(22)：8-13,6.DOI:10.7506/spkx1002-6630-201722002
基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性
Improving L-Asparaginase Activity from Bacillus licheniformis by Directed Evolution
摘要
Abstract
In order to improve its L-asparaginase activity,the L-asparaginase gene of Bacillus licheniformis was molecularly modified by directed evolution.Mutants S10,S16 and S21 were screened out of more than 19 100 mutants by two rounds of error-prone PCR and one round of DNA shuffling,whose specific activities were increased by 106％,74％ and 43％,respectively,as compared to that of the wild type,together increased Kcar/Km.The amino acid sequence of S10 showed three mutations,K43E,N67S and I269L.The results of three-dimensional simulation showed that amino acid mutations at position 43 for glutamic acid and at position 67 for serine may improve substrate affinity and catalytic efficiency,thereby increasing enzymatic activity.The circular dichroism analysis showed that the mutant enzymes contained less α-helix and more random coil than the wild enzyme,indicating a slight decrease in rigidity and an increase in flexibility.This study indicates that directional evolution can effectively improve the L-asparaginase activity from B.licheniformis.关键词
L-天冬酰胺酶/定向进化/酶活性Key words
L-asparaginase/directed evolution/enzyme activity分类
轻工纺织引用本文复制引用
邵泽香,焦琳舒,陆兆新,别小妹,赵海珍,张充,吕凤霞..基于定向进化技术提高地衣芽孢杆菌L-天冬酰胺酶活性[J].食品科学,2017,38(22):8-13,6.基金项目
国家自然科学基金面上项目(31671800) （31671800）
