Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from Klebsiella pneumoniae toward Higher Activity by Error-Prone PCR and Bioinformatics.
Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from Klebsiella pneumoniae toward Higher Activity by Error-Prone PCR and Bioinformatics.
复制标题
通过易错 PCR 和生物信息学指导肺炎克雷伯菌 1, 3-丙二醇氧化还原酶向更高活性的进化和解析机制。
DOI：
10.1371/journal.pone.0141837
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Fang B
中科院分区：
文献类型：
--
作者：
Jiang W;Zhuang Y;Wang S;Fang B
1，3-丙二醇氧化还原酶（PDOR）是甘油生物转化为1，3-丙二醇（1，3-PD）的关键酶，1，3-PD是一种有价值的化工产品，也是六大石油化工新产品之一。我们采用易错PCR和活性筛选来鉴定肺炎克雷伯氏菌的突变体（K。pneumoniae）PDOR。鉴定的突变体之一PDOR ′-24（其包括单个突变A199 S）的活性为48 U/mg，是野生型酶的4.9倍。对突变体进行分子对接分析，预测突变体中的氨基酸S103、H271、N366、D106、N262和D364与NADH结合。通过模拟突变体与底物和辅酶的相互作用机制，分析了PDOR ′-24以及其他三种突变体活性提高的原因。本研究提供了有用的信息，使用番红O板筛选的氧化还原酶的定向进化，确定感兴趣的网站，提高PDOR活性，并演示了利用分子对接分析突变体的相互作用机制的底物和辅酶，分别。
1, 3-propanediol oxidoreductase (PDOR) is a key enzyme in glycerol bioconversion to 1,3-propanediol (1, 3-PD) which is a valuable chemical and one of the six new petrochemical products. We used error-prone PCR and activity screening to identify mutants of Klebsiella pneumoniae (K. pneumoniae) PDOR with improved activity. The activity of one of the identified mutants, PDOR’-24, which includes a single mutation, A199S, was 48 U/mg, 4.9 times that of the wild-type enzyme. Molecular docking was performed to analyze the identified mutants; and amino acids S103, H271, N366, D106, N262 and D364 were predicted to bond with NADH. The origins of the improved activity of PDOR’-24, as well as three other mutants were analyzed by simulating the interaction mechanism of the mutants with the substrate and coenzyme, respectively. This research provides useful information about the use of safranine O plate screening for the directed evolution of oxidoreductases, identifies interesting sites for improving PDOR activity, and demonstrates the utility of using molecular docking to analyze the interaction mechanism of the mutants with the substrate and coenzyme, respectively.