基于理性设计提高1,3-丙二醇氧化还原酶的稳定性和活性

Rational design-based improvement of 1,3-propanediol oxidoreductase stability and activity

1,3-丙二醇氧化还原酶(1,3-propanediol oxidoreductase,PDOR)是甘油生物合成1,3-丙二醇(1,3-propanediol,1,3-PD)的关键限速酶之一。使用PoPMuSiC 2.1计算肺炎克雷伯菌(Klebsiella pneumoniae)来源PDOR(KpPDOR)突变的折叠自由能,结合HotSpot Wizard 3.0对催化中心附近氨基酸的功能热点分析和保守残基鉴定,理性选取突变位点,构建定点突变。经诱导表达、蛋白纯化和酶学性质研究,获得催化性能、热稳定性和pH耐受性提高的突变体。V155N突变体在37℃,pH 7.5下的还原活性为KpPDOR的1.7倍,pH 4.0时是KpPDOR的2.5倍。N262E突变体37℃下半衰期为KpPDOR的1.4倍。分析蛋白结构,发现Val155突变为Asn导致新形成两个氢键,稳定了Val155所在的β-折叠结构。而Asn262突变为Glu形所成的两个氢键,可增强催化中心刚性,提升酶稳定性。静息细胞转化甘油合成1,3-PD,发现突变体V155N工程菌株1,3-PD产能为1.16 mmol/L/OD_(600),高于野生型KpPDOR工程菌株的0.82 mmol/L/OD_(600)。此理性设计方法对改良其他工业酶的性能有一定参考价值。

1,3-Propanediol oxidoreductase(PDOR) is a key regulating enzyme which catalyzes the rate-determining step in the biosynthesis of 1,3-propanediol(1,3-PD) from glycerol.Rational design of site-directed mutations was performed to achieve better catalytic activity,thermal stability,and pH tolerance,through calculating the free folding energy of Klebsiella pneumoniae PDOR(KpPDOR) with PoPMuSiC 2.1 and identifying functional hotspot around the catalytic center,as well as sequence conservation analysis with HotSpot Wizard 3.0.Two improved variants were obtained after directed evolution,induced expression,and purification.Their enzymatic properties are further investigated.V155N showed 1.7-fold and 2.5-fold reducing activity,compared with that of KpPDOR at pH 7.5 and 4.0 respectively.While the half-life of N262E at 37 ℃ lengthened 40%.Protein structure analysis further illustrated the improved properties.V155N introduced two hydrogen bonds into the β-sheet where it's located,while N262E introduced another two hydrogen bonds,strengthening the rigidity of catalytic center.Thus,both variants improved the stability.The results of the conversion of glycerol into 1,3-PD by resting cells showed that the production capacity of 1,3-PD was 1.16 mmol/L of the engineered strain V155N,which was higher than that of the wild-type engineered strain(0.82 mmol/L).This study highlights a rational-design method which can be further applied in other industrial enzymes′ optimization.