酶动力学
催化效率
化学
突变体
饱和突变
合理设计
催化作用
产量(工程)
酵母
立体化学
酿酒酵母
生物化学
活动站点
纳米技术
材料科学
物理
热力学
基因
作者
Man Zhao,Bin Zhou,Xiaoli Jia,Meinan Wang,Zhi‐Qiang Liu,Yu‐Guo Zheng
标识
DOI:10.1016/j.jbiotec.2023.06.007
摘要
D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites. Semi-saturation, single and combined-site mutagenesis was performed on all six residues, and several mutants with improved enzymatic activities were obtained. Among them, the mutant SceCPRS158A/Y298H exhibited the highest catalytic efficiency in which the kcat/Km value is 2466.22 s-1·mM-1, 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPRS158A/Y298H had an expanded and increased hydrophilicity catalytic pocket, and an enhanced π-π interaction which could contribute to faster conversion efficiency and higher catalytic rate. The whole cell system containing SceCPRS158A/Y298H and glucose dehydrogenase (GDH), under the optimized condition, could reduce 490.21 mM D-PL with e.e.≧ 99%, conversion rate = 98%, and the space-time yield = 382.80 g·L-1·d-1, which is the highest level reported so far.
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