Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design
作者机构：The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral ChemicalsZhejiang University of TechnologyHangzhou 310014China Key Laboratory of Bioorganic Synthesis of Zhejiang ProvinceCollege of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhou 310014China
出 版 物：《Engineering》 (工程（英文）)
年 卷 期：2022年第19卷第12期
页 面：207-216页
核心收录：
学科分类：081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 070303[理学-有机化学] 0703[理学-化学]
基 金：This work was financially supported by the National Key Research and Development Program of China(2021YFC2102100 and 2018YFA0901400) the Fundamental Research Funds for the Provincial Universities of Zhejiang(RF-C2019005) the National Natural Science Foundation of China(32000898) the Natural Science Foundation of Zhejiang Province(LQ21B060006)
主 题：Lipase Sporisorium reilianum Site-directed mutagenesis Molecular dynamics simulation Rational design Moxifloxacin
摘 要：Lipase-catalyzed stereoselective resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate(cis-(±)-1)is an attractive route for the synthesis of(S,S)-2,8-diazobicyclo[4.3.0]nonane,an important chiral intermediate of the fluoroquinolone antibiotic,*** our previous study,a lipase from Sporisorium reilianum(SRL)was identified to possess excellent thermostability and pH ***,the low enzymatic activity of the SRL is a challenge that must be addressed.A rational design was initially employed for SRL tailoring according to the engineered Candida antarctica lipase B(CALB),resulting in a beneficial variant called SRL-I194N/***,two key amino acid residues in loop 6,L145 and L154,which might modulate the lid conformation between open and closed,were identified.A tetra-site variant,SRL-I194N/V195L/L145V/L154G(V13),with a significantly enhanced activity of 87.8 U∙mg^(−1) was obtained;this value was 2195-fold higher than that of wild-type *** V13 was used to prepare optically pure(2S,3R)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate((2S,3R)-1),resolving 1 mol∙L^(−1) cis-(±)-1 with a conversion of 49.9%in 2 h and absolute stereoselectivity(E200).Excellent stability with a half-life of 92.5 h was also observed at 50℃.Overall,the study findings reveal a lipase with high activity toward cis-(±)-1 at an industrial level and may offer a general strategy for enhancing the enzyme activity of other lipases and other classes of enzymes with a lid moiety.
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