Lipase-catalyzed stereoselective resolution of ciss-(±)-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 chi-ral intermediate of the fluoroquinolone antibiotic,moxifloxacin.In our previous study,a lipase from Sporisorium reilianum(SRL)was identified to possess excellent thermostability and pH stability.However,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/V195L Subsequently,two key amino acid residues in loop 6,L1 45 and L1 54,which might modulate the lid conformation between open and closed,were iden-tified.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 SRL Variant V13 was used to prepare optically pure(2S,3R)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate((2S,3R)-1),resolv-ing 1 mol·L1 cis-(±)-1 with a conversion of 49.9％in 2 h and absolute stereoselectivity(E＞200).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.

Xue Cai;Jiang-Wei Shen;Yu Qiang;Jing Hua;Zhang-Qi Ma;Zhi-Qiang Liu;Yu-Guo Zheng

The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals,Zhejiang University of Technology,Hangzhou 310014,China;Key Laboratory of Bioorganic Synthesis of Zhejiang Province,College of Biotechnology and Bioengineering Zhejiang University of Technology,Hangzhou 310014,China

工程（英文）

[1]Xue Cai;Jiang-Wei Shen;Yu Qiang;Jing Hua;Zhang-Qi Ma;Zhi-Qiang Liu;Yu-Guo Zheng-.Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design)[J].工程（英文）,2022(12):207-216

reilianum,Moxifloxacin,ciss,acetylpiperidine,diazobicyclo,I194N,V195L,L145V,L154G,resolv,lipases

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