2022, Volume 19, Issue 12
Engineering >> 2022, Volume 19, Issue 12 doi: 10.1016/j.eng.2022.03.020
Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design
a The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, China
b Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
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Abstract
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, 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, 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 SRL. Variant 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 (E > 200). Excellent stability with a half-life of 92.5 h was also observed at 50 °C. 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.
Keywords
Lipase ; Sporisorium reilianum ; Site-directed mutagenesis ; Molecular dynamics simulation ; Rational design ; Moxifloxacin
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