Efficient Activity Enhancement of a Lipase from Sporisorium reilianum for the Synthesis of a Moxifloxacin Chiral Intermediate via Rational Design
Xue Cai,
Jiang-Wei Shen,
Yu Qiang,
Jing Hua,
Zhang-Qi Ma,
Zhi-Qiang Liu,
Yu-Guo Zheng
Affiliations
Xue Cai
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
Jiang-Wei Shen
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
Yu Qiang
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
Jing Hua
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
Zhang-Qi Ma
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
Zhi-Qiang Liu
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; Corresponding author.
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
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.
