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.

脂肪酶催化的cis -(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate ( cis -(±)-1) 的立体选择性拆分是合成 ( S,S )-2,8-diazobicyclo的一条有吸引力的途径[4.3.0]壬烷，氟喹诺酮类抗生素莫西沙星的重要手性中间体。在我们之前的研究中，一种来自Sporisorium reilianum (SRL) 的脂肪酶被鉴定为具有出色的热稳定性和 pH 稳定性。然而，SRL 的低酶活性是一个必须解决的挑战。根据工程化的南极假丝酵母，最初采用合理设计进行 SRL 剪裁脂肪酶 B (CALB)，产生称为 SRL-I194N/V195L 的有益变体。随后，确定了环 6 中的两个关键氨基酸残基 L145 和 L154，它们可能调节打开和关闭之间的盖子构象。获得了四位点变体 SRL-I194N/V195L/L145V/L154G (V13)，活性显着增强，达到 87.8 U∙mg ^{−1 ；}该值比野生型 SRL 高 2195 倍。Variant V13 用于制备光学纯 (2 S ,3 R )-1-乙酰哌啶-2,3-二甲酸二甲酯 ((2 S ,3 R )-1)，解析 1 mol∙L ⁻¹ cis -(±) -1，2 h 转化率为 49.9%，绝对立体选择性 ( E > 200）。在 50 °C 下也观察到半衰期为 92.5 小时的出色稳定性。总体而言，研究结果揭示了一种脂肪酶在工业水平上对顺式 -(±)-1 具有高活性，并可能提供一种通用策略来增强其他脂肪酶和其他具有盖部分的酶类的酶活性。