Abstract: In our work，ester hydrolysis of chiral primary alcohols was adopted as model reaction. The results showed that those chiral primary alcohols with non-acyloxy oxygen atom were able to be resolved by Pseudomonas cepacia lipase (PcL) with a relatively high enantioselectivity，but those without the extra oxygen did not. Investigation by chemical modification of PcL by N-acetylimidazole indicated enantioselectivity towards primary alcohol with the non-acyloxy oxygen decreased significantly，but those without retained almost the same low value. Protein mass spectrometry analysis suggested that Tyr29 in PcL had a significant effect on selectivity inside the active center. Further investigation by molecular simulation indicated that phenolic hydroxyl hydrogen atom in PcL’s Tyr29 was able to serve as an extra hydrogen bond donor，and could form a hydrogen bond with the extra oxygen of primary alcohol. To a large extent，whether the substrate molecule could form a hydrogen bond with Tyr29 of PcL decided the lipase’s enantioselectivity. The mechanism of enantioselectivity towards chiral primary alcohols via study of chemical modification of PcL was proposed.

Key words: chemical modification, chiral primary alcohol, enantioselectivity, molecular dynamics, Pseudomonas cepacia lipase

摘要： 洋葱假单胞菌脂肪酶（PcL）催化多种手性伯醇酯的不对称水解反应显示其对结构中含有额外的非酯键氧原子的手性伯醇酯的对映体选择性较高，而对不含额外氧原子的选择性差。分别用多种氨基酸残基特异性的修饰剂修饰 PcL，结果发现经 N-乙酰咪唑（NAI）修饰酪氨酸残基（Tyr）后 PcL 对含额外非酯键氧原子的手性伯醇酯对映体选择性显著降低，而对不含额外氧的伯醇酯选择性几乎不受影响。蛋白质谱证实产生主要影响的是位于 PcL 活性口袋内的 Tyr29 残基，手性伯醇酯能否与 Tyr29 形成额外氢键决定了 PcL对其选择性。分子动力学模拟表明，Tyr29被修饰的 PcL 不能与底物形成氢键，因此选择性明显下降。这一发现成功揭示了脂肪酶对手性伯醇对映体选择性的分子识别机理。

关键词: 化学修饰, 手性伯醇, 对映体选择性, 分子动力学模拟, 洋葱假单胞菌脂肪酶