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。
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 Tyr^29 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 Tyr^29 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 Tyr^29 of PcL decided the lipase’s enantioselectivity. The mechanism of enantioselectivity towards chiral primary alcohols via study of chemical modification of PcL was proposed.
出处
《化工进展》
EI
CAS
CSCD
北大核心
2013年第11期2695-2700,2706,共7页
Chemical Industry and Engineering Progress
基金
国家973计划(2011CB710800)
国家863计划(2011AA02A209)
国家自然科学基金(20936002)
浙江省自然科学基金(2010C31127)项目
