利用结构多样性的对硝基苯酚羧酸酯和脂肪醇乙酸酯评估7个枯草芽胞杆菌酯水解酶的指纹图谱

基于GenBank公布的枯草芽胞杆菌168基因组序列,克隆表达了30个预测的酯水解酶基因。结果发现:其中7个酶对对硝基苯酚酯表现出明显的酯水解活力。它们在α/β水解酶家族中分属5个不同的亚家族。通过显色底物和pH指示剂进行的高通量筛选,分别绘制了这7个酶的底物指纹谱。考察了酶催化手性酯水解反应的对映选择性,结果表明:对硝基苄基酯酶PnbA和S-脱乙酰化酶Cah对手性醇的乙酸酯具有较广的底物谱,而PnbA和羧酸酯酶Nap分别对DL-薄荷醇乙酸酯和2-氯-1-苯乙醇乙酸酯/2-萘乙醇乙酸酯有极好的对映选择性(E>200)。此外,发现酯酶YitV催化2-氯-1-苯乙醇乙酸酯水解的反应遵循反-Kazlauskas规则。

Comparative evaluation of seven recombinant ester hydrolases from Bacillus subtilis 168 using structurally diverse p-nitrophenyl carboxylates and acetylated alcohols

Thirty putative hydrolase genes from Bacillus subtilis 168 were cloned and expressed based on the published genomic information in GenBank. Seven enzymes showed significant lipolytic activities towards p-nitrophenyl esters. Phylogenetic analysis revealed that these enzymes belong to five subfamilies of α/β hydrolase family. High-throughput screening methods involving chromogenic substrates and pH indictor were used to obtain activity fingerprint of these enzymes towards esters of carboxylic acids and acetates of alcohols. The enantioselectivity of these enzymes towards various ehiral substrates was also investigated and compared. It was shown that the esterase PnbA （ a para-nitrobenzyl esterase） and Cah （ an S-deacylase） accepted a broader range of acetyl esters of alcohols than the others. The enzymes PnbA and Nap （ earboxylesterase NP） exhibited excellent enantioseleetivity （ E 〉 200 ） in hydrolysis of dl- menthyl acetate 2-ehloro-l-phenethyl acetate and 2-naphthylethyl acetate. Moreover, a novel enzyme YitV was acted as an anti-Kazlauskas catalyst in hydrolyzing 2-chloro-l-phenylethyl acetate with moderate enantioselectivity.