Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
Sci Total Environ. 2021 Sep 1;785:147260. doi: 10.1016/j.scitotenv.2021.147260. Epub 2021 Apr 21.
A novel carboxylesterase gene estyz5 was isolated from a soil metagenomic library. The recombinant enzyme EstYZ5 is 298 amino acids in length with a predicted molecular weight of 32 kDa. Sequence alignment and phylogenetic analysis revealed that EstYZ5 belongs to the hormone-sensitive lipase (HSL) family with a deduced catalytic triad of Ser144-Glu238-His268. EstYZ5 contains two conserved motifs, a pentapeptide motif GDSAG and a HGGG motif, which are typically found in members of the HSL family. Esterolytic activity of the recombinant enzyme was optimal at 30 °C and pH 8.0, and the kcat/Km value of the enzyme for the optimum substrate p-nitrophenyl butyrate was as high as 1272 mM·s. Importantly, EstYZ5 showed activity toward di(2-ethylhexyl) phthalate with complex side chains, which is rare for HSLs. Molecular docking simulations revealed that the catalytic triad and an oxyanion hole likely play vital roles in enzymatic activity and specificity. The phthalate-degrading activity of EstYZ5, combined with its high levels of esterolytic activity, render this new enzyme a candidate for biotechnological applications.
从土壤宏基因组文库中分离到一个新型羧酸酯酶基因 estyz5。重组酶 EstYZ5 由 298 个氨基酸组成，预测分子量为 32 kDa。序列比对和系统发育分析表明，EstYZ5 属于激素敏感脂肪酶 (HSL) 家族，具有 Ser144-Glu238-His268 推导的催化三联体。EstYZ5 包含两个保守基序，一个五肽基序 GDSAG 和一个 HGGG 基序，这通常存在于 HSL 家族成员中。重组酶的酯酶活性在 30°C 和 pH 8.0 时最佳，酶对最佳底物对硝基苯丁酸的 kcat/Km 值高达 1272 mM·s。重要的是，EstYZ5 对具有复杂侧链的邻苯二甲酸二(2-乙基己基)酯表现出活性，这在 HSL 中很少见。分子对接模拟表明，催化三联体和氧阴离子穴可能在酶活性和特异性中发挥重要作用。EstYZ5 的邻苯二甲酸酯降解活性，加上其高酯酶活性，使其成为生物技术应用的候选酶。
