Biochemical characterization of an acetylesterase from Bacillus subtilis and its application for 7-aminocephalosporanic acid deacetylation.
Biochemical characterization of an acetylesterase from Bacillus subtilis and its application for 7-aminocephalosporanic acid deacetylation.
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枯草芽孢杆菌的乙酰酯酶的生化表征及其在7-氨基孢子孢酸脱乙酰基化中的应用。
DOI：
10.3389/fmicb.2023.1164815
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发表时间：
2023
影响因子：
5.2
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中科院分区：
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脱乙酰7-氨基头孢菌酸(D-7-ACA)可由7-氨基头孢菌酸(7-ACA)转化而来，是用于合成工业半合成β-内酰胺抗生素的重要起始原料。参与从 7-ACA 转化为 D-7-ACA 的酶是制药行业的关键资源。在本研究中，从枯草芽孢杆菌 KATMIRA1933 中鉴定出的推定乙酰酯酶 EstSJ 首次在大肠杆菌 BL21(DE3) 细胞中异源表达并进行了生化表征。 EstSJ 属于碳水化合物酯酶家族 12，对 p-NPC2 至 p-NPC6 的短链酰基酯具有活性。多重序列比对显示EstSJ也是SGNH家族酯酶，其N末端具有典型的GDS(X)基序和由Ser186-Asp354-His357组成的催化三联体。纯化的 EstSJ 在 30°C、pH 8.0 时表现出最高的比活性，为 1,783.52 U mg–1，并且在 pH 值 5.0–11.0 范围内稳定。 EstSJ可使7-ACA的C3′乙酰基脱乙酰生成D-7-ACA，脱乙酰活性为4.50 U mg–1。基于与 7-ACA 的结构和分子对接，揭示了 EstSJ 的催化活性位点（Ser186-Asp354-His357）以及四个底物结合残基（Asn259、Arg295、Thr355 和 Leu356）。这项研究提供了一种有前途的 7-ACA 脱乙酰酶候选物，可用于制药行业从 7-ACA 生产 D-7-ACA。
Deacetyl-7-aminocephalosporanic acid (D-7-ACA), which could be converted from 7-aminocephalosporanic acid (7-ACA), is a crucial starting material that is used for synthesizing industrial semisynthetic β-lactam antibiotics. Enzymes involved in the conversion from 7-ACA to D-7-ACA present critical resources in the pharmaceutical industry. In the present study, a putative acetylesterase, EstSJ, identified from Bacillus subtilis KATMIRA1933, was first heterologously expressed in Escherichia coli BL21(DE3) cells and biochemically characterized. EstSJ belongs to carbohydrate esterase family 12 and is active on short-chain acyl esters from p-NPC2 to p-NPC6. Multiple sequence alignments showed that EstSJ was also an SGNH family esterase with a typical GDS(X) motif at its N-terminal end and a catalytic triad composed of Ser186-Asp354-His357. The purified EstSJ displayed the highest specific activity of 1,783.52 U mg–1 at 30°C and pH 8.0, and was stable within the pH range of 5.0–11.0. EstSJ can deacetylate the C3′ acetyl group of 7-ACA to generate D-7-ACA, and the deacetylation activity was 4.50 U mg–1. Based on the structural and molecular docking with 7-ACA, the catalytic active sites (Ser186-Asp354-His357) together with four substrate-binding residues (Asn259, Arg295, Thr355, and Leu356) of EstSJ are revealed. This study provided a promising 7-ACA deacetylase candidate that could be applied to produce D-7-ACA from 7-ACA in the pharmaceutical industry.