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.

中文翻译：

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枯草芽孢杆菌乙酰酯酶的生化特性及其在 7-氨基头孢烷酸脱乙酰化中的应用

脱乙酰基-7-氨基头孢烷酸（D-7-ACA）可由7-氨基头孢烷酸（7-ACA）转化而来，是合成工业半合成β-内酰胺类抗生素的重要起始原料。参与从 7-ACA 到 D-7-ACA 转化的酶是制药行业的关键资源。在本研究中，推定的乙酰酯酶 EstSJ 从枯草杆菌KATMIRA1933，首先异源表达于大肠杆菌BL21(DE3) 细胞和生化特征。EstSJ 属于碳水化合物酯酶家族 12，对来自的短链酰基酯具有活性p-NPC2个到p-NPC6个. 多序列比对表明 EstSJ 也是一个 SGNH 家族酯酶，在其 N 末端具有典型的 GDS(X) 基序和由 Ser 组成的催化三联体186-天冬氨酸354-他的357. 纯化的 EstSJ 显示出最高的比活性 1,783.52 U mg–1在 30°C 和 pH 8.0 下，在 5.0–11.0 的 pH 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，催化活性位点 (Ser186-天冬氨酸354-他的357) 连同四个底物结合残基 (Asn259, 精氨酸295, 苏氨酸355, 和亮氨酸356) 的 EstSJ 被揭露。该研究提供了一种有前途的 7-ACA 脱乙酰酶候选物，可用于在制药工业中从 7-ACA 生产 D-7-ACA。