Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis
Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis
复制标题
嗜热细菌腾冲脂环酸芽孢杆菌新7-氨基头孢烷酸脱乙酰酶的鉴定和表征
DOI：
10.1128/jb.00471-15
复制
发表时间：
2016
影响因子：
3.2
通讯作者：
Huang Zun-Xi
中科院分区：
文献类型：
--
作者：
Ding Jun-Mei;Yu Ting-Ting;Han Nan-Yu;Yu Jia-Lin;Li Jun-Jun;Yang Yun-Juan;Tang Xiang-Hua;Xu Bo;Zhou Jun-Pei;Tang Hong-Zhi;Huang Zun-Xi
7-氨基头孢孢酸（7-ACA）在C-3位的去乙酰化为生产半合成β-内酰胺类抗生素提供了有价值的原料。然而，在此之前，很少有酶在这一过程中被表征。通过对嗜热细菌芽胞杆菌（aliicyclobacillus tenchongensis）基因组的比较分析，发现了一种具有典型酯酶特征的假设蛋白（EstD1）。从大肠杆菌BL21（DE3）中克隆、表达并纯化了EstD1蛋白。它确实表现出酯酶活性，在65°C和pH 8.5左右具有最佳活性，并优先选择具有短链酰基酯的酯（c2至C4）。测序结果表明，EstD1是一种SGNH水解酶，具有推测的催化三联体Ser15、Asp191和His194，属于糖类酯酶家族12。EstD1能水解7-氨基头孢孢酸（7-ACA） C-3位的乙酸，生成去乙酰基-7-ACA，是生产半合成β-内酰胺类抗生素的重要原料。当pH值为4 - 11，温度为65°C，孵育1小时时，EstD1保留了超过50%的初始活性。据我们所知，该酶是从嗜热菌中鉴定出的一种新的SGNH水解酶，能够水解7-ACA。去乙酰类头孢菌素是工业生产各种半合成β-内酰胺类抗生素的重要组成部分。这些化合物主要来源于7-ACA， 7-ACA是由头孢菌素c通过化学或酶促过程获得的。由于化学去酰化对环境的不利影响，7-ACA的酶转化是主要的方法。SGNH水解酶广泛存在于植物中。然而，用于鉴定和表征细菌，特别是嗜热菌中SGNH水解酶的工具相当有限。在这里，我们的工作表明EstD1属于SGNH家族，可以在7-ACA的C-3位置水解乙酸。此外，本研究可以丰富我们对该家族这些酶的功能的认识。
Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA.IMPORTANCEDeacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.