GDSL酯酶的定向进化与分子改造研究

Cephalosporins are the most widely employed antibiotics in clinical practice for the treatment of bacterial infection which used 7-Aminocephalosporaninc acid (7-ACA) as the primary precursor for synthesis. In recent years, development of new antibiotics is required due to the emergence of bacterial resistance and other problems. Deacetylation of 7-ACA at position 3 can form hydroxyl which can be used to transform various functional groups to synthesize new cephalosporins products. Deacetylases are needed in this process. In our previous work, we found that GDSL esterases from thermophiles have deacetylase activity. However, the similarities between the enzymes in our work and other esterases with the same functions are very low. So, they are deserved to be further studied. In this study, directed evolution will be applied for the modification of GDSL esterases to improve catalytic activity and substrate specificity. Meantime, combined with protein structure and site-directed mutagenesis, the catalytic mechanism will be intensively determined. This work not only can help better understand the catalytic mechanism of GDSL esterases but also can provide theoretical basis for the acquisition of industrial enzymes with higher catalytic activities.

头孢类抗生素是目前临床治疗细菌感染最常见的药物，主要以7-氨基头孢烷酸（7-Aminocephalosporanic acid, 7-ACA）为母体合成。近年来，随着细菌耐药性等问题的出现，开发新型抗生素成为趋势。7-ACA在3位上脱乙酰化形成羟基，能转化各种官能团，合成新型头孢类产品，此过程需要脱乙酰化酶参与完成。申请人在前期研究中发现来源于嗜热微生物的GDSL酯酶对7-ACA具有脱乙酰化作用，但与现有报道的具有此功能的所有酯酶有很低序列相似性，具有研究挖掘的潜力。课题拟采用定向进化策略对GDSL酯酶进行改造，提高酶催化活力、改善底物特异性。同时，对GDSL酯酶进行结构解析，并结合定点突变技术，解析其结构和功能关系及催化机理。该工作可以加深对此类酶催化机制的理解，为获得更高活力的工业酶奠定理论基础。

头孢类抗生素是目前临床治疗细菌感染最常见的药物，主要以7-氨基头孢烷酸（7-Aminocephalosporanic acid，7-ACA)为母体合成。近年来，随着细菌耐药性等问题的出现，开发新型抗生素成为趋势。7-ACA在3位上脱乙酰化形成羟基，能转化各种官能团，合成新型头孢类产品，此过程需要脱乙酰化酶参与完成。前期研究中，我们鉴定到了两个来源于GDSL家族的酯酶：Est8和EstD1，二者均具有7-ACA脱乙酰化活性，但活性较低。通过本项目的实施，我们利用定向进化-易错PCR手段，获得了一株对7-ACA脱乙酰化活性提高了2.7倍的突变子Est8-G45R（Est8第45位氨基酸由甘氨酸突变为精氨酸）；利用悬滴蒸汽扩散法得到了Est8和EstD1的蛋白晶体，经X-Ray衍射后分别收集到了2.3 Å和2.5 Å的衍射数据，解析了Est8和EstD1的蛋白三维结构；结合对Est8第45位氨基酸的定点突变及Est8的蛋白三维结构，解析了Est8第45位氨基酸的带电性与酶活性存在相关性，且带正电荷氨基酸显著提高酶活性，初步阐述了酯酶Est8与底物7-ACA结合的作用机制。通过该项目的实施，加深了对GDSL家族酯酶催化机制的理解，并为获得高活力的工业酶奠定了理论基础。

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