文献类型：期刊文献

中文题名：短链脱氢酶Lvchun的分子改造及其在氯霉胺合成中的应用

英文题名：Molecular Modification of the Short-chain Dehydrogenase Lvchun and Its Application in the Synthesis of Chloromycetin

作者：韩惠[1];张舰[1];任宇红[1]

机构：[1]华东理工大学生物工程学院生物反应器工程国家重点实验室,上海200237

年份：2023

卷号：39

期号：4

起止页码：81

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

语种：中文

中文关键词：氯霉胺;短链脱氢酶;分子改造;定点突变;甲酸脱氢酶;双酶共表达

外文关键词：chloromycetin;short-chain dehydrogenase;molecular modification;site-specific mutagenesis;formic acid dehydrogenase;dual enzyme co-expression

摘要：2-氨基-1-(4-硝基苯基)-1,3-丙二醇俗称氯霉胺(ANP),由于具有两个手性中心,且结构中的O原子和N原子有良好的配位能力,具有广泛的应用价值。针对化学合成法存在生产成本高、原子经济性低、环保压力大等诸多缺点,旨在通过化学水解法与生物催化法相结合的方式,构建以对硝基-α-乙酰氨基-β-羟基苯丙酮(p-NAH)为底物合成(1R)-ANP的新途径。首先采用化学法水解p-NAH制备1-(4-硝基苯基)-2-氨基-3-羟基苯丙酮(AHNA),并筛选对水解产物具有催化活性的羰基还原酶,通过分子改造提高该酶的催化活性,并对突变体mut-V112Y的酶学性质进行研究;然后构建mut-V112Y与甲酸脱氢酶的双酶共表达及融合表达重组菌株,并对重组菌株的催化效率进行比较;最后优化催化反应条件,并进行制备反应。结果表明,化学法可水解p-NAH生成AHNA,筛选到的短链脱氢酶Lvchun可催化AHNA生成(1R)-ANP,通过对该酶进行定点突变获得了催化效率提高3.47倍的突变体mut-V112Y,其最适温度为30℃,最适pH为7.5,具有良好的温度和pH稳定性。成功构建了mut-V112Y和甲酸脱氢酶CbFDH的双酶共表达和融合表达重组菌株,通过比较发现共表达菌株mut-V112Y-CbFDH的催化效率最高。通过优化催化反应条件,最终可在最适条件下反应30 min催化50 mmol/L AHNA生成14.56 mmol/L(1R)-ANP,收率为29.12%。化学水解法与生物催化法相结合的方式可有效地催化p-NAH合成(1R)-ANP,该方法为合成光学纯的ANP提供了新途径。
2-amino-1-(4-nitrophenyl)-1,3-propanediol,commonly known as chloromycetin(ANP),has a wide range of applications due to its two chiral centers and the good coordination ability of the O and N atoms in its structure.For responding to the many drawbacks of chemical synthesis such as high production cost,low atomic economy and high environmental pressure,here we aim to construct a new pathway for the synthesis of(1R)-ANP using p-nitro-α-acetamido-β-hydroxypropiophenone(p-NAH)as a substrate by combining chemical hydrolysis with biocatalysis.Firstly,1-(4-nitrophenyl)-2-amino-3-hydroxypropiophenone(AHNA)was prepared by chemical hydrolysis of p-NAH,and a carbonyl reductase with catalytic activity for the hydrolysis product was screened.The catalytic activity of the enzyme was improved by molecular modification,and the enzymatic properties of a mutant mut-V112Y were investigated.Then,a dual enzyme co-expression or fusion expression recombinant strain containing mut-V112Y and formic acid dehydrogenase was constructed,and the catalytic efficiencies of the recombinant strains were verified.Finally,the catalytic reaction conditions were optimized and the preparative reactions were performed.The results showed that the chemical method can be used to hydrolyze p-NAH to AHNA,the screened short-chain dehydrogenase Lvchun calatyzed AHNA to(1R)-ANP,and a mutant mut-V112Y with 3.47-fold increase in catalytic efficiency was obtained by targeted mutation of carbonyl reductase, which presented good temperature and pH stability with an optimal temperature of 30℃ and an optimal pH of 7.5. A dual enzyme coexpressionand fusion expression recombinant strain was successfully constructed, and the highest catalytic efficiency was found in strain mut-V112Y-CbFDH. The catalytic reaction conditions were optimized, 14.56 mmol/L(1R)-ANP was produced from 50 mmol/L AHNA in 30 minunder optimal conditions, with a yield of 29.12%. The combination of chemical hydrolysis and biocatalysis can effectively catalyze the synthesisof(1R)-ANP from p-NAH, which provides a new way to synthesize optically pure ANP.