Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production
Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production
复制标题
DOI：
10.1016/j.enzmictec.2005.08.029
复制
发表时间：
2006-05-02
影响因子：
3.4
通讯作者：
Shimizu, S
中科院分区：
文献类型：
--
作者：
Kataoka, M;Hoshino-Hasegawa, A;Shimizu, S
对马其顿假丝酵母AKU 4588的NADPH依赖性甲萘醌还原酶基因进行了克隆和测序。根据N-末端和内部氨基酸序列的一致性，确认1035 bp的核苷酸片段（mer）是编码该酶的基因。该片段编码345个氨基酸残基，与来自德巴利酵母、假丝酵母和酵母的假定蛋白以及来自接合酵母的酮还原酶的氨基酸序列具有高度的相似性。其N-末端区域包含NADPH结合基序GXXGXXA。这些发现表明，该酶属于二氢黄酮醇-4-还原酶超家族。构建了含有全长mer的表达载体pETMER。在T7启动子的调控下，大肠杆菌pETMER的甲萘醌还原活性比C. Macedoniensis的不对称还原反应合成（S）-4-氯-3-羟基丁酸乙酯（CHBE）。研究了共表达mer和葡萄糖脱氢酶基因的大肠杆菌细胞作为催化剂。形成的（S）-CHBE达到1680 mM（281 mg/ml），摩尔产率为92.2%。产物的光学纯度为（S）-异构体的91.6%对映体过量。加入到形成的CHBE中的NADP（+）的计算周转数为12，900 mol/mol。(c)2005年爱思唯尔公司All rights reserved.
The gene encoding an NADPH-dependent menadione reductase of Candida macedoniensis AKU4588 was cloned and sequenced. A 1035 by nucleotide fragment (mer) was confirmed to be the gene encoding the enzyme based on the agreement of N-terminal and internal amino acid sequences. The mer encodes 345 amino acid residues, and the deduced amino acid sequence shows high similarity with those of hypothetical proteins from Debaryomyces, Candida and Saccharomyces, and ketoreductase from Zygosaccharomyces. It includes NADPH-binding motif GXXGXXA in its N-terminal region. These findings suggest that the enzyme belongs to the dihydroflavonol-4-reductase superfamily. An expression vector, pETMER, which contains the full length of the mer, was constructed. Escherichia coli cells harboring pETMER exhibits a 127-fold increase in specific menadione-reducing activity under the control of T7 promoter as compared with that of C. macedoniensis.The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to (S)-4-chloro-3-hydroxybutanoate ethyl ester (CHBE) with E. coli cells, in which both the mer and the glucose dehydrogenase gene were co-expressed, as a catalyst was investigated. The (S)-CHBE formed amounted to 1680 mM (281 mg/ml), the molar yield being 92.2%. The optical purity of the product was 91.6% enantiomeric excess for the (S)-isomer. The calculated turnover number of NADP(+) added to CHBE formed was 12,900 mol/mol. (c) 2005 Elsevier Inc. All rights reserved.