Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Appl Microbiol Biotechnol. 2012 Feb;93(4):1619-25. doi: 10.1007/s00253-011-3664-x. Epub 2011 Nov 15.
Ketopantoic acid (KPA) reductase catalyzes the stereospecific reduction of ketopantoic acid to D-pantoic acid. Based on the N-terminal amino acid sequence of KPA reductase from Stenotrophomonas maltophilia 845, the KPA reductase gene was cloned from S. maltophilia NBRC14161 and sequenced. This gene contains an open reading frame of 777 bp encoding 258 amino acid residues, and the deduced amino acid sequence showed high similarity to the SDR superfamily proteins. An expression vector, pETSmKPR, containing the full KPA reductase gene was constructed and introduced into Escherichia coli BL21 (DE3) to overexpress the enzyme. Bioreduction of KPA using E. coli transformant cells coexpressing KPA reductase together with cofactor regeneration enzyme gene was also performed. The conversion yield of KPA to D-pantoic acid reached over 88% with a substrate concentration up to 1.17 M.
酮戊二酸（KPA）还原酶催化酮戊二酸的立体特异性还原为 D-泛酸。基于嗜麦芽寡养单胞菌 845 的 KPA 还原酶的 N 末端氨基酸序列，从嗜麦芽寡养单胞菌 NBRC14161 中克隆了 KPA 还原酶基因并进行了测序。该基因包含一个 777bp 的开放阅读框，编码 258 个氨基酸残基，推导的氨基酸序列与 SDR 超家族蛋白具有高度相似性。构建了一个包含完整 KPA 还原酶基因的表达载体 pETSmKPR，并将其导入大肠杆菌 BL21（DE3）中以过表达该酶。还使用共表达 KPA 还原酶和辅因子再生酶基因的大肠杆菌转化细胞进行了 KPA 的生物还原。当底物浓度高达 1.17 M 时，KPA 到 D-泛酸的转化率超过 88%。
