Wada M, Kataoka M, Kawabata H, Yasohara Y, Kizaki N, Hasegawa J, Shimizu S
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Biosci Biotechnol Biochem. 1998 Feb;62(2):280-5. doi: 10.1271/bbb.62.280.
A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced alpha-, beta-keto esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.
通过包括蓝琼脂糖亲和层析在内的四个步骤，从大孢假丝酵母AKU4643中纯化出一种依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的羰基还原酶，使其达到同质。该酶催化4-氯-3-氧代丁酸乙酯立体选择性还原为相应的（S）-醇，对映体过量率为100%，这是药物化学合成中一种有用的手性砌块。在高效凝胶过滤色谱上，该酶的相对分子质量估计为76,000，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上为32,000。除了4-氯-3-氧代丁酸乙酯外，该酶还能还原α-、β-酮酯和共轭二酮。该酶的活性受到槲皮素和氯化汞的抑制，但不受乙二胺四乙酸（EDTA）的抑制。该酶的N端氨基酸序列与其他氧化还原酶的序列没有明显相似性。
