来自酮还原短杆菌ATCC21914的2-酮糖酸还原酶的纯化与特性分析

Purification and characterization of the 2-ketoaldonate reductase from Brevibacterium ketosoreductum ATCC21914.

作者信息

Yum D Y, Bae S S, Pan J G

机构信息

Bioprocess Engineering Division, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon, Korea.

出版信息

Biosci Biotechnol Biochem. 1998 Jan;62(1):154-6. doi: 10.1271/bbb.62.154.

DOI:10.1271/bbb.62.154

PMID:9501529

Abstract

2-Ketoaldonate reductase, which is involved in ketogluconate catabolism, was purified to homogeneity from Brevibacterium ketosoreductum ATCC21914. The enzyme was found to catalyze the reduction of 2,5-diketo-D-gluconate to 5-keto-D-gluconate, and to a lesser extent, 2-keto-D-gluconate to D-gluconate, and 2-keto-L-gluconate to L-idonate. The molecular mass of the reductase was 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 72 kDa by gel filtration, indicating that the native enzyme may exist as a dimer. The reductase was optimally active at pH 6.0 with NADPH as a preferred electron donor. The pI of 4.7 was measured for the enzyme. The apparent Km for 2,5-diketo-D-gluconate and NADPH were 5 microM and 10 microM, respectively. The amino-terminal amino acid sequence was NH2-Ala-Ser-Ile-Ser-Val-Ser-Val-Pro-Ser-Ala- Arg-Leu-Ala-Glu-Asp-Leu-Ser-Asp-Ile-Glu.

摘要

参与酮葡糖酸分解代谢的2-酮醛糖还原酶，从酮还原短杆菌ATCC21914中纯化至同质。发现该酶催化2,5-二酮-D-葡糖酸还原为5-酮-D-葡糖酸，在较小程度上催化2-酮-D-葡糖酸还原为D-葡糖酸，以及2-酮-L-葡糖酸还原为L-艾杜糖酸。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，还原酶的分子量为35 kDa，通过凝胶过滤测定为72 kDa，表明天然酶可能以二聚体形式存在。以NADPH作为首选电子供体时，还原酶在pH 6.0时具有最佳活性。测得该酶的pI为4.7。2,5-二酮-D-葡糖酸和NADPH的表观Km分别为5 microM和10 microM。氨基末端氨基酸序列为NH2-Ala-Ser-Ile-Ser-Val-Ser-Val-Pro-Ser-Ala-Arg-Leu-Ala-Glu-Asp-Leu-Ser-Asp-Ile-Glu。