肠球菌 V583 中重组 FAD 非依赖性降解乙酰乳酸合酶的特性研究。

Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.

机构信息

Department of Chemistry and Research Institute of Natural Sciences, Hanyang University, Seoul 133-791, South Korea.

出版信息

Enzyme Microb Technol. 2013 Jan 10;52(1):54-9. doi: 10.1016/j.enzmictec.2012.10.006. Epub 2012 Oct 18.

DOI:10.1016/j.enzmictec.2012.10.006

PMID:23199739

Abstract

The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60 kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent K(m) for pyruvate was calculated to be 1.37 mM, while the K(c) for thiamin diphosphate (ThDP) and Mg(2+) were found to be 0.031 μM and 1.27 mM, respectively. Negligible absorbance at 450 nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.

摘要

粪肠球菌 V583 的分解代谢乙酰乳酸合酶（cALS）被克隆、在大肠杆菌中表达并纯化至均一性。纯化的蛋白质分子量为 60 kDa。粪肠球菌的 cALS 与其他 cALS 高度同源，而与合成酶的同源性较低。粪肠球菌的 cALS 在 37°C 和 pH 6.8 的温度下表现出最佳活性。根据酶的特性，计算出丙酮酸的表观 K(m)为 1.37 mM，而硫胺素二磷酸（ThDP）和 Mg(2+)的 K(c)分别为 0.031 μM 和 1.27 mM。在测定缓冲液中加入黄素腺嘌呤二核苷酸（FAD）时几乎没有 450nm 的吸光度且没有增强活性，表明粪肠球菌的 cALS 不是依赖 FAD 的。该酶对有机溶剂二甲基亚砜（DMSO）表现出极强的稳定性，而在存在丙酮和乙醇的情况下，活性下降到 50%以下。