THERMOSTABLE ALANINE RACEMASE FROM BACILLUS-STEAROTHERMOPHILUS - MOLECULAR-CLONING OF THE GENE, ENZYME-PURIFICATION, AND CHARACTERIZATION
THERMOSTABLE ALANINE RACEMASE FROM BACILLUS-STEAROTHERMOPHILUS - MOLECULAR-CLONING OF THE GENE, ENZYME-PURIFICATION, AND CHARACTERIZATION
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DOI：
10.1021/bi00359a028
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发表时间：
1986-06-03
期刊：
影响因子：
2.9
通讯作者：
SODA, K
中科院分区：
文献类型：
--
作者：
INAGAKI, K;TANIZAWA, K;SODA, K
以嗜热脂肪芽孢杆菌的L丙氨酸脱氢酶基因为载体，克隆了嗜热脂肪芽孢杆菌的丙氨酸消旋酶基因(EC 5.1.1.1)，并在大肠杆菌C600中进行了表达。采用L-丙氨酸脱氢酶与四氮唑盐联用的方法直观地检测克隆中丙氨酸外消旋酶的活性。在携带pICR4的克隆中过量生产的丙氨酸外消旋酶，通过包括热处理在内的5个步骤，从细胞提取液中纯化了约340倍的纯度。通过与嗜热脂肪芽孢杆菌的免疫化学交叉反应，证实该高产酶来源于嗜热脂肪芽孢杆菌。纯化的酶分子量约为78000，由两个相同的亚基组成，分子量为39000。在最适温度(50℃)下，该酶的比活力为1800U/mg(V_(Max)，D-到L-丙氨酸)。拆分和重组实验以及酶的吸收光谱表明，嗜热脂肪芽孢杆菌的丙氨酸外消旋酶是一种吡哆醛5‘-磷酸酶。
The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50.degree.C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconsititution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5''-phosphate enzyme.