Ashiuchi M, Tani K, Soda K, Misono H
Research Institute of Molecular Genetics Kansai University, Suita, Osaka, 564-0073, Japan.
J Biochem. 1998 Jun;123(6):1156-63. doi: 10.1093/oxfordjournals.jbchem.a022055.
We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.
我们在枯草芽孢杆菌IFO 3336的细胞提取物中发现了谷氨酸消旋酶活性，该菌株能大量产生聚γ-谷氨酸。在生长的稳定期早期获得了最高活性。该消旋酶被纯化至同质。该酶是一种分子量约为30 kDa的单体，不需要辅因子。它几乎只催化谷氨酸的消旋作用；其他氨基酸，包括丙氨酸和天冬氨酸，但不包括高半胱氨酸亚磺酸盐，作为底物或抑制剂均无活性。尽管该酶对L-谷氨酸的Vmax值比对D-谷氨酸高21倍，但L-谷氨酸的Vmax/Km值几乎与D-对映体相等。消旋酶基因glr被克隆到大肠杆菌细胞中并进行了测序。通过将glr基因的起始密码子TTG替换为ATG，该消旋酶在大肠杆菌克隆细胞的可溶部分中过量产生。在枯草芽孢杆菌IFO 3336细胞中未检测到D-氨基酸转氨酶活性。枯草芽孢杆菌CU741是枯草芽孢杆菌168的leuC7衍生物，其谷氨酸消旋酶活性和聚γ-谷氨酸的生产率均低于枯草芽孢杆菌IFO 3336。这些结果表明，谷氨酸消旋酶主要参与枯草芽孢杆菌IFO 3336中聚γ-谷氨酸生产所需的D-谷氨酸合成。
