Enzymatic synthesis of high-molecular-mass poly-γ-glutamate and regulation of its stereochemistry
Enzymatic synthesis of high-molecular-mass poly-γ-glutamate and regulation of its stereochemistry
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DOI：
10.1128/aem.70.7.4249-4255.2004
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发表时间：
2004-07-01
影响因子：
4.4
通讯作者：
Misono, H
中科院分区：
文献类型：
--
作者：
Ashiuchi, M;Shimanouchi, K;Misono, H
首次利用枯草芽孢杆菌的酶相关细胞膜，成功地在体外合成了大分子质量(~gt；1000 kDa)的聚谷氨酸(PGA)。郑国江。然而，在胶束中测试的洗涤剂的临界浓度存在时，PGA合成的活性很容易丧失。该反应的最适pH与7.0相近。我们考察了一些二价阳离子对PGA合成的影响，发现镁离子是催化所必需的，锌离子还能促进PGA的合成。而Fe~(2+)和Ca~(2+)则起抑制作用。Mn2+对PGA的体外形成无明显影响。D-谷氨酸(D异构体含量为60%~80%)为最佳底物，D-谷氨酸优于L异构体为底物。当用D-谷氨酸和L-谷氨酸进行反应时，PGA的伸长链分别由D-和L-异构体组成。我们的结果表明，酶法合成的PGAs的立体化学性质很大程度上取决于谷氨酸作为底物的立体化学(DL比)。此外，遗传分析表明，所有负责枯草杆菌细胞产生PGA的pgsB、-C和-A基因产物也是酶促合成PGA所必需的。
For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (> 1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookiang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be similar to7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg2+ was essential in catalysis and that Zn2+ additionally boosted the activity. In contrast, Fe2+ and Ca2+ acted as inhibitors. Mn2+ did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; D-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.