Enzymatic Synthesis of High-Molecular-Mass Poly-γ-Glutamate and Regulation of Its Stereochemistry

Abstract

For the first time, we succeeded in synthesizing in vitro poly-γ-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang . 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 ∼7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg ²⁺ was essential in catalysis and that Zn ²⁺ additionally boosted the activity. In contrast, Fe ²⁺ and Ca ²⁺ acted as inhibitors. Mn ²⁺ 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.