Synthesis of super-high-molecular-weight poly-γ-glutamic acid by Bacillus subtilis subsp. chungkookjang

Poly-γ-glutamic acid (PGA) with high molecular weight is a most promising biomaterial in industrial uses; however, it generally diverse in molecular structure and co-produced with polysaccharides and various other biopolymers. In this study, it was ascertained that Bacillus subtilis subsp. chungkookjang cells are superior to B. subtilis (natto) cells as the biocatalyst for the synthesis of super-high-molecular-weight PGA (over 2000 k). We effectively purified PGA and fractionated according to its molecular weight by anion-exchange chromatography, and further developed a simple method for determination of the molecular weight of PGA on the basis of numbers of glutamate monomers generated by hydrolysis and a free amino group quantified with 1-fluoro-2,4-dinitrobenzene (FDNB). The molecular weight determination with FDNB was available even for a super-high-molecular-weight PGA, e.g. the 2000-k polymer. Super-high-molecular-weight PGAs (average 2000 k and 7000 k), which were synthesized by the use of B. subtilis subsp. chungkookjang cells in the presence of a high concentration of ammonium sulfate, were rich in l-glutamate rather than in the d-enantiomer.