Microbial production of a poly(γ-glutamic acid) derivative by Bacillus subtilis

Bacillus subtilis C1, a bacterium capable of producing a glycerol and γ-PGA bioconjugate, was studied. C1 is a glutamate independent bacterium, which produces the γ-PGA derivative in the absence of l-glutamate. A large amount of the bioconjugate, 21.4 g/l, was produced when the bacteria were cultured in medium T at 37 °C, 150 rpm for 6 days. The number-average molecular weight (Mn) of the bioconjugate was over 1 × 107 as determined by gel permeation chromatography, amino acid analysis showed only glutamic acid peak and the 1H NMR spectrum showed chemical shifts of both γ-PGA and glycerol. The d-glutamate content was over 97% in every bioconjugate produced under the conditions used and the d-glutamate content was indifferent to the Mn2+ concentrations under study. During bioconjugate production, an intracellular glutamate racemase activity was detected, suggesting the enzyme is involved in the d-glutamate supply. The molecular weight of the bioconjugate varied with salt concentration in the medium; the molecular weight decreased by a factor of approximately 10 from 7.94 × 106 to 0.73 × 106 Da at 4-day cultivation time for cultures, which contained 0.05 and 5% NaCl, respectively. The viscosity of the bioconjugate produced by C1 is significantly higher than that of γ-PGA itself. This bioconjugate is the first example of γ-PGA derivative directly produced by microbes; thus, it is a unique biomaterial.