Bacillus subtilis Cl, a bacterium capable of producing a glycerol and gamma-PGA bioconjugate, was studied. C1 is a glutamate independent bacterium, which produces the gamma-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 (M) of the bioconjugate was over 1 x 107 as determined by gel permeation chromatography, amino acid analysis showed only glutamic acid peak and the H-1 NMR spectrum showed chemical shifts of both gamma-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 x 10(6) to 0.73 x 10(6) 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 gamma-PGA itself. This bioconjugate is the first example of gamma-PGA derivative directly produced by microbes; thus, it is a unique biomaterial. (c) 2005 Elsevier Ltd. All rights reserved.