The Regulatory Mechanism Of Calcium On Glutamate Dehydrogenase In Production Of Poly-γ-glutamic Acid By Bacillus Subtilis Natto

Poly-γ-glutamic acid (y-PGA) is an environment-friendly anionic biopolymer, which is comprised of L-and D-glutamic acid units connected by amide bonds between a-amino and y-carboxylic acid groups. It is water-soluble, biodegradable and edible towards human and environment. Possessing these properties, y-PGA has been utilized such as food, medicine and agriculture industries. The previous studies found that the glutamate dehydrogenase (GDH) activity was significantly improved with addition calcium ions into the fermentation broth of y-PGA by Bacillus subtilis natto, so that more a-ketoglutarate was synthesized to L-glutamic, and more y-PGA was formed.Using the regulatory mechanism of glutamic acid substrates as the breach, this study break through the previously limited studies on the y-PGA synthesis by the regulation of y-PGA synthetase. This project clarified that the-regulating mechanism of calcium on glutamate dehydrogenase, and provided theoretical and experimental evidence for the synthesis mechanism of y-PGA. Further more, it provide reference for artificial control in the production of γ-PGA. It is expected y-PGA would be synthesized in large quantities and play a significant role in the adsorption material, food ingredients, pharmaceutical engineering fields.The main results were as follows:(1) In this paper, the Bacillus subtilis natto gdhA glutamate dehydrogenase gene was cloned, recombined to prokaryotic expression vector pET28a(+), and expressed by E. coli BL21(DE3) successfully. The SDS-PAGE electrophoresis was used to identify this protein and its coding genes.luGlutamate dehydrogenase activities changed little while calcium of different concentrations were added in vitro. The result showed that there was no direct activation of calcium on GDH. On the other hand, the transcription level of gdhA gene was measured by real-time quantitative PCR while calcium of different concentrations were added in the culture during fermentation process. The results showed that with the calcium concentration increasing, the expression abundance of gdhA gene also increased. Glutamate dehydrogenase activities data verified the result. It was indicated that calcium can regulate the expression level of glutamate dehydrogenase in Bacillus subtilis natto, and its expression level increase with calcium concentration increasing.(3) OD600of bacterium solution and y-PGA yield were determined after fermentation of B. subtilis natto for24h with calcium of different concentrations added. The results showed that with the increasing calcium concentration, OD600showed a downtrend and the y-PGA production increased firstly and then decreased. When the calcium concentration was0.1%0, O.y-PGA could reached the highest yield,9.68g/L. It was indicated that y-PGA yield was related to the combined effect of calcium regulation on glutamate dehydrogenase and its affect growth of bacteria.The study identified and verified gdhA glutamate dehydrogenase gene in Bacillus subtilis natto, laying the foundation of further study of the regulatory mechanism of glutamate dehydrogenase in fermentation processing of y-PGA. Meanwhile, we found that the regulation of calcium on glutamate dehydrogenase was a in vivo regulation action, and calcium concentration played a decisive role on the cell growth and y-PGA production. These detailed experimental evidence was conducive to provide human control technical strategy to further improve the level of y-PGA yield.