Study Of Glutamate Dehydrogenase And Poly-γ-Glutamic Acid Hydrolase In Bacillus Licheniformis

Poly-γ-glutamic acid (γ-PGA in short) is a multi-functional biopolymers, which is featured by its unique biological characteristics of edible、non-toxic and biodegradable. Bacillus licheniformis WX-02(WX-02in short) is a kind of poly-γ-glutamic acid producing strain relying on adding extracellular glutamate. In the process of producing poly-γ-glutamic acid, much glutamic acid is needed, which increases the cost of fermentation, and is not propitious to the industrial development of poly-γ-glutamic acid. At present,the function of the essential enzymeofpoly-γ-glutamic acidbiosynthesisin WX-02remains unknown.Based on the metabolic pathways,the characterization of key enzyme, intracellular glutamic acidbiosynthesisand γ-PGA degradationof Bacillus licheniformis WX-02were analyzied. Besides, HPLC, GC-MS, and Q-RT-PCR are also applied to the constructed engineering strains to examine their specific physiological and biochemical changes.The major findings of this research are listed as follows:1.Three enzymes, Glutamine synthetase, glutamate synthase and glutamate dehydrogenase (encoded by glnA, gltAB and rocG or gudB genes), are involved in intracellular glutamic acid biosynthesis. The WX-02ΔrocG and WK-02ΔgltA strains were obtained through knockout of rocG and gltA gene respectively. And the WX-02P43rocG was obtained by enhancing rocG gene in WX-02. Transcription of rocG and gltA were not detected in WX-02ΔrocG, WK-02ΔgltA respectively. Transcription of orcocG was1.54times higher in WX-02P43rocG than that in WX-02through Q-RT-PCR. Transcription of gudB was not detected in WX-02. Transcription of gudB was4.72times higher in WX-02ΔrocGthan that in WX-02ΔgltA. The γ-PGA yield was9.82g/L of WX-02in culture without external glutamate added. The γ-PGA productivity of WX-02ArocG and WX-02P43rocG were54.70%and105%, compared with WX-02. The difference between γ-PGA yield of WX-02ΔgltA and WX-02was not significant. It was implied that glutamate dehydrogenase was the major enzyme which was responsible for glutamic acid biosynthesis.2.The rocG gene was cloned based on B. licheniformis WX-02genome and was inserted into pET-28(+) vector. Recombination vector pET-28b(+)-rocG was transformed into E. coli BL21(DE3). The RocG was obtained after inducing by IPTG and purificating. Optimal conditions for activity were pH8.0and a temperature of40℃and RocG was stable in buffers ranging from pH6.0to8.5and a temperature below40℃. Km for substrate a-ketoglutarate, NADPH and glutamate were4.727mmol/L,0.111mmol/L, and23.296mmol/L. The Kcat/Km of a-ketoglutarate, NADPH and glutamate were1.923mmol-1?L?min-l,100.09mmol-1?L?min-1, and0.159mmol-1?L?min-1. It was noteworthy that catalytic reaction efficiency of glutamate synthesis was higher than that in reverse reaction. It was certified that glutamate dehydrogenase was responsible for glutamic acid biosynthesis in vitro.3.Glyoxylate cycle can partly complement TCA and thus increase intracellular glutamic acid biosynthesis. There were two key enzymes of isocitrate lyase encoded by aceA gene and malate synthase encoded aceB gene in glyoxylate cycle. WX-02AaceA and WX-02P43aceA were obtained through knockout and enhancing aceA gene based on WX-02respectively. Transcription of aceA was not detected in WX-02AaceA. Transcription of aceA and aceB were3.6and2.8times higher than that in WX-02. The y-PGA yield of WX-02PA3aceA and WX-02AaceA were115%and66.8%of WX-02respectively. It was implied that the complement of glyoxylate cycle was required in intracellular glutamic acid biosynthesis and y-PGA productivity can be improved through enhancing glyoxylate cycle. Transcription of aceA decreased by14%and28%after glyoxylate cycle inhibiter malic acid or succinate added, whereas culture without external glutamate and y-PGA yield increased by13.4%and16.5%respectively. It was noted that the metabolism from a-ketoglutarate to glutamic acid was enhanced to improve y-PGA production, although malic acid or succinate inhibit glyoxylate cycle inhibiter were added, which is due to the increasing additive of the intermediate of TCA.4.The molecular weight of poly-γ-glutamic acid can be decreased through degradation of poly-γ-glutamic acid hydrolase. WX-02ΔpgdS was constructed by knockout of pgdS gene based on WX-02and WX-02/pHYpgdS, which was obtained through enhancing pgdS gene with WX-02/pHY as CK. The relative molecular weight of WX-02ΔpgdS was the highest with GPC detection. The relative molecular weight of WX-02/pHYpgdS was lower than WX-02/pHY with same results compared with WX-02. It was implied that molecular weight of poly-γ-glutamic acid can be decreased by poly-γ-glutamic acid hydrolase. There was no difference of γ-PGA yield between WX-02and WX-02/pHY. The γ-PGA productivity of WX-02ApgdS decreased to83%in WX-02. However, the γ-PGA productivity from WX-02/pHYpgdS improved54%compared with WX-02/pHY. There was no transcription of pgdS in WX-02ΔpgdS and transcription of gltT decreased to86%compared with WX-02. The transcription of pgdS and gltT was 9.86and1.8times higher in WX-02/pHYpgdS than that in WX-02/pHY. It was certified that there was certain connection between PgdS and GltT which changed intracellular glutamic acid concentration and γ-PGA yield.