Screening Of GlmS And Gna1 For N-acetylglucosamine Productiom And Katalytic Kinetics Analysis

N-acetylglucosamine(Glc NAc), derivative of glucosamine(glucosamine, Glc N), is widely used in the field of health food and pharmaceutical. Glc NAc are currently produced by acid or enzymatic hydrolysis of chitin extracted from shrimp shells, chemical synthsis and microbial fermentation. In our previous work, we obtain the production of Glc NAc through microbial fermentation and a recombinant Bacillus subtilis strain for production of Glc NAc was constructed. In this work, to enhance the production of Glc NAc, two key enzymes,Glucosamine-6-P Synthsis(Glm S) and Glucosamine-6-P N-acetyltransferase(Gna1)in the Glc NAc synthesis pathway were optimized. Main contents of research is as follows:(1) On the basis of recombinant Glc NAc-producing B. subtilis, we overexpressed Glm S with different originations, finding that Bsglm S, originating from B. subtilis, can better accumulate Glc NAc, which enhanced Glc NAc titer to 5.86 g·L-1 in the engineered B. subtilis. Further investigation results showed that the recombinant Bsglm S has higher affinity and catalytic efficiency for Gln and Fru-6P compared with other four originations though purification and dynamics analysis. Therefore, we choose Bsglm S for the producing of Glc NAc, which originating from B. subtilis.(2) On the basis of recombinant Glc NAc-producing B. subtilis with the integration of Bsglm S, we overexpressed Gna1 with different originations, finding that Cegna1, originating from Caenorhabditis elegans, can better accumulate Glc NAc, which enhanced Glc NAc titer to 7.31 g·L-1 in the engineered B. subtilis. Further investigation results showed that the recombinant Cegna1 has higher affinity and catalytic efficiency for Glc N-6P and Ac-Co A compared with other six originations though purification and dynamics analysis. Therefore, we choose Cegna1 for the producing of Glc NAc, which originating from Caenorhabditis elegans. By fed-batch fermentation in 3 L bioreactor, Glc NAc titer further increased to 24.23 g·L-1, which is 24.69% higher than the reference strain.(3) The basic enzymology properties of Bsglm S and Cegna1 were investigated, including the optimal catalytic temperature, p H, thermal stability, the influence of metal ions on enzyme activity and so on. And the mechanism of the double substrates for Bsglms and Cegna1 are sequential reactions. Finally, it is found that the Glc N-6P has obvious inhibition on the the recombinant Bsglm S and Cegna1. Identified Glc N-6P inhibition of Bsglm S and Cegna1 provides future directions for improvement of Glc NAc production by directed evolution of Bsglm S and Cegna1 for alleviating substrate inhibition.