Modification Of GlmS Enzyme Activity And Its Effect On Glucosamine Production By Escherichia Coli

GlcN is the main component of glycoprotein,chitosan and chitin.Glucosamine synthase（GlmS）convertsfructose-6-phosphateglutamineinto glucosamine-6-phosphate and glutamic acid.Overexpression of GlmS in Escherichia coli increased the synthesis of glucosamine-6-phosphate,which was dephosphorylated and secreted into growth medium in the form of glucosamine.In this study,the glmS gene of Escherichia coli was modified by site-directed saturation mutation mediated by PCR to develop a microbial strain suitable for fermentation of glucosamine.Glucosamine synthase（GlmS）overexpression can increase glucosamine production.The recombinant plasmid pET-28a-glmS was obtained by linking the glmS gene of Bacillus subtilis to the expression vector pET-28a.Then the recombinant plasmid was transformed into E.coli BL21 competent cells,and the engineering strain E.coli BL21-pET-28a-glmS,whose glucosamine production increased significantly during fermentation,was successfully constructed.The engineering bacteria were fermented in shake flask.When fermented for 16 hours,the maximum glucosamine production in fermentation broth was 1.01g/L.The enzymatic properties of GlmS were determined.The optimum reaction temperature and pH were 37℃and 7.5respectively.Computer simulation analysis was used to predict the glucosamine synthase space.Low conservative amino acid sites were selected through rational analysis.Then mutation primers were designed according to glmS sequence to carry out site-directed saturation mutation of the constructed plasmid pET-28a-glmS.The mutant plasmid was transformed into E.coli BL21 competent cells and induced to express.By detecting the activity of glucosamine synthase,four strains（L601H,A602C,K603T,S604G）were screened and the enzyme activity was improved by43.3%,34.6%,55.3%and 50%,respectively.The enzymatic properties of mutant glucosamine synthase were determined.It was found that the optimum reaction temperature was 37℃and the optimum reaction pH was 7.5.Fermentation experiments were carried out with strains E.coli BL21,E.coli BL21-pET-28a-glmS,mutant strains L601H,A602C,K603T and S604G.The optimized fermentation medium was screened out as follows:50 g/L of glucose,10g/L of peptone,20 g/L of yeast extract and 20 mg/L of MnCl₂.The production of GlcN in fermentation broth was detected by shaking flask fermentation.The production of GlcN in fermentation broth of L601H reached 1.45g/L.GlcN production in A602C fermentation broth reached 1.3 g/L.GlcN production in K603T fermentation broth reached 1.1 g/L.GlcN production in S604G fermentation broth reached 1.0 g/L.