Construction Of Genetic Engineering Bacteria To Express Recombinant Glucosamine Synthase And Enzymology Properties Of The Enzyme

Glucosamine (GlcN) is structural monomer of some complex oligosaccharides and polysaccharides, which has an effective role in the treatment of rheumatoid arthritis in the cartilage tissue. Besides, it is regarded as the natural harmless ingredients for food and health products. And GlcN synthase plays a key role in the hexosamine biosynthesis pathway. Biosynthetic pathway of GlcN starts at Fructose-6-phosphate (Fru-6-P) of EMP. GlcN synthase catalyzes amino of L-glutamine (Gln) to the corresponding locus of Fru-6-P to synthesize glucosamine-6-phosphate (GlcN-6-P), and catalyzes transference of the ammonia of GlcN-6-P to Fru-6-P. Then, a mutase transfers generated GlcN-6-P into glucosamine-1-phosphate (GlcN-1-P) and further becomes the UDP-GlcNAc. UDP-GlcNAc is a precursor of macromolecules, including chitin and mannoprotein in fungi, peptidoglycan and lipopolysaccharide in bacteria, and glycoproteins in mammals. Moreover, GlcN synthase in mammals is thought to induce insulin resistance in diabetes. Besides, fungal GlcN-6-P synthase is considered to be a potential target for antifungal therapy. At present, more in-depth studies of the structural properties of Escherichia coli GlcN synthase were completed, but few studies on eukaryotic GlcN synthase, and reported findings were related to GlcN synthase derived from Candida albicans.The present work first realized the overexpression of Saccharomyces cerevisiae GlcN synthase gene gfal in prokaryotic expression system (E. coli BL21 (DE3), pET-28a) and eukaryotic expression systems (Pichia. pastoris SMD1168, pPICZaA), and purification of high purity GlcN synthase GFA1. Moreover, the structural properties of the enzyme and effect of heterologous expression of GFA1 in E. coli on intracellular synthesis of glucosamine were investigated. The method of Elson-Morgan was used to the process of enzymatic reaction to determinate GlcN concentration, because of simple reaction system. And the use of a HPLC method was to measure the intracellular GlcN concentration. Quantitative results are ideal.The results show that gfal could not only be implemented in a prokaryotic expression system expression but also be expressed in eukaryotic expression systems. The expression of S cerevisiae GFA1 could enhance intracellular GlcN content in E. coli, which maintained at a lower level, because of hexosamine pathway inhibition of GlcN. S cerevisiae GFA1 enzymatic properties with E. coli and C. albicans had some differences, but had high degree of similarity with Zygosaccharomyces bailii ISA 1307. The enzyme was purified by gel chromatography to near homogeneity. Optimal activities were at pH 5.5 and 55℃, respectively, at which the highest specific activity was 6.8 U mg protein-1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60℃. The Km and Vmax of the GlcN-6-P synthase towards D-fructose 6-phosphate were 2.8 mM and 6.9 μmol min-1 mg-1, respectively.