Preparation Of Glucosamine-6-Phosphate With Immobilized Kinase

There are three ways to prepare glucosamine-6-phosphate, including chemical synthesis, microbial fermentation and enzymatic synthesis. One enzymatic synthesis method, in early stage, the fructose-6-phosphate, glutamine and adenosine triphosphate used as materials, catalysed by glucosamine-6-phosphate synthase, another enzymatic synthesis method use glucosamine and adenosine triphosphate as materials catalysed by hexokinase. Because of convenient source of enzymes, the price of ATP declining, recycling using ADP and immobilized enzyme used, this method is possible used in large scale manufacture.A high glucosamine kinase activity bacteria strain has been isolated from the soil through the experiment, the medium and culture conditions of the kinase fermentation were optimized. In the medium optimization process, focus on the medium investigated the carbon source, nitrogen source, and inorganic ions. Carbon source study glucosamine, maltose, sucrose, mannitol, glucose, nitrogen source including corn steep liquor, beef extract, yeast extract, peptone, ammonium sulfate; also investigated effect of CaCl2·2H2O, MgCl212H2O, FeSO4·7H2O, CuSO4·5H2O, MnSO4·5H2O, glucosamine is selected as the best carbon source, corn steep liquor as the best nitrogen source and the enzyme activity promoted by MgCl2·12H2O. The orthogonal experiment carried out to get the optimum medium composition:glucosamine3%, corn steep liquor0.5%, MgCl2·12H2O0.03%; optimum fermentation conditions as:stirring speed600rpm, the fermentation temperature is30℃, gas flow rate is5L/min,2%inoculum quantity, the enzyme activity reached145U/ml broth(glucosamine HC1as substrate).The cell disruptions were researched with grind. SDS and toluene. The results indicate that disruption with SDS was the best. The operation conditions were0.1%SDS, EDTA concentration3mmol/L extraction time4hours, after ultrafiltration and purified with DEAE cellulose chromatography, get430purity times, and then immobilized by polyacrylamide, in the presence of5%glucose yield45%of the enzyme, it can be reused10times and the enzyme activity still retained more than50%. It was investigated that the optimal reaction conditions of enzymatic synthesis of glucosamine-6-phosphate, the final optimal conditions for glucosamine2mmol, ATP1.8mmol,1.5mmol MgCl2the yield of ATP more than90%, after simple isolation glucosamine-6-phosphate obtained, which had the purity more than98 %,while the main by-product ADP contains no ATP, simplified the separation and purification ADP, reduce production costs significantly.