Investigations On Purification Characterization And Cloning Of Glucose Dehydrogenase

Fructoolingosaccharide(FOS),As a functional sweetener, it has been found in various fruits and vegetables in nature, and has two characteristic properties, nondigestibility and selective utilization by beneficial intestinal bacteria, which make them useful as low-calorie dietary fiber for relief of constipation, improvement of blood lipid composition, cholesterol reduction, and suppression of intestinal putrefactive substances. Because of these beneficial physiologic functions, FOS has attracted more and more attentions worldwide.Glucose was accumulated as byproduct when Fructosytransfrase(FTase) converted sucrose to fructooligosaccharide(FOS) and inhabit the reaction moved forward further. One strain of microorganism isolated from soil in our lab, designated as GX-0013, was found to be capable of producing glucose dehydrogenase(GDH).The GDH producing from GX-0013 was able to convert the glucose resulted from the FOS synthesis into gluconate acid which could be easily removed from the mixture products, thus the high-content FOS was produced.The strain GX-0013 was cultivated for GDH. The cells harvested were disrupted by ultrasonic , followed by leaching and centrifugalizing to obtain a crude enzyme solution. The activity of the GDH was assayed by determining the content of gluconate acid formed in the reaction mixture of glucose and the crude enzyme by employing both regular high-performance liquid chromatography (HPLC)and high-performance anion-exchange chromatography (HPAEC).The crude GDH was further purified by using weak anion-exchangechromatography of DEAE-Sephadex A-50 and Mono?Q column. Further investigations indicated the enzyme was an membrane-bound enzyme. The enzymatic properties of GDH was characterized and found that it had pH optimum of 6.0 and were stable over a pH range of 5.0-7.0. The optimal temperature enzyme was 30 癈.To produce the GDH in sufficient quantity, the gene for the GDH from GX-0013 has been amplified by PCR and transformed to an Escherichia coli strain JM109. The PCR production had been ligated in plasmid p GEM-T Easy vector. We will study the gene sequence and expression.