The High Activity Of Sorbitol Dehydrogenase Oxidation Of Glucose Acid Bacteria Breeding And Bio-catalytic Synthesis Of Mig Column Alcohol

Miglitol, a kind of N-hydroxyethyl ramification of 1-Deoxynojirimycin can strongly inhibit a-glucosidases. It has been first chosen as therapeutic drugs for treatment of type 2 diabetes mellitus. Up to day, there are chemical and combined biotechnological-chemical two methods to synthesize miglitol.The former is more complicated than the latter, and synthetic cost higher. In the latter method, for membrane-bound sorbitol dehydrogenase of Gluconobacter oxydans catalyticly oxidizing N-(2-hydroxyethyl)-glucamine to 6-(2-droxyethyl)amino-6-deoxy-a-L-sorbofiuanos is the key step. Sorbitol dehydrogenase total activity and storage stability of the cells produced by feimentation is small. In this study a high sorbitol dehydrogenase activity strain was obtainied. To raise the productivity of miglitol the combined biotechnological-chemical technology was improved with the high sorbitol dehydrogenase activity whole cells being catalyst.By studying cultivation of G. oxydans A1 and characterization of the sorbitol dehydrogenase activity, sorbitol being carbon source restrained the microorganism from growing. When the catalytical reaction was carried out by the sorbitol dehydrogenase, the more oxygen in the reactive system, the more cells required, the react rate was faster. At the same quantity of oxygen, when the concentration of the resting cells was the critical value, the react rate was the highest. The optimum temperature was 28℃and the optimum pH was 5.5.By UV mutation to G. oxydans A1, a high sorbitol dehydrogenase activity strain named as G. oxydans Gouv2007 which biomass was raised 11.2% was screened by grads breeding with sorbitol dehydrogenase activity per boimass being the same as G. oxydans A1 and cultivating time being shortened 6 hours. The kinetics were established for G. oxydans A1 and G. oxydans Gouv2007, and the kinetic models of the two strains were substrate restrain,μmax being 0.2165h-1 and 0.2507h-1, Ki being 1.5069g/L and 3.9663g/L, respectively. G. oxydans Gouv2007 abolished restrain in part.By optimizing the conditions of G. oxydans Gouv2007 by single experiment, the optimum carbon source sorbitol, nitrogen source yeast extract powder, minerals MgSO4 and K2HPO4, rotating speed 200r/min, liquid volume 20%, cultivating temperature 28℃and initial pH nature were obtained. Using central composite design and response surface analysis, the optimum concentration of sorbitol, yeast extract powder and K2HPO4 was 18.0g/L, 10.0g/L and 3.0g/L respectively with the biomass (1.333g/L) being raised 34.6% and enzyme activity per boimass being the same as G. oxydans A1.G. oxydans Gouv2007 was cultivated in 2L bioreactor with the biomass being 5.580g/L, sorbitol dehydrogenase activity per boimass was 1.1 U/mg.The enzymatic characteristics about the sorbitol dehydrogenase of G. oxydans Gouv2007 was studied. When pH of the reactive system was maintained 5.5 and the temperature was 28℃sorbitol dehydrogenase activity per biomass was the highest. At the course of reaction, the sorbitol dehydrogenase almost did not lose its activity. When sorbitol was almost consumed and feed-beatch was carried out twice, the sorbitol dehydrogenase lost its activity little each time. Being reused once, it lose activity a little, but being reused twice, it lost 40% activity.Glycerol was added gradiently in sorbitol medium cultivating G. oxydans Gouv2007 to increase sorbitol dehydrogenase activity. The concentration of glycerol being 20.0g/L, sorbitol dehydrogenase activity per biomass was 1.9U/mg being raised 63%. The storage stability was raised, too. After 30 days storage, the enzyme activity still remained 75%, not-inducted 30%. Ks was modificated from 51mmol/L to 38 mmol/L, signalling of a 1.34-fold increase in affinity toward sorbitol. So the technology with which to produce high sorbitol dehydrogenase activity by glycerol inducting was established.Glucose and ethanolamines were catalytically hydrogenated to N-(2-hydroxyethyl)-glucamine with a transformation rate of 89%. N-(2-hydroxyethyl)-glucamine was then dehydrogenated to 6-(2-droxyethyl)amino-6-deoxy-a-L-sorbofiuanos by the high activity sorbitol dehydrogenase of G. oxydans Gouv2007 through aeration. At last 6-(2-droxyethyl)-amino-6-deoxy-a-L-sorbofiuanose was hydrogenated catalytically to miglitol. From the transformation of N-(2-hydroxyethyl)-glucamine to miglitol, the total transformation rate was 77.3% and 73.6% respectively by separating and identifying with TLC and ion exchange chromatography and the product rate was 62.7%. After purifying the sample of miglitol, it was white powder and its melting point was 142～147℃. At last, it was confirmed with IR and 1HNMR.