Study Of Gluconate Transformation And Enzyme System In Gluconobacter Oxydans Strain DHA3-9on Glucose Metablization

Gluconobacter oxydans is widely used in industrial application for its character of incomplete oxidizationr of sugars and sugar alcohols. This character is based on the excellent dehydrogenase system located on cell membrane. Dioxyacetone, D-gluconate, vitamin G and migltol can be biotransformed by G.oxydans. D-gluconate is an important intermediate for product of medicine, chemistry and food industry. The great marketing demand promotes people to improve the technic of D-gluconate production. Compared with the traditional D-gluconate transformation by Aspergillus niger, G.oxydans with better tolerance on low pH and high glucose concentration has the advantages of low environmental contamination and simple simple to operate in the gluconate transformation processIn this study, a strain of G.oxydans named DHA3-9was isolated from flowers. The tramsformation of D-gluconate and the enzymes involved in D-glucose metabolism were studied. The main results are as follows:1. Cloning and knocking out of the D-gluconate dehydrogenase gene gadH:Partial sequence of D-gluconate dehydrogenase gene was amplied by PCR. Based on its phylogenetic analysis, the similarity with gadH in G.oxydans621H was85%. After knocking out of the gene gadH, no2-keto-D-gluconate(2KGA) was produced any more by the strain DHA3-92. High concentration D-gluconate produced by strain DHA-AgadH:Under the condition of the high concentration of initial glucose medium without CaCO3to reduce accumulation of5-keto-D-gluconate(5KGA),142g/L D-gluconate accumulated after72h cultivation.3. Cloning and knocking out of the D-glucose dehydrogenase gene mgdH:Partial sequence of membrane bound D-glucose dehydrogenase gene was amplied by PCR. Based on its phylogenetic analysis, the similarity with mgdH in G.oxydans621H was85%. After knocking out of the gene mgdH, gluconate transformation by the strain DHA-AmgdH was decreased98%.4. Study of glucose metabolic enzyme system in DHA-AmgdH:Ion chromatography analysis indicated that more dioxyacetone and pyruvate was produced by the strain△mgdH. Acetate, which didn’t exsit in that of wild strain was detected in medium of strain△mgd. High concentration of acetate suppressed glucose metabolism of strain△mgdH. These results indicated that there was another glucose metabolic pathway, that is glycolytic pathway(EMP), in△mgdH strain that transformed glucose to acetate...