Optimizing Of Conditions For Catalyzing Validoxylamine A Glycosylation By The Engineering Strain Of Valg And Enzymatic Characterization Research

In the process of validamycin fermentation,the precursor Validoxylamine A will accumulate in quantity,which greatly affected the quality of products.In order to solve this problem,,the engineering strain of glycosyltransferase that use for catalyzing validoxylame A glycosylation have constructed and preliminary applied in the biotransformation of Validoxylamine A glycosylation to generate validamycin A,however,the result is not ideal.In this research,the glycosyltransferase will be purified and further the enzymatic properties will also be investigated.Based on the results of enzymatic properties superposition,the condition for catalyzing validoxylame A glycosylation by the engineering strain will be optimized.Nickel column chromatography was proved to be a effectively and quickly method that used for purification of the ValG,the enzyme gets the highest activity in 30? but its stability to temperature is not good,the optimal pH of the enzyme is between 8?9,the glycosyl transferase has preferable adaptability to pH.In addition,K+?Ca2+?Mg2+?Mn2+ and Co2+all have measurable promotability to enzyme reaction.Based on the research of the kinetics of enzyme reaction,it shows that the glycosyl transferase has strong affinity to zymolyte Validoxylamine A but the affinity to UDPG is correspondingly weak.At the same time,several groups of experiment results indicate that the enzyme catalysis mechanism conforms to the "double-replace" model.It can also get the conclusion from the enzyme structure characteristics.Moreover,the dates show that the optimal buffer solvent for the whole-cell catalysis Validoxylamine-A to glycosylate is 50 mM pH 6.0 Na2HPO4-NaH2PO4 and the cells used for catalytic has best capacity after being induced by 100 mM lactose,the bacteria concentration in the catalytic system should be controlled between 0.04?0.08 g/mL.Under the conditions above,the mole yield of Validoxylamine-A can reach more than 95%to theoretical value,the highest concentration reachs 1386?g/ml.Furthermore,to join in catalytic systems 10 mM Mg2+,Mn2+,0.1%Tween-80 can accelerate the catalytic process and shorten the catalytic time.During the catalytic process,another factor that may influence the catalytic efficiency is the Synthetic quantity of intracellular glycosyl donor UDPG,so we also do many jobs to strengthen the anabolism of UDPG by genetic engineering means:Cloned UDPG anabolism key enzyme(UDP-glucose pyrophosphorylase)gene from JM109 genome and successfully built a engineering bacteria(named pET28-valG-galU BL21(DE3)which can both express glycosyl transferase and UDP-glucose focal phosphorylase through series connection.However,The intracellular quantity of UDPG synthesized by the engineering bacteria can get 1.36 times than starting strain pET28-valG BL21(DE3),what all above,provides a new train of thought to realize efficient catalytic validamycin to glycosylate.This research provides a new train of thought and tactics for soling the problem of precursor accumulation during antibiotic production.Besides,the results also provide the basis for the further theoretical study and application of the glycosyltransferase in Glycosylation modification of antibiotics.