The Biochemical Information Research Of Resveratrol Synthase From Grape

In this paper, an improved procedure was described for separating and purifying resveratrol synthase(RS) from the peel of fresh grape(Vitis vinifera Linn.). At the same time the chief biochemical information of RS was studied. The experiment showed that: (1) The extraction program was refined by orthogonal design. The optimal conditions for RS extraction were: the type of buffer was potassium phosphate buffer at PH7.5 (0.1M/L, including 5mM mercaptoethanol); the solid-liquid ratio(g/ml) was 1 : 2. After extraction for twelve hours at 4℃, the supernate was the crude enzyme solution by centrifugation at 20000g, 4℃ for 15 minutes.(2) The purification procedure of RS was as follows: the crude enzyme was fractionally precipitated by 65%,90% degree of saturation; after dialysis and desalination ,then RS was isolated on DEAE-cellulose 52 column and hydroxylapatite column successively, the columns were equilibrated with buffer D (0.01 M/L potassium phosphate buffer at PH7.5, including 1mM mercaptoethanol)and done gradient elution with 0.01-0.1 M/L potassium phosphate buffer at PH7.5 (respectively including 1mM , 5mM mercaptoethanol), the elution speed were 0.6ml/min, 1.2ml/min.After the process, the electropherogram and chromatographic profile of RS showed one band and one peak; the specific activity reached 36.4 U/mg from 0.35 U/mg, the purification fold was from 1.0 to 104. (3) The relative molecular weight was 87 000D by applying gel filtration chromatography; RS had higher activity at neutral or slightly basic conditions, its optimal PH was 7.5; the Km,Vmax of the two substrates, 4-coumaroyl-CoA and malonyl-CoA were respectively 1.93μM,4.84μM/min and 9.8μM, 4.95μM/min, so 4-coumaroyl-CoA was the optimal substrate of RS. We could tentatively consider that RS was a Michaelis enzyme for the V-[S] curves of the two substrates were both similar to the one of Michaelis enzyme.(4) Applying biological analysis software, databases and internet resources, the bioinformaticanalysis of RS indicated: RS included two domains, Chal_stil_synt and Chal_stil_syntC. The amino acid sequences of five RS from grape had over 95% homology, we obtained the conservative sequences of RS and its three-dimensional model as well. The distribution probability of RS was respectively 88%, 36%, 31.3%, 28% in nucleus, mitochondrial matrix space, microbody(peroxisome) , chloroplast thylakoid membrane.