Expression,Purification And Enzyme Characterization Of Xyn-E18 And DFR

Xylanase is a kind of enzyme that can hydrolyze xylan.The source of xylanase is very extensive.Because of different enzyme activities and action characteristics,the xylanase studied in this paper comes from corn green storage feed,called Xyn-E18.In this experiment,E.coli was used as the carrier of protein expression.Firstly,a large amount of soluble protein Xyn-E18 was obtained by induced expression.Then,the protein was separated and purified by Ni affinity column and molecular sieve.Finally,the target protein with high purity and concentration was obtained.The relative molecular weight was determined to be 38 kDa.The methylation experiment was carried out on protein,and the hydrophobicity of protein was improved by substituting methyl for hydroxyl.It was found that the methylated protein had a tendency to form a crystal structure at neutral pH,which indicated that the methylated protein had a certain tendency to crystallize.In order to determine the activity and enzymatic characteristics of xylanase Xyn-E18,the experiment was carried out by DNS chromogenic method.The results showed that the enzyme activity of Xyn-E18 could reach 1782U,with the highest catalytic reaction rate at pH 6.5 and better catalytic efficiency at 50℃ or so.As for thermal stability,if the enzyme was stored in an environment below 50℃,it could maintain better activity.As for pH stability,if the enzyme is preserved in neutral and alkalescent environment,it can maintain good activity.Dihydroflavonol-4-reductase(DFR)is an important enzyme in the process of anthocyanin production.It can use dihydromyricetin as substrates and combine NADPH to produce anthocyanin,thus making plants form different colors.The flavanone-4-reductase(DFR)of chrysanthemum morifolium ramat studied in this paper is derived from chrysanthemum morifolium.It belongs to membrane protein,and its water solubility is not high.In the experiment,the induction time and IPTG concentration were first screened to obtain a large amount of soluble protein.Finally,the induction time was about 10 h,and the IPTG concentration was 0.2 mmol/L.Furthermore,the target protein was obtained by the same separation and purification method.The purity of the purified protein solution was over 90%,and the molecular weight of the protein was about 38 kDa.The enzyme activity of DFR protein was determined by HPLC,and the in vitro activity of DFR protein was preliminarily determined.The corresponding product delphinidin was produced by using dihydromyricetin as substrate.Both of these proteins belong to enzyme proteins.In the experimental method,the expression and purification of the research enzyme and the activity properties of the enzyme can be summed up together.Xyn-E18 is a conventional enzyme possessed by bacteria in natural state.Protein is extracted through experiments to detect whether it is also active in vitro,which can provide help for the development and utilization of feed additives.DFR protein in chrysanthemum morifolium has different substrate specificity from conventional enzymes.Therefore,the activity and structural characteristics of this enzyme need to be studied clearly in the experiment,and the reason for its substrate specificity needs to be analyzed structurally.