The Study Of Expression And Purification Of ?-Amylase-Trehalose Synthase Bifunctional Fusion Enzyme And Analysis Of Enzymatic Properties

Trehalose is a non reducing two sugar,and its unique properties give it special protective properties.Trehalose has good water retention,can stabilize the enzyme protein,food,cosmetics and drugs in high temperature conditions,to maintain cell survival,protein degeneration in drying and freezing,because of these characteristics,the commercial prospects of trehalose is considerable.The production process of trehalose is complicated,which leads to high production cost,which limits the application prospect of trehalose.Based on the dual functional expression in Escherichia coli with beta amylase gene and trehalose synthase gene fusion protein as the basis,the removal of protein through separation and purification,enzymatic characteristics analysis of enzymatic properties of fusion enzyme,then optimized by means of connecting peptide enzyme fusion than the double function is relatively stable and efficient.In this study,the culture medium was optimized by single factor orthogonal method and named M9-MZ on the basis of M9 medium,and the induced expression conditions were optimized.The optimized concentration of treucose was 18.52 g/L,the conversion rate of trehalose was 30.87%,and the concentration of crude enzyme was30.87%after 6 h incubation with 6%amylose at pH 7.0 and 45?.Of the enzyme activity of 13.6 U/mL.Compared with the results obtained by M9-ZJ-LM medium,the activity of fusion enzyme increased by 28.5%.According to the special structure of the fusion enzyme with 6×His-tag tag affinity chromatography with anion exchange chromatography firstly obtained fusion enzyme relatively pure by Ni-NTA.The final purification ratio was 12.97 and the recovery rate was 25.37%.The optimum reaction temperature was 40?,the optimum pH was 8.0,shifted to 0.5 units in alkaline direction,the thermal stability was higher than that of single enzyme,and the alkali resistance was higher than that of single enzyme.3mmol/L metal ions Ba²⁺,Co²⁺,Cu²⁺in the presence of three kinds of enzyme activity was significantly inhibited,Mg²⁺on the enzyme activity to promote the role.The enzyme activity of the fusion enzyme BT3 was 92.4 U by HPLC.The yield of the purified trehalose was 33.72 g/L,the yield was 56.2%,which was 26.7%higher than that before the purification?18.52 g/L,30.9%?,and the fusion enzyme BT3 Of the trehalose yield compared with the mixed enzyme system increased by 6%.This study through the replacement of different length of connecting peptide?EAAAK?n,n=2-4,to get the three kinds of fusion enzymes BT3-A,BT3-B,BT3-C The trehalose concentration was three 30.36 g/L,32.29 g/L and 37.99 g/L by the comparison of the purification of the two new fusion enzymes to catalyze the amylose solution of 6%.Among them,BT3-C catalyzed high trehalose concentration,trehalose yield reached 63.32%.The yield of trehalose was 7.12%higher than that of the fusion enzyme BT3?33.72 g/L,56.2%?,which indicated that the length of the ligated peptide had different effects on the single enzyme at both ends,and the appropriate length was selected,The enzyme activity of the fusion enzyme can be appropriately increased.