Heterogeneous Expression,Enzyme Properties And Preparation Of AA-2G Of Cyclodextrin Glycosyltransferase

2-O-α-D-glucopyranosyl-l-ascorbic acid（AA-2G）,as a derivative of L-ascorbic acid（L-AA）,has better antioxidant activity than L-AA,and can be decomposed into L-AA and D-glucose in vivo under the action ofα-glucosidease,thus playing the physiological role of L-AA.Therefore,it is the best substitute of L-AA so far.It is the excellent physical and chemical properties of AA-2G that make it widely used in food and feed,medical drugs,cosmetics industry.Now there are two ways to produce AA-2G,such as chemical synthesis and biosynthesis.But the chemical method has the disadvantages of high production cost and complex operation process.The enzymatic synthesis of AA-2G is the main way to synthesize AA-2G.Among the five enzymes for biosynthesis of AA-2G,cyclodextrin glucosyltransferase（CGTase）is generally considered to be the most efficient enzyme for AA-2G synthesis due to its high substrate specificity and high yield.Therefore,the enzymatic synthesis of AA-2G by CGTase has become a research hotspot.In this study,my20 gene encoding cyclodextrin glucosyltransferase was obtained from marine microorganism metagenome in Mariana Trench,which was cloned into the vector pET-24a after artificial synthesis.The high-efficiency heterologous expression of E.coli BL21（DE3）was successfully achieved.At the same time,the preliminary application experiment of enzymatic preparation of AA-2G was also carried out.The main results are as follows:To increase the expression of recombinant CGTase in E.coli BL21（DE3）,the fermentation conditions such as time,inducer and temperature were optimized.The results showed that the cyclization activity of recombinant CGTase was the highest when the induction time was 18 h.If the induction time is too long,the cyclization activity of recombinant enzyme will gradually decrease,which may be caused by cell death and protein degradation.When the concentration of IPTG was 0.4 mM,the cyclization activity of recombinant enzyme was the highest.Although the expression vector pET-24a is strongly induced by IPTG,on the one hand,high concentration of IPTG will lead to the rapid translation of recombinant protein,which will lead to protein misfolding.On the other hand,the accumulation of high concentration of IPTG will also produce toxic effects on cells.When the induction temperature was 20℃,the cyclization activity of recombinant enzyme was the highest.When the induction temperature is too high,the amount of soluble protein will decrease.Therefore,when OD₆₀₀ value reached 0.6-0.8,0.4mM IPTG was added and induced at 20℃for 18h,the cyclization activity of recombinant CGTase reached the highest value.The crude enzyme was purified by Ni²⁺column.SDS-PAGE showed that the purified enzyme reached electrophoretic purity.The molecular weight of CGTase is consistent with the theoretical molecular weight of 75k Da.After purification by Ni²⁺column,the specific activity of the enzyme increased from 0.94 U/mg to 63.3 U/mg.When the soluble starch was used as substrate,the optimal reaction temperature of the recombinant enzyme was 80℃,and the recombinant CGTase had good heat resistance.The cyclization activity of the recombinant CGTase remained almost unchanged after 2 hours at 10℃-80℃.The optimum reaction pH is 7,and the enzyme can still maintain high activity in a wide range of pH.By exploring the enzymatic properties of the recombinant enzyme,the recombinant enzyme has better thermal stability and pH stability than most of the reported.When applied to industrial production,it can prolong the working time of recombinant enzyme,reduce the risk of pollution.In addition,it can also increase the temperature of the reaction system and further increase the concentration of the reaction substrate.Therefore,the recombinant enzyme has potential application value in the industrial production of AA-2G and cyclodextrin.K⁺,Mg²⁺,Ba²⁺,Mn²⁺and Li⁺had little effect on the activity of recombinant enzyme,while Fe²⁺,Cu²⁺could inhibit the activity of recombinant enzyme,Ca²⁺and EDTA could improve the activity of recombinant enzyme.According to Hanes Woolf plot method,K_m is 5.1 g/L and V_max is 0.65μmol/min at the optimum reaction condition.Whenα-cyclodextrin,β-cyclodextrin,γ-cyclodextrin and soluble starch were used as glycosyl donor substrates to synthesize AA-2G,α-cyclodextrin as substratss the production of AA-2G was the highest,followed by that ofβ-cyclodextrin,and the soluble starch was the lowest.However,considering the high price ofα-cyclodextrin,it is not suitable for industrial production of AA-2G.Therefore,β-cyclodextrin was selected as the donor substrate for the follow-up experiment.In this study,in order to further improve the yield of AA-2G catalyzed by the recombinant enzyme,β-CD and L-AA were used as glycosyl donors and glycosyl receptors respectively.The single factor optimization experiments were carried out on the reaction time,temperature,reaction pH,substrate concentration and enzyme concentration to improve the production of AA-2G.The results showed that when the reaction time was 24 h,temperature was 40℃,the reaction pH was 4,the substrate concentration was 50 g/L,and the enzyme concentration was 75 U/gβ-CD,the highest concentration of AA-2G was 28 g/L,and the yield of AA-2G was higher than most reported CGTase.