Gene Mining,Recombinant Expression And Compound Application Of Enzyme In Preparation Of Gentiooligosaccharide

Gentiooligsaccharide is a new type of functional oligosaccharide formed by the connection of glucose through?-1,6-glycosidic bonds,including gentiobiose,a small amount of gentiotriose and tetrasaccharide.It has the characteristics of low calorie,low sweetness and the function of cleaning the intestine,and is widely used in the dessert and beverage industries.?-glucosidase?Bgl?can produce gentiooligsaccharide from glucose by reverse hydrolysis reaction,however,except for a small amount of by-products such as cellobiose,laminbiose,and sophorose,the product basically contains only gentiobiose,it is impossible to detect oligosaccharides with a degree of polymerization of trisaccharide and above.The data shows that?-1,6-glucanase?Bgn16?can use gentiobiose as the donor and acceptor to synthesize gentiotriose,even the gentiotetraose.This study explored the mechanism of reverse hydrolysis of?-glucosidase in GH3 family by sequence analysis,expression verification,site-directed mutation,and identified key amino acid that affected the reverse hydrolysis activity of enzyme.Based on the above research results,Talaromyces cellulolyticus?-glucosidase TcBgl3A with high reverse hydrolysis activity was excavated from the database,and then,T.cellulolyticus?-1,6-glucanase TcBgn16was selected to synergistically produce gentiooligsaccharide with TcBgl3A.Finally,P.pastoris KM71/pPIC9K-tcbgl3a and P.pastoris KM71/pPIC9K-tcBgn16 were subjected to3.6 L tank high-density fermentation to realize the efficient preparation of enzymes.The main results are as follows:?1?Three different sources of GH3 family?-glucosidase were mined from the database,and the abilities to produce gentiobiose by reverse hydrolysis reaction were evaluated.Among them,the?-glucosidase TpBgl3A derived Talaromyces piceae had the best application effect,it could use 800 g·L^-1 glucose as the substrate to produce gentiobiose with the enzyme amount of 300 U·g^-1 glucose,the maximum yield was 15.62%.Around the+1 receptor site,the reverse hydrolysis reaction mechanism of GH3 family?-glucosidase was explored by site-directed mutation using TpBgl3A as the research object.The results showed that the disaccharide yields of Q34G,Q34M and Y200F were respectively 22.19%,24.53%and20.52%,among them,the gentiobiose yields were 17.61%,17.02%and 16.62%,which all improved compared with the wild type.Kinetic parameters indicated that the introduction of hydrophobic amino acids at 34 and 200 sites could increase the affinity of enzyme with glucose glycosyl receptor and the hydrophobicity of+1 receptor site to enhance the ability of reverse hydrolysis.?2?Based on the predominant characteristic sequence at+1 receptor site of the?-glucosidase,TcBgl3A derived from T.cellulolyticus with high reverse hydrolysis activity was obtained from the database.The effect of substrate concentrations and the amount of enzyme in preparation of gentiobiose by TcBgl3A were investigated at 60? and pH 4.5.In800 g·L^-1 glucose solution,when the enzyme amount was 400 U·g^-1 glucose,the conversion rate of gentiobiose could reach 19.36%,which was currently the highest level of single enzyme in preparation of gentiooligsaccharide.?3?T.cellulolyticus?-1,6-glucanase TcBgn16 was heterologously expressed,and the compound system of TcBgn16 and TcBgl3A to produce gentiooligsaccharide was constructed.When the enzyme amount of TcBgn16 and TcBgl3A both were 400 U·g^-1 glucose,the conversion rate of gentiobiose could reach 23.89%at 60? and pH 4.5.Among the product,the yield of gentiotriose was 4.00%,and the yield of gentiobiose was almost the same as TcBgl3A.It showed that TcBgn16 could indeed produce gentiotriose from gentiobiose and increase the content of gentiooligsaccharide overall by transglycoside activity.?4?A preliminary study on 3.6 L tank high-density fermentation of P.pastoris KM71/pPIC9K-tcbgl3a and P.pastoris KM71/pPIC9K-tcBgn16 were executed.When the induction temperature was 25?,the initial induced cell concentration of OD₆₀₀ was 150,and the methanol concentration was 1.0%,the enzyme activity could respectively reached 350 and1795 U·mL^-1,which were 10.48 and 6.01 times of the shake flask.