Study On The Transformation And Expression Of Dextransucrase Molecule Based On Transglycosyl Function And Its Catalytic Performance

The transglycosylation of glycosyltransferase(GT,EC 2.4.x.y)catalyzes the transfer of glucose to the acceptor moleculeto glycosylation of the receptor molecule to form a glycosylated derivative including a non-natural carbohydrate and functional oligosaccharides.Non-natural carbohydrate compounds can change the physical and chemical properties of the original natural compounds,functional oligosaccharides as the regulation of intestinal balance of the bifidus factor used in medicine,food,health products and other fields.In this paper,dextranuclease(Dextransucrase,DSR)was modified by molecular means to construct a DSR mutant enzyme with high activity and to reveal the high activity of transgenic glycosylation.The glycosylation of different carbohydrate receptors and vitamin C(VC)was carried out by using the modified transglycosylase,and compared with the original enzyme,and the catalytic rule of the mutant enzyme to glycosylation of the receptor was obtained.First,the C-terminal 1494 bp fragment of dextransucrase was truncated by molecular truncation to obtain mutationenzyme DSR-S1-ΔA with the 1-3087 bp gene fragment,and the expression conditions of the mutant enzyme were optimized.To study the enzymatic properties: the optimal pH was 5.4,the pH stability was 4.4-7,the optimum temperature was 25 ℃,and the activity of DSR-S1-ΔA was completely lost in the absence of receptor;when the receptor was present,the activity of DSR-S1-ΔA was 71.4% of that of the original enzyme,and the activity of DSR-S1-ΔA was 5.3 times lower than that of the original enzyme,at 30 ℃ for 1 h there is only 20.8% of the enzyme activity.Maltose as a receptor study showed that the activity of DSR-S1-ΔA was higher when the receptor concentration was 200 m M,and the enzyme activity increased with the increase of sucrose concentration.The results show that the main activity of DSR-S1-ΔA is transglycosyl function,and the polysaccharide polymerization ability is low,which clarified the transglycosyl functional region of DSR.The transglycosylation of different carbohydrate receptors such as maltose,cellobiose,raffinose and stachyosewas studied.The results showed that:maltose is the best receptor for the transglycosylation of the enzyme,and it is active for both the cellobiose,the cotton trisaccharide and the water sugar,but the transglycosyl group is weakened;the transglycosylation of different natural oligosaccharides tends to produce low molecular weight sugars,transfer 1-2 glucosyl to receptor molecules,and the smaller the molecular weight of the receptor,the more easily transglycosylated,the α bonds compounds are more readily transferable than glycosidic compounds than β bonds,indicating that the reconstituted enzyme can synthesize more oligosaccharides with less maltose.At the end of this paper,VC was used to study the transglycosylation of DSR-S1-ΔA enzyme,and the glucosyl derivative of VC was obtained.In summary,this study clarifies the molecular area of the transoglycoside function of dextransucrase,which is helpful to further understand the catalytic mechanism of dextran sucrase.It is also important for the preparation of functional oligosaccharides and the study of glycosylation of natural products.