Directional Synthesis Of Oligosaccharides In The Acceptor Reaction Catalyzed By Dextransucrase

Functional oligosaccharides can be used as prebiotics and have physiological functions such as adjusting the structure of human intestinal flora,reducing blood lipid and improving immunity,which are widely used in functional food,medicine,feed and other fields.Traditional preparation methods such as direct extraction method,acid hydrolysis method or enzymatic hydrolysis method have the problems of poor product quality and low yield,complicated operation steps,high cost and the like.Enzymatic synthesis of oligosaccharides has the advantages of simple reaction system and high yield.Dextransucrase can catalyze the hydrolysis of sucrose to D-glucose residue and continuously transfer to sugar chain to form dextran.However,dextransucrase can catalyze D-glucose residue to connect with the acceptor to synthesize low molecular weight oligosaccharide products in the presence of small molecular weight saccharide acceptor.In order to solve the problems in oligosaccharide synthesis process,the polymerization mechanism of oligosaccharide products catalyzed by different dextransucrase acceptor reactions was studied through the intervention of small molecule saccharide acceptor in the reaction system,and the influence law of exogenous acceptor on enzymatic synthesis of dextran was revealed.On this basis,dextranase was introduced to study the regulation mechanism of molecular weight and its distribution of acceptor reaction products by double enzyme method,so as to lay theoretical and experimental foundation for targeted synthesis of oligosaccharides by enzyme-catalyzed acceptor reaction.The main research contents and experimental results are as follows:(1)Construction of analytical detection method for catalytic products of dextransucrase acceptor reaction.Based on the discovery of low molecular weight products in the process of dextran synthesis from single substrate sucrose catalyzed by dextransucrase,two detection methods of gel filtration chromatography column and amino hydrophilic interaction chromatography column were constructed.Through comparative analysis,it is found that gel filtration chromatography column with double columns connected in series can effectively separate polysaccharide,oligosaccharide and monosaccharide in dextransucrase acceptor reaction,track and monitor the changes of molecular weight and distribution of products.Amino hydrophilic interaction chromatography column can separate,identify and quantitatively analyze oligosaccharides with different polymerization degrees and isomers of certain saccharides.Meanwhile,the precision,reproducibility and stability of the two chromatographic column detection methods are proved by repeatability,precision and standard addition recovery experiments.(2)Study on the mechanism of oligosaccharide synthesis by dextransucrase acceptor reaction.Three kinds of small molecular carbohydrate receptors(glucose,maltose and lactose)were used to interfere with the reaction system of enzymatic synthesis of dextran respectively.The regulation and polymerization rules of dextransucrase catalyzed different acceptor reactions to synthesize oligosaccharides were discussed.The effects of donor-acceptor ratio,dextransucrase amount and substrate concentration on the molecular weight and distribution of oligosaccharides and dextran,oligosaccharide yield and yield,sucrose conversion rate and acceptor conversion rate were studied.The experimental results show that dextransucrase can catalyze two transglycosylation reactions simultaneously under the action of different acceptor molecules,namely acceptor reaction and polysaccharide chain extension reaction,and polymerize into two products(oligosaccharide and dextran)with greatly different molecular weights.The strength of the acceptor molecule determines the degree of competition between the two reactions,but the presence of the acceptor has little influence on the molecular weight(Mw>106 Da)and structure of dextran,which is a byproduct of the sugar chain extension polymerization,indicating that the acceptor action will not change the active site of dextran polymerization catalyzed by dextransucrase.The larger the proportion of acceptor,the stronger its ability to compete with receptor products and dextran sugar chains for D-glucose residues,and the easier it is to synthesize products with low degree of polymerization.Therefore,the proportion of small molecular weight fragments(3.6×102 Da?103 Da and 103 Da?104 Da)keeps increasing and the average molecular weight gradually decreases.However,the polymerization degree of different acceptor reaction products is different.The acceptor products of glucose and maltose can be used as new receptor substances to continuously connect D-glucose residues to synthesize oligosaccharide products with higher polymerization degree.The average molecular weight of oligosaccharide has negative correlation with acceptor ratio,substrate concentration and enzyme addition amount.However,under different reaction conditions,lactose acceptor reaction only synthesizes one oligosaccharide with polymerization degree of 3 and molecular weight of 504 Da,which indicates that the polymerization degree of oligosaccharide produced by different acceptor products is closely related to the structure and properties of receptor molecules.Acceptor molecules with glucose group as sugar unit are more easily recognized and utilized by dextransucrase to synthesize series of oligosaccharides with different polymerization degrees.In addition,compared with the acceptor products of glucose and lactose,the small molecular weight fragments(3.6×102 Da?103 Da and 103 Daa?104 Da)of maltose acceptor products account for a larger proportion and a higher maltose conversion rate,which indicates that the affinity recognition force between maltose molecules and dextransucrase is stronger,and more dextransucrase can be adsorbed to promote the Glycosylation polymerization reaction,so that the yield of oligosaccharide is higher.However,the proportion of large molecular weight fragments(105 Da?106 Da and>106 Da)in the reaction product of glucose and lactose receptor is relatively large,the yield of oligosaccharide in the acceptor product and the conversion rate of the acceptor are both relatively low,and more by-products dextran are synthesized in the system,so maltose is a strong acceptor substance for dextransucrase,and glucose and lactose are weak receptor substances.(3)Study on the regulation of oligosaccharide synthesis by dextranase degradation.The addition of small molecule carbohydrate acceptor can realize the directional preparation of oligosaccharide to some extent,but there will be more by-product dextran in the system.In order to maximize the utilization of by-products,dextranase was introduced to regulate its molecular weight.The results showed that dextranase mainly degrades high molecular weight dextran but has little effect on maltose acceptor reaction,which tends to synthesize oligosaccharide with lower polymerization degree.With the increase of the amount of dextranase enzyme,the large molecular weight fragments(>105 Da)in the reaction products are fully degraded,the average molecular weight is continuously reduced and the molecular weight distribution is gradually narrowed,and the proportion of small molecular weight fragments(<103 Da)is continuously increased,and oligosaccharide products with richer and more diverse structures are produced.At the same time,the conversion rate of donor and acceptor remained almost unchanged,and the yield of oligosaccharide had a positive correlation with the amount of dextranase enzyme.In addition,in the reaction system of 0.3 M sucrose solution and different ratios of donor and acceptor,the average molecular weight change of the product oligosaccharide follows the classic Malhortra model,and the fitting relationship is good.