Directional Synthesis Of Dextran By Dual Immobilized Enzyme And Its Regulation Mechamnism

Dextran is produced from sugar by Leuconostoc mesenteroides, was widely used in medical field as a natural polysaccharides, is one of the hot research of sugar polysaccharide, and the character and use of the dextran are closely related to its molecular weight, molecular weight distribution and its structure. Small and medium molecular weight dextran, the molecular weight within 104-105 Da, can augmented blood volume, anticoagulant, reduce blood viscosity role; and the molecular weight of less than 104 Da dextran can be used to synthesis dextran iron and other dextran derivatives used for clinical purposes, also can be used as prebiotics for health care, is a hotspot of current research. However, dextran produced by existing production methods, has a wide distribution in the molecular weight, the content of medium and small molecular weight dextran is very low, and has many impurities, so that the quality is bad, must through a series of purification processing to get the product, and this progress need to be consumed in the process consumpted large amounts of acid and organic solvents. Therefore, establish a method to simplify the operation and reduced the cost, to produce specific molecular weight dextran, is very important to improve the industrialization development and application of dextran.Immobilized enzyme used to produce dextran has advantages of easy separation, enzyme preparation can be reused. This topic using immobilized dextransucrase(DS) and immobilized dextranase(DN), join in the sucrose solution at the same time, by controlling the concentration of the substrate (sucrose), the amount of immobilized DS and DN, regulating the molecular weight and molecular weight distribution of dextran, and explored the regulation of this method, as well as dynamic analysis and modeling. The main research contents and results of this article are as follows:(1)Through orthogonal test to verify the feasibility of dual immobilized enzymatic directional synthesis dextran, preliminary screening the reaction conditions of dextran with molecular weight (Mw<105 Da). When the enzyme activity ratio(DN:DS), the DS enzyme concentration and sucrose concentration are different in reaction system, the average molecular weight, homogeneous degree, and the percentage of each molecular weight segments are different. Compared with the early research which using single dextransucse catalytic sucrose to produce dextran, the molecular weight distribution of dextran is closer to target molecular weight in dual immobilized enzyme system, and the percentage of molecular weight Mw<105 Da increased significantly. Show directional synthesis special molecular weight dextran by dual immobilized enzyme is feasible. The reaction conditions of molecular weight range of Mw<105 Da dextran preliminary screening as follows:enzyme activity ratio of DN:DS is 1:8, DS concentration is 0.4 U/mL, substrate concentration is 0.2 M.(2) Explored the influence regulation of the substrate concentration, the DS concentration and enzyme activity ratio to the substrate sucrose conversion, yield of dextran and the percentage of target molecular weight dextran (Mw<105 Da). When DS concentration and enzyme activity ratio(DN:DS) were 0.4 U/mL and 1:8, increased the substrate concentration, the sucrose conversion increased at first and then declined, dextran yield increased, and the percentage of target molecular weight dextran(Mw<105 Da) increased and then reduced. When the substrate(sucrose) concentration and enzyme activity ratio(DN:DS)kept as 0.2 M and 1:8 respectively, as DS concentration increased, sucrose conversion and dextran yield increased while the percentage of target molecular weight dextran(Mw<105 Da) decreased, but in 0.3 U/mL and 0.5 U/mL has a slight fluctuation. The substrate concentration and DS concentration kept as 0.2 M and 0.4 U/mL, when enzyme activity ratio(DN:DS) decreased, the sucrose conversion increased, dextran yield decreased first and then increased, and the percentage of dextran molecular weight less than 105 Da increased.(3) Based on the dual immobilized enzyme system, we analysis the sucrose consumption rate, dextran yied and the percentage of target molecular weight (Mw<105 Da) fragment by mathematical method, and construct relevant mathematical models and expressions.Sucrose consumption rate is conformed to the first order reaction, the reaction rate is proportional to sucrose concentration, and the rate constant is different according to the change of the ratio of enzyme activity, showing that the reaction rate is also related to the concentration of dual enzyme. In the range of enzyme activity ratio (DN:DS) 1:12～1:4, percentage of target molecular weight (Mw<105 Da) segment increases with dual enzyme enzyme activity exponentially, the relationship of dextran yield and percentage of target molecular weight (Mw<105 Da) segment with enzyme activity ratio (DN:DS) can be determined by the curve fitting, showing that when substrate concentration is 0.2 M, DS concentration is 0.4 U/mL, dual enzyme activity (DN:DS) between 1:12～1:4 scale, directional synthesis process of dextran is controllable, and reaction kinetics of immobilized dual enzyme system is still follow the general rule of chemical reaction kinetics.