Study On The Polymerization Process Of Dextran With Double Enzyme (DS/DN) System

Dextran is a typical polysaccharide drug,the molecular size and distribution of which are closely related to the property and pharmacological activity.The traditional method of dextran production by fermentation-acidolysis-alcohol precipitation is not ideal for the molecular weight control of dextran,which is not suitable for medical application.There is also the research on the degradation of macromolecular dextran using the cavitation effect of ultrasound.However,.there exists a limit about ultrasonic degradation,that is,the effect of ultrasonic degradation is more obvious when the molecular weight of dextran is above the limit.The synthesis of pharmaceutical dextran by double-enzyme method can control the molecular weight well and obtain a series of dextran preparations with a large molecular weight distribution range required by clinical medicine.The dextran preparations have a more uniform product and a higher yield,of which the industrial application prospects is good.At present,there are very few commercial grade dextransucrase,and the control of the molecular weight of the synthetic product dextran by synthetases is also not specific.The directed synthesis of double-enzyme system has good control over the molecular weight and homogeneity of the product,however,the rule of this synthetic process is not yet clear,and the process regulation and mechanism need to be further understood.In this study,the synthesis route of dextran was constructed by using sucrose as polymerization substrate,DS as polymerization catalyst and dextranase as polymerization regulator in vitro experiment.The dextran polymerization process was quantitatively studied to explore the polymerization of repeating units,polymerization methods and characteristics of dextran synthesis by double-enzyme system.The influencing factors and control ways of highly synthetic flux-oriented synthesis process was revealed.And the kinetics of dextran polymerization process were studied.The enzymatic degradation and ultrasonic degradation process of dextran with different initial molecular weights were also compared.The regularities and characteristics of the two regulatory pathways were analyzed and evaluated,which helps to develop a theoretical reference for the directional preparation of dextran.The main research contents and conclusions are summarized as follows:(1)The polymerization process of dextran by single enzyme(DS).The synthesis system of dextran was constructed by using sucrose as polymerization substrate and dextransucrase as polymerization catalyst.The experimental results showed that in the single enzyme(DS)synthesis system of this experiment,the molecular weights of the dextran products in each reaction period were mainly concentrated in the two ranges of>106 Da and 103-104 Da with different substrate sucrose concentrations and different initial enzyme activities.And as the reaction proceeded,the other molecular weights of dextran product were not detected,indicating that the molecular weight increasing mode of dextran may be the same as the slow mode in which a simple glucosyl group is linked to a low molecular weight dextran fragment and the fast mode in which an oligosaccharide is linked into a high molecular weight dextran fragment.(2)The polymerization process of dextran with double enzyme(DS/DN)system.The DS/DN synthesis system was constructed with sucrose as substrate,dextransucrase as polymerization catalyst and dextranase as polymerization regulator.The experimental results showed that in DS/DN synthesis system,the molecular weight of dextran products mainly concentrated in the same molecular weight range under the same experimental conditions.However,when sucrose concentration is constant and enzyme activity ratio is different,the molecular weight range of the dextran products are different from each other.When the double enzyme activity ratio is different and the sucrose concentration is same,the molecular weight of dextran products are mainly concentrated in the same molecular weight range,indicating that dextranase well regulates the molecular weight of dextran product when the double enzyme synergetic system directs the synthesis of dextran.As the reaction progressed,the homogeneity of the dextran product also increased.Under the same substrate concentration and dextransucrase activity,the double enzyme synergy system with dextranase preferentially detected the dextran molecular fragment compared with the single enzyme synthesis system,and the concentration range of the main concentration was different,indicating that the synergistic effect of the double enzyme system,dextranase not only regulated the molecular weight of dextran product,but also promoted the polymerization of dextran by dextransucrase.According to the kinetic characteristics of enzyme catalysis,the kinetic equations of synthesis of dextran are in accordance with Malhotra model.(3)Enzymatic degradation process of dextran by dextranase.The single enzyme degradation system was constructed by using the different initial molecular weight as substrate and DN as catalyst.The experimental results show that not only low molecular weight dextran can be obtained by enzymatic degradation,but also the homogeneity of dextran can be improved.Degradation was even more pronounced in the initial stages of enzyme degradation,especially for larger initial molecular weights and lower concentrations of dextran samples.During enzymatic degradation,high molecular weight dextran segments are preferentially degraded.During enzymatic degradation,high molecular weight dextran are preferentially degraded.In addition,under the existing experimental conditions,the percentage of 104-105 Da fragments in Dex-270,Dex-1000,Dex-2000 and Dex-F samples tended to increase with increasing degradation time.Under different initial molecular weights,different substrate concentrations and different enzyme concentrations,the kinetics of dextran molecular weight degradation are in accordance with the second-order kinetic equation.(4)Comparative study on the degradation process of enzymatic degradation and ultrasonic degradation.The experimental results showed that,for both the rule of degradation process,the higher the initial molecular weight of the sample,the lower the initial substrate concentration,the more obvious the degradation,the molecular weight decreased most rapidly at the beginning of degradation(0-10 min).And the dextran molecular weight degradation kinetics were in accordance with the second-order kinetic equation.However,for low molecular weight dextran,enzymatic degradation is more pronounced than ultrasonic degradation.At the same time,the D value of dextran also decreased with the decrease of molecular weight under ultrasonic field degradation.The D value of dextran firstly increased and then decreased under the conditions of enzymatic degradation trend,and the larger the molecular weight,the easier it is to reach the maximum of D value.As both reactions proceed,the molecular weight homogeneity of dextran degradation products both increased.In the last stage of degradation of Dex-F,under the conditions of enzymatic degradation,large molecular weight(>106 Da)dextran was completely degraded,while dextran with large molecular weight(>106 Da)still exists under the conditions of ultrasonic degradation.In comparison,enzymatic degradation is easier to obtain different low molecular weight dextran,and the molecular weight range is more concentrated.