Fusion Modification Of Dextransucrase Based On Low Molecular Weight Dextran Biosynthesis And Its Molecular Mechanism

Dextran is a glucan composed of several glucose linked byα（1,6）bonds.Low-molecular-weight dextran is widely used in food and medicine,and its specific functions are classified according to its molecular weight.The synthesis and isolation of low molecular weight dextran is chemically difficult,environmentally unfriendly,and expensive and time-consuming.However,there are few studies on the one-step enzymatic synthesis of low molecular weight dextran with uniform molecular weight,and the catalytic mechanism needs to be further investigated.In this paper,we obtained dexYG,a dextransucrase derived from Leuconostoc mesenteroides 0326,and fused it with dextranase based on its structural and catalytic properties to obtain a fusion enzyme capable of one-step catalytic synthesis of low-molecular-weight dextran,and to elucidate its catalytic mechanism.We also performed structural domain fusion to obtain a variant enzyme with higher thermal stability.The main results are listed as follows:（1）Construction and expression of cascade fusion enzymes.Based on the cascade catalytic relationship and molecular structure between dextransucrase and dextranase,a bifunctional fusion enzyme（YnrlD,rl is a rigid linker,n=0,1,2,3,5,8）was constructed for the one-step synthesis of low molecular weight dextran from sucrose.The fusion enzyme library was constructed by changing the length of the linker between the two enzymes.The recombinant fusion plasmids involved in this paper were constructed by seamless cloning technique,and the purified enzymes were obtained by molecular sieving after expression.The purity and molecular weight（265 k Da）of the purified enzymes were examined by high performance liquid chromatography,and the enzymatic properties of these purified fusion enzymes were studied.The results showed that the fusion modification of the structural domain of dextransucrase resulted in a more stable enzyme structure,which in turn improved the temperature and pH stability of the enzyme.（2）Mechanistic studies on the synthesis of molecular weight-concentrated dextran by fusion enzymes.The one-step enzymatic conversion of constructed and expressed cascade fusion enzymes to low molecular weight dextran was studied and the corresponding substrate channel mechanism was revealed.Different lengths of linkers change the distance of the active center of the fusion enzyme,which in turn leads to the control of the homogeneous molecular weight of the product.The intramolecular channels of the fusion enzymes were demonstrated by product behavior analysis and the lag time required for the intermediates to reach steady state,and the substrate channel adaptation of this series of fusion enzymes was investigated.The results showed that the addition of appropriate linkers to the fusion enzymes produced dextran with homogeneous molecular weight（the weight average molecular weight is about 13050±1341 Da）.And the YrlD fusion enzyme had a high degree of substrate channelization and the smallest distribution coefficient(D_YrlD=1.36).In addition,the temperature kinetics of YrlD was combined to adjust the catalytic efficiency of the two enzymes in a"matched"state to increase the content of the target product to 62%.（3）Study on the mechanism and physiological activity of fusion enzyme synthesis of low micro-molecular weight dextran.A fusion enzyme（YD）that can catalyze the directional synthesis of low micro-molecular weight dextran with homogeneous molecular weight in one step was screened from the fusion enzyme library,and its catalytic mechanism was studied.The results showed that temperature acts as a"toggle switch"for the catalytic efficiency of the two-stage fusion enzyme and regulates the catalytic product of the fusion enzyme;under the conditions of catalytic temperature of35℃,sucrose concentration of 500 m M and final enzyme activity of 25 U/m L,the enzyme can effectively utilize 100%of sucrose and convert 70%of the glucose moiety into homogeneous low micro-molecular weight dextran.The product was purified and characterized as having a concentrated molecular weight of 1000±125 Da and containing almost noα（1,3）glycosidic bonds.Besides,the low micro-molecular weight dextran obtained in this study had significantly better probiotic function and anti-inflammatory activity than other oligosaccharides.（4）Fusion modification based on improved stability of dextransucrase.The paper concludes with a fusion modification of the structural domain of dextransucrase with the aim of improving the stability of the enzyme.By fusion extending the C-terminus,six structural domain fusion mutants of different sizes were constructed to obtain a new enzyme with higher stability.The experimental results showed that the optimal temperature of the structural domain fusion mutants was increased by 10℃compared with the original enzyme.dexYG-fdx（D-F）showed an increased half-life of 280%and200%at 35℃and 45℃,respectively,and a wider pH tolerance range.The structural simulation and molecular docking showed that the increase of structural domains reduced the terminal degrees of freedom of the enzyme to some extent,and reducing the structural domain degrees of freedom could improve its stability.In summary,the results of this thesis provide a strategy for the enzymatic targeted preparation of low molecular weight dextran and for obtaining stable dextransucrase with certain application value;meanwhile,they are scientifically important in the construction of fusion enzymes,the elucidation of catalytic mechanism and the controlled synthesis of polysaccharide polymers.