The Thermoduric Effects Of Site-directed Mutagenesis And The Introduction Of Disulfide Bond On Dextransucrase

Dextransucrase（DSR,EC2.4.5.1）from Leuconostoc mesenteroides 0326 is a glucansucrase,producing dextran with sucrose as substrate.It has been widely used in food and pharmaceutical industries.However,the kinetic stability of this enzyme at higher temperature is poor,which restricts the development of industrial applications.The aim of this study is to improve the enzyme activity and thermal stability of dextransucrase by site-directed mutagenesis and the introduction of disulfide bonds.In this work,on the basic of the cloning dex-YG gene of dextransucrase from Leuconostoc mesenteroides 0326.The 3D model structure of dextransucrase is established by homologous modeling,and the mutation sites are selected with the semi-rational design carried out.Pro-473,Pro-678,and Pro-856 were selected as engineering targets and replaced with serine,Lys-378,Lys-725,and Lys-955 were replaced with threonine,respectively.Mutant P473S/P856 S was chosen as the highest enzymatic activity mutant from fourteen mutants.Specifically,the mutant P473S/P856 S showed a 2.86-fold increase in enzyme activity,a significant increase in the half-life of thermal inactivation with a 7.4-fold increase at 35℃and a 2-fold increase in catalytic efficiency compared with the wild-type and the thermal stability is obviously improved.The results of structural simulation demonstrate that the new intramolecular hydrogen bonds in mutated enzymes may increase the structural stability of the protein,thereby increasing the thermal stability.The disulfide bonds play an important role in stabilizing the protein structure and maintaining its active function.114 potential disulfide bonds were obtained by Disulfide by Design^TM,and the disulfide bonds were introduced using the plasmid of P473S/P856 S as a template.8 disulfide bonds were constructed,respectively,such as H323C-D351 C and L341C-S345 C.The enzyme activities of all the mutants decreased after mutagenesis.The optimum temperatures of L838C-V887 C and A948C-A1013 C were improved to 35℃;the products of D739C-F932 C and A948C-A1013 C catalyzed synthesis of dextran contain 15.8%α（1-4）glycosidic bonds and 2.5%α（1-2）glycosidic bonds,indicating the catalytic specificity of these mutants were changed.Bioinformatics modeling showed that disulfide bonds formed after mutagenesis and more hydrogen bonds were introduced,but the thermal stabilitities of the mutants were not improved significantly.It may be due to the introduction of disulfide bonds,the conformation of the enzyme has changed greatly that effects the enzyme stability.This study provided the basis and strategy for improving the thermal stability of recombinant dextransucrase.The three-dimensional structure of the enzyme still has some hot spots that has not been found,and it is expected to obtain the dextransucrase with good heat resistance and low hydrolysis reaction.The mutant dextransucrase has an enormous potential of application in food and pharmaceutical industry.