Cloning,Modification And Bioinformatics Analysis Of Ferulic Acid Esterase Gene

Ferulic acid esterase(FAE)belongs to the hemicellulose-degrading enzyme system,which can hydrolyze the ester bond between the polysaccharide and polysaccharide,the polysaccharide and lignin.It is beneficial to hydrolyze the cell polysaccharide from the cell wall.FAE has a wide range of applications in bioenergy,food,pulp paper and biosynthesis.At present,the activity,reaction conditions and stability of FAE has a large space for improvement.There are relatively few mature commercial FAE preparations in the market,so the application is not large-scale in industry.So the significance of cloning and modification of FAE are very important.The heterologous expression of enzyme often forms inclusion bodies without biological activity.In this study,three fusion protein tags such as maltose binding protein(MBP)are added to the front of the Aspergillus clavatus FAE gene in order to increase the solubility.The activity and solubility of the esterase is analyzed.It has been determined that AcFae with maltose binding protein(MBP)showed a characteristic of FAE-D.At present,these recombinant plasmids with fusion labels are still under study.In this study,bioinformatics softwares were used to study the physicochemical properties,secondary structure,tertiary structure and evolution history of bifunctional xylanase-ferulic acid esterase of Prevotella ruminicola 23.By homologous alignment,the enzyme has been identified containing two domains,which provided theoretical basis for follow-up study.In addition,in order to obtain FAE with high viability and stability,the bifunctional Xylanase-Ferulic acid esterase gene has been cloned from Prevotella ruminicola 23 genome and expressed in E.coli BL21(DE3)successfully.The results showed that the optimal temperature of the enzyme was between 35 ? and 40 ?,and the optimum pH was about 7.0.The effects of various solutions on activity were different.For studying the catalytic mechanism and structure of bifunctional enzyme,two parts of bifunctional enzyme gene were cloned by bioinformatics analysis and PCR.The results showed that the optimal conditions of enzyme did not change,but the activity of single enzyme decreased after separation,and FAE lost the ability of decomposing ethyl ferulate.In this study FAE was modified by added 17 amino acids,signal peptides before the sequecne and truncated domains for improving the activity and stability.The enzymatic properties of the modified FAE were also studied.The results showed that the truncated FAE lost activity;the optimal conditions of FAE added 17 amino acids and signal peptide did not change,but the activity is further reduced.