Modification And Immobilization Of Xylanase Xyn? From Trichoderma Reesei And Construction Of Xylan Metabolic Pathway

Xylanase?EC 3.2.1.8;endo-?-1,4-D-xylanase?,a hydrolytic enzyme demanded in depolymerization of xylan with?-1,4-xylanolytic linkages,finds application in many industrial processes such as prebleaching of paper pulp,fruit juice clarification,bioconversion of agricultural residues,extraction of oils from plants,and improvement of animal feed stock digestibility.The xylanase Xyn II from Trichoderma reesei has the advantages of high catalytic activity and good stability,and has a good application prospect.In this study,heterologous expression,enzyme immobilization and molecular modification of XynII were performed.Based on this,Escherichia coli was used as the expression host to construct a new xylan metabolic pathway,which laid the foundation for the comprehensive utilization of biomass resources with xylan as the main component.Firstly,in this study,a magnetic agarose metal microsphere carrier was designed and prepared,and the xylanase Xyn II was purified and immobilized for the first time.The effects of the immobilization conditions,enzyme concentration,temperature,and pH on the catalytic activity of the enzyme were studied systematically.The results showed that the immobilized xylanase showed better thermal stability than that of the free enzyme,its thermal stability was higher than that of the free enzyme,and the immobilized enzyme had good storage stability,and retained 80%activity after 35 days of storage.The immobilized enzyme maintained a very high recycling rate.Under repeated use,the remaining enzyme activity was about 80%.Interestingly,the immobilized xylanase has higher organic solvent stability than the free enzyme.For example,in the 25%ethanol,acetone and butanol solution,the immobilized enzyme activity is 1.5,1.8,and 1.8 times of the free enzyme,respectively.It showed that the enzyme immobilized on the magnetic polymer metal microspheres should be particularly suitable for catalyzing reactions in organic solvents.Secondly,in the present study the curdlan,Ca²⁺and Co²⁺were used for the preparation of magnetic curdlan metal microspheres for the firest time,and the purification and immobilization of the xylanase Xyn II were achieved simultaneously.The effects of the immobilization conditions,enzyme concentration,temperature,and pH on the catalytic activity of the enzyme were studied.The results showed that the immobilized enzyme had a very good storage stability and retained 80%of its activity after 35 d of storage.In addition,the immobilized enzyme retained a very high recycling rate.The remaining enzyme activity was 80%when the enzyme was used repeatedly 5 times.The immobilized xylanase has higher organic solvent stability than the free enzyme.For example,in the 25%ethanol,acetone and butanol solution,the activities of immobilized enzyme were 1.5,2.83 and 17.4times that of the free enzyme,respectively.It showed that the the magnetic polymer metal microspheres based on curdlan,Ca²⁺and Co²⁺should be particularly suitable for immobilizing enzymes used in organic solvents.In particular,it is worth mentioning that magnetic curdlan Co²⁺microspheres immobilized enzymes were significantly superior to magnetic curdlan Ca²⁺microspheres and free enzymes in terms of thermal stability,indicating that the immobilized enzyme was more stability.In addition,the efficiencies of Ni-NTA and magnetic curdlan metal microspheres were compared for purification of proteins.The results showed that the magnetic curdlan metal microspheres had similar ability as Ni-NTA for protein purification,but the magnetic curdlan metal microspheres had better reusability than Ni-NTA.These results indicated that the magnetic curdlan metal microspheres might have a very wide range of application.In addition,by comparing the magnetic agarose metal microspheres and the magnetic curdlan metal microspheres,it was found that themagnetic curdlan metal microspheres had 1.5 times more immobilized amount of the enzyme than the magnetic agarose metal microspheres,indicating that the themagnetic curdlan metal microspheres have better carrier characteristics.Thirdly,we will adopt a new method of molecular modification of the xylanase Xyn II by the metagenomic segment shuffling srategy.In this study,we will identify the conserved segments of the enzyme by comparing the xylanase genes,and then use the CODEHOP principle to design degenerate primers in two adjacent conserved regions.With metagenomic DNA used as a template,these primers were used to specifically amplify DNA sequences between two conserved regions.Then the amplify DNA sequences was used to replace the corresponding segment of Xyn II by overlapping extended PCR to modify Xyn II.Through screening,8 mutant gene genes were obtained,which laid the foundation for the subsequent determination of enzyme activity,and also provided a reference for further construction of mutant enzymes.Compared with the traditional molecular engineering methods,the metagenome segment shuffling method can fully utilize the DNA resources of the metagenome library,and has the advantages of being simple,efficient,and short in the new gene screening period.Therefore,this method may find wide application in the future.Fourth,in this study,the xylose isomerase gene Dd-XIase from Dicheya dadantii was cloned for the first time and performed heterologous expression in E.coli.On this basis,a xylan metabolic pathway containing a xylanase gene,a xylosidase gene,and a xylose isomerase gene was constructed.The results showed that Dd-XIase has good temperature stability,and strong alkali resistance in pH research.Therefore,it may have a very high application value.In addition,studies have shown that 10%n-butanol and 10%ethanol solution can promote the enzyme activity of Dd-XIase,and the reason is worth further analysis.In terms of metal ion tolerance,Ag⁺,Ca²⁺,Co²⁺,Cu²⁺,K⁺,Li⁺,Mg²⁺,Mn²⁺,Rb²⁺,and EDTA all play a role in promoting enzyme activity.These results showed that Dd-XIase may have a high application value in harsh environments.The Dd-XIase had highest substrate specificity to D-glucose substrate,followed by D-xylose.On this basis,the xylanase gene,xylosidase gene and xylose isomerase gene were cloned into the same expression vector to construct a metabolic pathway that can metabolize xylan to produce D-xylulose.It has layed a solid foundation for the subsequent efficient use of xylan.