Study On The Catalytic Reaction Mechanism Of Xylanase

Xylanase is a kind of biological enzyme widely existing in nature.It mainly comes from bacteria,actinomycetes and fungi.Xylanase acts on the glycosidic bonds of oligosaccharides and polysaccharides by hydrolysis,which degrades them into xylo-oligosaccharides,but also produces a small amount of xylose.Xylanase is widely used in paper industry,food industry,feed industry and bio-energy industry.It is because of the broad industrial application prospects of xylanase that more and more researchers devote themselves to the development and research of xylanase.With the development of genetic engineering,protein engineering,bioinformatics,enzymology,crystallography and computational biology,xylanases with different enzymatic characteristics have been discovered.Because of the mild reaction conditions of xylanase and the need for high temperature and acid and alkali resistance in industrial production conditions,the application of xylanase in industrial production is very difficult.Therefore,in order to make xylanase play a greater role in industry,we urgently need to study the mechanism of xylanase enzymatic reaction more deeply,and guide protein modification according to the catalytic reaction mechanism,so as to improve the application effect of xylanase in industry.A xylanase from Trichoderma reesei has been studied in this paper.The wild type and mutants of xylanase with high quality were obtained by cloning xylanase gene,expression in prokaryotic cells,affinity chromatography and ion exchange chromatography.In this paper,xylanase was purified by the principle of specific adsorption of His tag and Ni column,and the target protein was obtained by innovative two-step purification of Ni column.This method shortens the time of traditional purification method and improves the purification efficiency.In this experiment,a solubilizing protein Nusa was added to the N-terminal of xylanase,which increased the soluble expression of xylanase in E.coli and reduced the formation of inclusion bodies.Protein crystallography was used as a means to study the catalytic mechanism of xylanase.High quality protein crystals were obtained by suspension drop method and sitting drop method.The required crystals were X-ray diffracted to obtain volume diffraction patterns,and the spatial structure of protein molecules was calculated by the crystal diffraction patterns.This method studies the mechanism of xylanase catalytic reaction by observing the changes of the internal space structure of protein molecules in the most intuitive way.The residues Y88 and Y77,which directly form H bonds with E86 and E177,are very important for catalysis.The functions of these residues have been studied by theoretical pKa calculations using high resolution X-ray crystallography.Our results show that these conserved tyrosine residues not only change the pKa value of catalytic glutamic acid,but also change the conformation of xylose in the active site to promote catalysis.The results of high resolution X-ray crystals obtained in this study support the energy travel of 1 glycan ring in the xylanase hydrolysis process as 1S3?4H3?4C1,which indicates that the flexibility of 1 glycan ring is very important for breaking glycoside bonds.Since the amino acid residue N44 is close to the cleavage position of the glycosidic bond,it is hypothesized that whether D44 can be used as a proton donor like E177 after mutating N44 to D44 or E44 is even possible to form a coupled dicarboxylic acid system-enhancing enzyme in mutant N44D.live.However,this speculation was negated by the binding of the mutant structure to the enzyme activity.Based on the crystal structure of wild xylanase and its mutants,the mechanism of xylanase enzymatic reaction was systematically studied,and the mechanism of xylanase enzymatic reaction was improved,It provides a theoretical basis for more efficient utilization of xylanase in industry.