Molecular Modification Of Xylanase And Construction Of Xylanase@MOFs For Transformation Of Xylosyl Substances From Plant

Xylanases are glycoside hydrolases that degrade xylan,among which,endoxylanase andβ-xylosidase are two key enzymes,since they can degrade the backbone of xylan to produce xylooligosaccharides or xylose.Xylose is an important sugar that is converted to xylitol,lactic acid,ethanol,and other chemicals.Xylanase also hydrolyzes the xylosylated substances from plants to produce bioactive substances,and xylosylates sugars,alcohols,phenols and other substances through transxylosylation to generate various xylosylated substances with potential functions.However,natural xylanase would be inactivated,unstable and unrecyclable in industrial application.Enzyme engineering is difficult to improve activity and stability of enzyme at the same time,and it depends on the structural data of enzyme,while the structures of many enzymes have not been resolved.In this work,in order to obtain the rubost xylanases which convert xylan and notoginsenoside R1 efficiently,the molecular modification and immobilization by metal organic frameworks（MOFs）were used to improve the activity and stability of endoxylanase andβ-xylosidase derived from thermophilic microorganisms.The research contents include:（1）The fusion of xylanases and its application in conversion of corncob xylan.Theβ-xylosidase（xln-DT,derived from Dictyoglomus thermophilum）and endoxylanase（Xyn F,derived from Herbinix hemicellulosilytica）were obtained by heterologous expression.Determination of biochemical properties showed that the two enzymes had similar optimal reaction conditions,but xln-DT showed some superior performances.In this work,xln-DT was fused into Xyn F with different peptides and directions to promote chimeras to perform better.The result showed that the catalytic efficiencies of chimeras Xln-L0-Xyn F and Xln-L3-Xyn F were 31-fold and 20-fold enhancement than that of Xyn F respectively,and maintained the same high stability as parental enzymes.This might be attributed to the appropriate linkers and the direction which decrease the adverse interactions between the two modules.These two chimeras could hydrolyze corncob xylan to produce approximately 30%XOSs with polymerization of 2-6.The XOSs exhibited higher antioxidant activity.（2）The mutagenesis of xln-DT and its application in the extraction of saponins.Five mutants of xln-DT were constructed semi-rationally,then their biochemical characteristics and corresponding structural features were investigated in detail.These mutants all exhibited higher catalytic efficiency toward p NPX compared with the wild type.Among them,the catalytic efficiencies of S160N-R333Q and S160N-R333H were 14 times and 12 times than that of the wild type,respectively.S160N-R333H exhibited a wider optimum temperature range and higher stability.Structural analysis proved that the amino acids in 160 and 333positions were critical for the substrate binding and stability of xln-DT.S160N-R333Q and S160N-R333H were combined with endoxylanase Xln-L0-Xyn F to extract ginsenoside Rg1from Panax notoginseng coupled with ultrasonic-assisted technology.After the optimization of conditions,the extraction yield of ginsenoside Rg1 reached 135.2 mg g^-1,which was mainly attributed to the conversion of notoginsenoside R1 to ginsenoside Rg1 by theβ-xylosidase mutants.（3）Construction ofβ-xylosidase-MOF catalytic system and its application in the conversion of notoginsenoside R1.The mutant S160N-R333H（R333H）was immobilized on a metal-organic framework Ui O-66-NH₂.The biochemical properties of immobilized R333H were investigated in detail.The results showed that improved performances were achieved,which was attributed to the change of the secondary structure ratio of the enzyme and the adsorption of MOF.The reusibility of immobilized R333H in the conversion of notoginsenoside R1 was further explored.The results showed that the conversion rate remained higher than 80%after six recycles in pure water and Mc Ilvaine buffer.In the water system,the xylose produced by hydrolysis of notoginsenoside R1 was converted sequentially by uncovered Ui O-66-NH₂composition to produce furfural.The furfural yield was increased with the increase of cycles,since the catalytic sites of the MOF were exposed due to enzyme desorption.The furfural yield obtained by uncovered Ui O-66-NH₂reached 69%.（4）Construction ofβ-xylosidase/endoxylanase-MOF catalytic system and study of its enzymatic properties.Improving the activity ofβ-xylosidase in high temperature and acetone is important for the conversion of xylan,phytochemicals and some hydroxyl-containing substances to produce xylose and bioactive substances.In this study,theβ-xylosidase mutant R333H and endoxylanase Xln-L0-Xyn F（Xyn F′）were simultaneously coimmobilized on the MOF of Ui O-66-NH₂.Compared with single R333H immobilization system,the optimum p H of coimmobilized R333H shifted from 6.0 to 5.5;the coimmobilization enhanced the activity of R333H in high temperature and high concentration of acetone,which suggested that Xyn F′had an impact on the structure or microenvironment of R333H.After 5 cycles,the relative activities of the co-immobilized enzymes towards p NPX and corncob xylan were 52%and70%respectively,and the accumulated amount of reducing sugars obtained by co-immobilized enzymes degrading corncob xylan in 30%（v/v）acetone solution was 1.7times than that with no acetone.