A Preliminary Study On The Specific Mechanism Of Hydrolysis Of Styntomyces Rameus L2001 GH11 Xylanase XynA

Xylanase(EC 3.2.1.8)is a type of hydrolase that cleaves xylan?-1,4 xylosidic bonds,and its industrial applications are extensive.In order to meet the needs of industrial production,it is necessary to conduct in-depth research on the characteristics of xylanase,especially the mechanism of its hydrolysis characteristics.GH11 is a recognized typical xylanase with the smallest?-sheet structure.Taking it as a research object is typical for exploring the inherent mechanism of xylanase characteristics.To this end,this study selected the GH11 xylanase XynA produced by Streptomyces rameus L2001 as the research object to explore the influencing factors of its hydrolysis characteristics,thus providing a theoretical basis for explaining its hydrolysis specificity.Using Discovery Studio 2018 software,using Xln B2(ID:5ej3)and thermophilic GH11 xylanase(ID:3zse)as templates to simulate the three-dimensional structure of XynA,and using molecular docking analysis to simulate its docking with the hexahexaose molecule the key sites for enzyme-catalyzed production of specific hydrolysates.After referring to the results of relevant literature and structural analysis,4 N-terminal sites(N-terminal sequence substitution,R₂ region substitution,T30E,S33P),5 functional domains site(Y97A,R121A,P125A,T100C-Y117C,Q135A)and1 C-terminal site(Y179A)were designed.After obtaining the corresponding mutant gene by overlapping extension PCR technology,it was connected to the expression vector p ET-28a,and introduced into E.coli BL21(DE3)to obtain the mutant enzyme,and the crude enzyme solution was obtained by induced expression,which was pre-tested on the hydrolysis of beechwood xylan.It showed that the hydrolysis products of N-terminal mutants(XynAN,XynAR,XynAS),C-terminal mutant XynA179 and functional domain mutant XynA121 had significant changes.The original enzyme XynA and five mutant enzymes were induced to express and purified by nickel column to obtain pure enzyme.The systematic analysis of the nature,the results are as follows:(1)N-terminal mutants:Compared with XynA,the optimal temperature of XynAS and XynAR is increased by 10?and 15?,respectively,to 70?and 75?,XynAR is kept at 80?for 30 minutes and the residual enzyme activity still reaches 90%above,the half-life of XynAS increased by 2.5 times at 60°C,reaching 210.0 min.The temperature stability of both was significantly improved,while the optimal temperature of XynAN was reduced by 20°C to 40°C,and its half-life was 21.9 s,a decrease of231.5 times,its heat resistance is significantly reduced;the specific enzyme activity of XynAS and XynAR is increased,and the specific enzyme activity of XynAN is reduced;the efficiency of XynAS and XynAR hydrolysis of xylan is improved,of which XynAR is mainly reflected in the shortened hydrolysis time and the production of xylobiose and xylotriose Significantly increased,indicating that the five sites in the R₂ region are the key non-functional regions that affect the hydrolysis of xylan to form xylobiose and xylotriose;XynAS is mainly reflected in the shortened hydrolysis time and the significant increase in the production of xylotetraose,indicating that position 33 is affects the hydrolysis of xylan to produce key non-functional areas of xylotetraose;XynAN hydrolyzes beechwood and oat-spelt xylan,the ratio of hydrolysate is changed,and the efficiency of hydrolysis of birchwood xylan is improved;The N-terminal mutant enzyme is the same as the original enzyme,neither hydrolyzes xylobiose and xylotriose,and has the same mechanism of hydrolysis of xylotetraose and xylopentose.The N-terminal is the unfolding start and is located at the non-reducing substrate recognition site.It plays a key role in the structural stability of the enzyme and substrate recognition.Changing the N-terminal site will affect its enzymatic properties,including hydrolysis properties.(2)C-terminal mutant:the optimal temperature of XynA179 is 60?;the specific enzyme activity is reduced;and the ratio of hydrolyzed xylan product is different from that of the original enzyme.Decreased,which indicates that the 179 site is the key site for hydrolyzing xylan to generate xylobiose;XynA179 does not hydrolyze xylobiose and xylotriose,and the mechanism of hydrolyzing xylotetraose and xylopentose is different from that of the original enzyme.The C-terminal is at the site of product release at the reducing end,which may play an important role in product release.(3)Functional domain mutant:XynA121 has a half-life of 45.0 min at 60°C,which is 1.9 times lower than the original enzyme;the specific enzyme activity is reduced;its main product of hydrolyzing xylan is the same as N-terminal and C-terminal mutant enzymes,and its hydrolysis Increased efficiency;XynA121 does not hydrolyze xylobiose and xylotriose.The mechanism of hydrolyzing xylotetraose and xylopentose is the same as the original enzyme.The 121 position of the functional domain is located near the catalytic crack,which has a certain effect on substrate binding.This paper comprehensively analyzes the effects of the N-terminal,C-terminal and functional domains of GH11 xylanase XynA on the hydrolysis characteristics.The results show that in addition to the sites near the functional domain that affect the hydrolysis characteristics of xylanase,other non-functional domains It also has an impact on its hydrolysis characteristics.This research result is conducive to a comprehensive and systematic exploration of the mechanism of xylanase hydrolysis to produce specific products,and provides important research ideas and theoretical guidance for improving the hydrolysis specificity of GH11 xylanase.