Molecular Modification Of Thermostability On The Xylanase XynZF-2 From Aspergillus Niger XZ-3S

BackgroundXylanases?EC3.2.1.8?are glycoside hydrolases that hydrolyze ?-?1,4?-D-xylosidic linkages of xylans,which are widely used in food processing,pulp bleaching,feed processing,and other area of industries.In general,Xylanases belong to the mesophilic xylanase.Because of the low activity and thermostability of xylanases,the applicability of xylanases in industrial processes is restricted under the high temperature conditions.Therefore,it has become a hot topic for transformation of the xylanase on the thermostability.ObjectiveIn order to improve the thermal stability of xylanase,the xylanase gene was oriented transformed.Methods1 The bioinformatics analysis on xylanasesThe Prot Param was used to analyze the physicochemical properties of xylanases.The homology sequence and the 3D structure of xylanases were performed using the phyre2.The 3D structure of xylanases were viewed and analyzed by DS Viewer Pro 6.0.The disulfide bridge was predicted by the Di ANNA 1.1 web server.The active center of xylanases was predicted by PROSITE.Through the analysis of bioinformatics on xylanases,the mutated sites of xylanases were acquired on themostability.2 Site-directed mutagenesis and the construction of the recombinant vectorThe mutated genes were amplified by overlap extension PCR method.Then,the expression vector pET-28 a and the genes gained by PCR were double enzyme digested.The mutated genes,were respectively inserted into pET-28 a,followed by transforming them into Escherichia coli BL21?DE3?.The resulting recombinant Escherichia coli were screened and aquired.3 Expression and purification of the variant xylanasesThe transformants were incubated with IPTG and expressed to aquired obtain the crude enzyme solution.The recombinant xylanases contained the His-tag protein were purified and separated with metal chelate affinity chromatography.4 Enzymatic properties analysis of the recombinant xylanasesOptimal temperature,thermal stability,optimal pH and pH stability of the purified recombinant enzymes were measured,of which effects were analyzed on the activity of xylanase.Results1 The active sites of xylanase XynZF-2 was successful predicted by the software of bioinformatics.The active sites were substituted by site-directed mutagenesis?E103D and E194D?and the mutated gene xynED was obtained.The amino acids residues?Val1 and Glu27?at the N-terminal domains were substituted into cysteine and the mutated disulfide bridge xynDC-encoding genes were constructed.The amino acids residues in the ?-helix and Cord domains were substituted into the hydrophobic amino acids residues?V125A,K178 M and G180A?,and the mutated hydrophobic xynMA-encoding genes were constructed.The N-terminus of xylanase XynZF-2 was substituted with the corresponding sequence of a hyperthermostability family GH11 xylanase Ev Xyn11.The corresponding sequence of xylanase Ev Xyn11 was introduced into aromatic residues?P9Y and H14F?,and the hybrid xylanase gene xynEV-34 was constructed.All of the mutated genes were expressed in Escherichia coli BL21?DE3?.2 The recombinant transformants,BL21/pET-28a-xynED,BL21/pET-28a-xynDC,BL21/pET-28a-xynMA and BL21/pET-28a-xynEV-34,were constructed expressed with IPTG.Then all of the mutated enzymes were analyzed by SDS-PAGE.It is confirmed that the mutant xylanase gene was expressed successfully in Escherichia coli BL21?DE3?.3 Compared to the native xylanase,the residual activity of the mutated xylanase XynED was 0.17%.4 With Ni²⁺ column affinity chromatography purification mutation enzyme,the level of electrophoresis pure enzymes were obtained.5 The optimum temperature and pH of variant XynDC was 45? and 5.0.The optimum temperature and pH of variant XynMA was 48? and 5.0.The optimum temperature and pH of variant XynEV-34 was 48? and 4.8.6 After treatment at 40? for 60 min,the XynZF-2,XynDC,XynMA and XynEV-34 retained 44.36%,66.77%,74.71% and 77.96% residual activity,respectively.Compared to the xylanase XynZF-2,t1/245 ? of the variant XynDC,XynMA and XynEV-34 were increased by 7 min,12 min and 16 min,respectively.The pH stability of the mutant enzymes were basically consistent with that of the original enzyme XynZF-2.Conclusions:1 The catalytic activity center of xylanase XynZF-2?Glu103 and Glu194?was predicted and confirmed.2 The variant xynED,xynDC,xynMA and xynEV-34 could be expressed in Escherichia coli BL21?DE3?.3 The introduction of two disulfide bonds,hydrophobic amino acids and aromatic amino acids,and N-terminal replacement could significantly improve themostability of the xylanase XynZF-2.