Phylogenetic Analysis Of The GH43 Family And Modification Of The XynZT-2 Gene From Alteromonas Macleodii

BackgroundXylanases were a class of enzyme systems that degrade plant cell walls.Thus,Xylanases had a wide range of industrial applications.Xylanases were derived from the GH10,GH11,GH43 and other Glycoside Hydrolase families.Because of their small molecular weight and simple spatial structure,the GH10 and GH11 families have been extensively studied,while the GH43 family has been less studied.In order to determine the evolutionary pattern,the genetic analysis of the gene population was carried out on the GH43 from different species sources.Then,the molecular modification of the enzyme was conducted to investigate the differences in enzyme activity between the recombinant enzyme and the original enzyme.This evolutionary relationship analysis and the study of amino acid sites near the active center of xylanase might be a guidance for the later molecular modification of GH43 xylanase.Objectives1.It was identified by bioinformatics analysis that the population evolution pattern of the GH43(Glycoside Hydrolase 43)family and the non-strictly conserved sites near the active center of the GH43 family.2.The targeted modification of Xyn ZT-2,xylanase from Alteromonas macleodii was conducted to explore the evolutionary relationship between GH43 family gene sequences and their functions Methods1.A phylogenetic tree was constructed based on the structural domain of the GH43 family xylanase gene.The xylanase gene xyn ZT-2 was used as an example to compare homologous sequences of closely related species using MEGA and select mutation sites.2.Homologous sequence comparison of xylanase gene xyn ZT-2 was performed by MEGA.Three-dimensional modeling and analysis of Xyn ZT-2 were performed by SWISS-MODEL and Py MOL.3.In base of using the xylanase gene of Alteromonas macleodii from marine as a template,the mutant gene was constructed by using the overlap extension PCR method.After the mutant gene and plasmid p ET28 a were double digested,ligated,and transformed into E.coli,the mutant strain was obtained.The enzyme was induced from the mutant bacteria.Then,the enzymatic properties of the mutant strain were determined after purified.Results1.Evolution: The GH43 genes arose from bacteria,archaea,fungi,and plants prior to differentiation.At the same time,the GH43 genes were not present in animals.2.Homologous sequence comparison revealed the differences of the amino acid near the active center of the near-derived GH43 xylanase from different species sources.3.The sites which were near conservative areas of the five groups of ?-folded lamellae of Xyn ZT-2,xylanase from Alteromonas macleodii were identified.Then they were mutated with T28 I,A33V,M97 L,A102C,S148 T,A152G,A238 G,S239P,M241 L,T287S,and I291 V to obtain the corresponding mutant genes.After ligating to vector p ET28 a,the corresponding mutants were successfully transferred into BL21(DE3)to obtain the recombinant bacteria.4.After the mutant bacteria were incubated and induced,the specific activities of 11 mutants were obtained.Compared with the original specific activity of 1.068 U/mg,six mutant enzymes Xyn ZT-2A33 V,Xyn ZT-2S148 T,Xyn ZT-2A152 G,Xyn ZT-2A238 G,Xyn ZT-2S239 P,Xyn ZT-2T287 S had increased specific activities,and the specific activity could be increased up to 10.472 U/mg.The specific activities of five mutant enzymes were decreased,and the lowest specific activity could be decreased to 0.13 U/mg.5.The enzymatic properties of the enzymes showed that the optimum temperatures of T28 I,A33V,M97 L,A102C,S148 T,A152G,A238 G,S239P,M241 L,T287S,and I291 V were 45?,40?,80?,50?,45?,65?,40?,45?,40?,45?,60?,respectively;while the optimum p H of which were p H 7.0,p H 7.0,p H 8.0,p H 7.0,p H 7.0,p H 6.5,p H6.5,p H 6.5,p H 7.5,p H 7.0,p H 6.0,respectively.Compared with the optimum temperature45? and optimum p H 6.5 of the original enzyme Xyn ZT-2,all mutation sites showed changes in enzymatic properties.Conclusions1.Evolution: With widely distributed,the GH43 family had an early origin and conserved active centers.There were amino acid differences near the active center of the near-derived GH43 xylanase from different species sources.2.Amino acid alterations in closely related species caused differences not only in enzyme activity,but also in optimum temperature and/or optimum p H.3.The phylogenetic laws could be used for the modification of GH43 family xylanases on the basis of ensuring the presence of enzyme activities.