Construction And Expression Of Thermostable Xylanases And Determination Of Its Enzymatic Properties

Xylanase is one of the most important digestive enzyme and widely used in paper pulp,feed and biofuel industrial.Especially,in feed industrial,xylanase can improve the animal performance and reduce pollution.However,most of the natural isolated xylanaes can not competent in the conditions of high temperature and the extreme pH in industrial.Thus,thermo-stable xylanase has gain much attention in the last decades.Especially,the GH 11 family xylanases have gain the most foucs due to its high specific activity,substrate specificity and simple structure.In this work,we are aiming to improve the thermostability of Xyn2 by genetic engineering.As the previous study indicated that the domain with high B-factor value was defined as unstable.Thus,in this work,the two unstable regions in Xyn2 was decided by the B-factor and stabilized by introduced disulfide bond respectively.The two mutant genes named Xyn2C14-52 and Xyn2C59-149 are subsquently expressed in E.coli for enzymatic properties analysis.And then,the mutant genes were introduced to the P.pastoris expression system for high-efficiency expression.Furether more,the most effective xyn2C59-149 was used in broiler.The main results of this work were listed as flowing:Experiment 1:Cloning of Xyn2 and constructing of mutant Xyn2The T.ressei were induced with beech-Xylan and then collected for isolation of total RNA.The cDNA was reverse transcribe by RT-PCR based on the template of total RNA.Then the gene Xyn2 was successfully amplified by a pair of specific primers and cloned to the T-vector.The nucleotide sequences of the Xyn2 were sequenced by Sangon Co.And the amino acid sequences are speculate based on the nucleotide sequences.The three-dimensional(3D)structure of Protein Data Base code 3akq was chosed for template of the Xyn2 by sequence alignment on Protein Data Base(PDB).Then we determined the unstable domain between the N-terminal and the ?-core based on the B-factor of 3akq.Thus,the mutant Xyn2C14-52 with the mutantion of F14C and Q52C were designed to form a disulfide bond in the unstable domain.In addition,based on the previous studty in the region of a-helix,we determined a more efficient approach to fixa-helix by introducing a disulfide bond between thea-helix and the p-core with the mutantion of V59C and S149C(Xyn2C59-149).According to the molecular interaction analysis,the Cys14-52 and Cys59-149 have the possibility to formed two disulfide bonds in the Xyn2 respectively.Experiment 2:Expression the Xyn2 and its mutants in E.coliThe gene Xyn2,Xyn2C14-52 and Xyn2C59-149 were successfully constructed into the expression vector pET32a.And the recombinant proteins were successfully expressed in E.coli Origami(DE3)by each recombinant expression vector with the inducer IPTG.The specific activity of Xyn2,Xyn2C14-52 and Xyn2C59-149 were 617.31?313.97?524.17 U/mg.Comparing to the wild type of Xyn2,the mutants of Xyn2c 14-52 and Xyn2C59-149 were improved a lot in thermostability and alkali-acid resistance.In brifly,incubating the Xyn2 and its mutants in 70 ? for 10min,the wild type Xyn2 almost lost all of its activities whereas the mutants Xyn2C14-52 and Xyn2C59-149 were all remained over 20%relative activities.In addition,the mutants Xyn2C14-52 and Xyn2C59-149 shown a better resistance to the acid condition and the Xyn2C59-149 also shown a better resistance to the alkali condition.However,the introducing disulfide bonds have no influence in the optima pH and temperature of Xyn2,except the optima temperature of disulfide bond in Xyn2c14-52.Experiment 3:Expression the Xyn2 and its mutants in P.pastorisThe genes Xyn2,Xyn2C14-52 and Xyn2C59-149 were successfully integrated to the genome of P.pastoris by the shuttle plasmid pPICZaA and expressed a 20 kDa recombinant protein with the inducer of 1%methanol of each recombinant P.pastoris.According to the glycosylation prediction and validation,indicated that there is no difference of glycosylation between the Xyn2 and its mutants.In addition,the glycosylation have no influence to the trend of the optima pH and temperayure alos the pH stability and thermostability comparing to the E.coli.However,after a treatment of glycosidase the thermostability of the Xyn and its mutants were significant decreased which indicated that the protein glycosylation have an advantage in protein thermostability.Experiment 4:The condition optimization of the Xyn2C59 149 expression in P.pastoris and analysis of enzymatic performanceThe Xyn2C59-149 were chosen for the expression condition optimizing.The results indicated that the concentration of methanol are the most important factor in expression of Xyn2C59-149 in P.pastoris.And the expression level is up to 420.1 U/ml in the optima expression condition at 29.43 ?,pH 5.93,methanol concentration(v/v)1.28%.Moreover,after feeding broiler with 5000 U/kg uncoated Xyn2C59-149 for 24 h the chyme were remained 2297,2472,1680 U/kg residual enzymatic activity in duodenum,jejunum and ileum respectively.That indicated that the xylanase Xyn2C59-149 have a better resistance to the physicochemical environment and protease in the alimentary canal.In conlusion,in this work,we obtain two thermostable xylanases Xyn2C14-52 and XynC59-149 by site-directed mutagenesis.The Xyn2C14-52 and XynC59-149 have a better thermostability and alkali-acid resistance comparing to Xyn2.And the recombinant P.pastoris reduced the difficulties of purification and improved the biological activities(specific activity,substrate binding capacity and thermostability)of recombinant Xyn2C14-52 and Xyn2C59 149.In addition,after feeding broiler with uncoated Xyn2C59-149 the results indicated that the xylanase have a nice resistance to the physicochemical environment and protease.Thus our study provides an efficient route to create a thermostable xylanase and obtained a xylanase Xyn2C59-149 possed Industrial application value.