Improving The Alkalophilic Performances Of Family 11 Xylanase By Molecular Modification

Xylanase is the key enzyme that can degrade xylan,the main component of plant hemicellulose.It was widely used in animal feed,food,paper,bio-energy and other industries.Xylanase could reduce the use of toxic chemical bleach agents in pulp bleaching process,thereby reducing the toxicity in wastewater and the pollution of the environment.Because of smaller molecular mass and cellulase-free activity,family 11 xylanases are more suitable in pulp bleaching process.The pulp is at the environment of high temperature and alkaline p H in pulp bleaching process.To reduce energy consumption and simplify the process,xylanases are required to be active at high temperature and alkaline p H.But the wild-type family 11 xylanases that isolated from nature are mostly neutral,and the number of alkaliphilic xylanases are rare.Therefore,it is necessary to carry out molecular modification of the existing wild-type family 11 xylanases to obtain the mutant xylanase improved alkalophilicity.The present research randomly mutated xyn11A-LC gene to establish mutation library by utilizing Gene Morph II Random Mutagenesis kit,and then screened mutant xylanase with improved alkalophilicity.The results were as follows:1.A mutation library containing 5000 clones was obtained by randomly mutated xyn11A-LC gene.2.By screening the mutation library for significantly improved alkalophilicity,mutant M52-C10 was found.The optimum p H of mutant M52-C10 is pH8.0,which is higher than the the wild type by 0.5 units,the relative enzyme activity of 10-15% was improved compared with the wild type in p H8.5-p H10.0,the optimum temperature of M52-C10 is 55?,and it have better alkali-tolerance and thermostability.3.Analysis of the sequencing results of M52-C10 showed that it contained two amino acid mutations,which were E135 V and V116 A,respectively.E135 V has a major effect on the alkaline improvement of the M52-C10.The optimum p H of E135 V is p H8.0,the relative enzyme activity of 10-20% was improved compared with the wild type in p H8.5-p H10.0,the optimum temperature of E135 V is 55?,and it have better alkali-tolerance and thermostability.4.E135 K and E135 R were obtained by site-directed mutagenesis.The optimum p H of E135 K is p H8.0,and the relative enzyme activity of 10-19% was improved compared with the wild type in p H8.5-p H10.0;the optimum p H of E135 R is p H8.0,which is higher than the the wild type by 0.5 units,and the relative enzyme activity of 15-25% was improved compared with the wild type in p H8.5-p H10.0,the optimum temperature of E135 K and E135 R is 55?,and it have better alkali-thermo stability.5.Three mutants,E135 V,E135K and E135 R,were obtained in this research,which were significantly alkalophilic higher than the wild type xylanase xyn11A-LC.