Study On Improving The Alkali Resistance Of Xylanase By Combination Mutation

Xylanase,as a naturally occurring enzyme,has a wide range of distribution.It has been found in the stomach of herbivores,marine microbes,terrestrial microbes,etc.Its main role is to reduce the amount of hemicellulose,leading to wide application in feed production,food processing,bioenergy manufacturing and paper bleaching industries.The application of G / 11 family xylanase to bleach paper can significantly reduce the dependence of chlorine on white matter in papermaking processes because of its smaller molecular weight and its specific degradation of hemicellulose,which influence the pulp whiteness.Paper industry has it's own requirements in the process,the pulp bleaching process always doing in high temperature and high pH environment,therefore,it`s required that the xylanase added into the pulp must have a relatively stable activity at a higher temperature and higher alkalinity.At present,the utilization of gene recombination and protein engineering means to improve the performance of xylanase has been reported successfully in many reports,but the alkali-resistant characteristic of xylanase improvement was rarely reported.This is because the different types of xylanase have a significant difference in the alkali resistance mechanism.Although in the same family of glycoside hydrolase,but because of its long-term evolution in the different alkaline environment,the adaptation strategy and alkali-resistant mechanism is different,too.At present,the study of alkali-resistant of G / 11 family xylanase is still underway,and the mechanism of alkali-resistant in homologous xylanase have no unified understanding.In this study,a random mutant library of target gene was established by the method of error-prone PCR and double digestion.The library capacity was 11063 monoclones.After two rounds of screening,four mutants of D89 R,E109R,E135 R and E160 R were selected,and their relative enzyme activities were about 15% higher than those of wild-type strains in the range of pH 8.0-9.5.The E135 site had a relative enzyme activity of 20% higher than that of the wild-type strain at pH 9.0.The optimal pH value of E135 A,E135M,E135 T,E135Y,E135 H and E135 Q mutant enzymes were found to be 8.0.E135 P mutant enzyme optimal pH value was 6.0.The optimum pH of the other mutant enzymes were consistent with that of wild-type enzymes.On the basis of that,we combined the four mutant sites,and the substrate affinity and catalytic efficiency of each mutant enzyme was higher than that of the wild type one.The optimum temperature was consistent with the wild type enzyme,and the pH stability was also better than that of the wild type enzyme,which was stable at pH 4.5-10.5.The optimum pH value of the four-site mutant enzyme was 8.5 which was 1.0 pH higher than the wild type.The optimum pH of the three-site mutant enzyme is 8.0,which is 0.5 pH higher than the wild type.Preliminary analysis,suggesting that its alkali resistance is enhanced with the distribution of negatively charged amino acid residues on the surface of the enzyme molecule that was mutated to positively charged amino acid residues.Three-dimensional simulation of protein structure revealed that the mutated E135 R site may form an arginine guanidine group with D89 R,which changes the molecular structure of the enzyme and increases the alkali resistance of the mutant enzyme.This research provides a feasible method for the modification of G / 11 family xylanase of Xyn11A-LC,and also provides some help for the study of alkali resistance of xylanase.