Recombinant Expression Of Thermophilic Xylanase Gene Umxyn10A In E.Coli And Its Thermal Stability Modification

Xylan is an heterogeneous polymer,it is one of the main components of hemicellulose.In the past decades,xylanase,a major member of the biomass degradation enzyme system,has become a hot spot of research,which has been widely used in many industrial fields such as textiles,food,feed,paper making and medicine.The xylanase mainly used for the production of oligosaccharides and xylose with different molecular weights by acts on the ?-1,4-glycosidic bonds of xylan.At present,most of the cloned expressed xylanase genes have poor thermal stability at high temperature,which cannot meet the demand of industrial production and greatly limits the application of xylanase.Therefore,it is of great significance to study the thermal stability of xylanase.The xylanase UmxynlOA(Gen Bank serial number ABL73-883.1)was screened from uncultured microorganisms in compost by our laboratory and belongs to the GH 10 family.The transmembrane domain of UmxynlOA was remove and the codon was optimized to clone the expression in E.coli,the results show,xylanase UmxynlOA optimum reaction temperature is 80?,but poor thermal stability,enzyme activity dropped substantially when the temperature exceeds 60 ?,the half-life of under 65 ? for 15 min,under 75 ?heat 4 min is almost the deactivation,under 75 ? heat inactivation almost 4 min.The optimum pH of the xylanase Umxyn10A is 6.0,which can maintain more than 80%of the enzyme activity in the range of pH 4.5 to 10.0.Besides,it has strong hydrolysis ability to xylan,this enzyme has no enzyme activity to carboxyl methyl cellulose,avicel,starch,chitin,lichenan 2-hydroxyethyl cellulose,and methylcellulose.CuCl2,AL2(S04)3,AgN03 and SDS have a strong inhibitory effect on the enzyme activity of xylanase Umxyn10A,while other metal ions have little effect on its enzyme activity.The hydrolytic products of xylanase UmxynlOA to xylo-oligosaccharide are mainly xylobiose,which has potential commercial application value.The Km and Vmax value were 3.14 mg/mL and 370.37?mol/(min·mg),respectively,with different concentrations of xylanose as the substrate.The results showed that the recombinant enzyme Umxyn10A had excellent enzymological properties,but poor thermal stability Therefore,site-specific mutation was used to mutate the enzyme in order to improve its thermal stability.The multiple sequences of the amino acid sequences of the xylanase UmxynlOA were compared with the reported amino acid sequences of the four heat-resistant xylanases in the GH10 family and homologous modeling of three-dimensional structures,10 amino acid sites were selected for site-specific mutagenesis.Recombinant expression plasmids of 10 mutated genes of the enzyme were constructed,expression and protein purification were performed in E.coli.The enzymatic properties of 10 recombinant enzymes were determined and two mutant enzymes with significantly increased thermal stability,Umxyn10AA31F and Umxyn10AL307V were obtained.Mutation enzyme Umxyn10AA31F optimum reaction temperature is 85 ?,the 5? higher than that of wild enzyme,the half-life of under 65 ? and 70? are 105 min and 15 min,respectively,compared with wild enzyme raised 6 times and 2 times respectively.The mutant enzyme Umxyn10AL307V optimum reaction temperature is 80?,consistent with the enzyme in the wild,the half-life of under 65 ? are 40 min,compared with wild enzyme raised 1.5 times.In addition,when the thermal stability of mutant enzymes Umxyn 10AA31F and Umxyn 10AL307V were improved,their pH tolerance,influence of metal ions on enzyme activity and hydrolytic products retained excellent properties of wild enzymes.In order to further improve the thermal stability of the xylanase Umxyn 10A,the 31 site amino acids and 307 site amino acids of the enzyme were mutated,the expression and protein purification were performed in the E.coli.The results showed as follows mutation enzyme Umxyn 10AA31F/L307V optimum temperature is 85?,the 5? higher than that of wild enzyme,it has a half-life of 35 min than wild enzyme extend for 20 min under the 65 ?,however,the effect did not show superposition effect on thermal stability.