Improving Thermostablity Of Family 11 Xylanase With Molecular Engineering

Xylanase can degrade xylan into oligosaccharides and xylose,which is one of the key enzymes in the degradation process of polysaccharide.Xylanase is widely used in the production of paper,feed,food and bioenergy.Different industrial processes require different types of xylanase,such as the acid and high temperature resistant xylanase in the feed industry,while the paper industry requires alkaline and high-temperature resistant xylanase.Xylanase is mostly active in medium temperature.The family 11 xylanase has high enzymatic activity,but its optimum temperature is 55? and the optimum pH is 5.0.Thus,it is difficult to meet the requirements of industrial conditions.Protein engineering is a powerful technology to improve thermostability,which is widely applieid in modern biotechnological industry.Protein structure is closely related to function.There are many factors affecting the thermal stability of proteins,including hydrogen bond,salt bridge,disulfide bond,hydrophobic interaction and so on.In recent years,a lot of protein structures,calculation softwares,and the experimental database makes protein engineering more and more efficient.In this paper,family 11 xylanase was used as the research target to improve its thermal stability by using two strategies based on semi-rational design and computer-aided screening.The semi-rational design method using the molecular dynamics simulation to find the unstable resign based on the high B-factor paratmeter.According to the reported homologous xylanase on the N terminal,a disulfide bond T2CT28C was built into a similar position.In the 152 position,according to the hydrophobic and spatial structure we designed a single mutation T152F.Our results showed that the optimal temperature of T2CT28C mutations increased by 15 degrees,and the enzyme activity increased 50%.The single mutation T512F activity under 70? is 27%higher than the wild type.We applied the Modeller homology modeling to build the whole sequence of xylanase saturated mutant library,then we used FoldX to calculate the free energy of folding ? G,finally,we use the HotSpot Wizard sequence analysis.Finally,we obtained 28 mutations.Verified by molecular dynamics simulation and enzyme activity experiments,in the 28 mutations,five mutations' activity under 70 ? was higher than the wild type.In the top ten mutations obtained by molecular dynamics simulation,four mutants' activity under 70? was higher than the wild type.Our results show that above two strategies are effective to improve the thermal stability of family 11 xylanase.These two strategies greatly reduced the number of screening task,quickly abtained disulfide bond and many single mutations with improved thermal stability.It has accumulated experience for mutagenesis of xylanase in the future and provided ideas for thermal stability modification of other similar proteins.