Improving The Thermal Stability Of The GH11 Xylanase By Rational And Semi-Rational Transformation Methods

Xylan is the main component of hemicellulose,which is a renewable and rich resource in nature.Through the degradation of xylan by xylanase,it could get the agricultural solid waste fully utilized and create huge economic benefits.Xylo-oligosaccharides and xylose would be generated after the β-1,4-glycosidic bond in the main chain of xylan being specifically hydrolyzed by xylanase,which are widely applied in food,paper making,feed and bio-energy,etc.The GH11 family xylanases,among all the family members,has attracted much attention due to its strict substrate specificity,high catalytic efficiency and wide temperature and pH range.Xylanase with good thermal stability is very important in industrial production.Therefore,it is meaningful to modify the xylanase structure to improve their thermal stability.In this paper,the main two research objects are GH 11 family xylanase XynⅡ from the fungus Trichoderma reesei and GH 11 family xylanase XynM1 from the fungus thermophilic Paecilomyces sp.The former is a typical mesophilic enzyme,while the latter is a commercial thermophilic enzyme.In this study,the relationship between several important regions in the structure of XynⅡ and its thermal stability and activity was explored in detail through in-depth analysis of the crystal structure of XynⅡ,and combing molecular dynamics simulation technology.The thermotolerance and high catalytic efficiency mechanism of XynⅡ could be explained from the perspective of structural biology.A rapid screening method for thermostable mutants of XynⅡ was established and successfully applied to commercial xylanase XynM1,and combined mutants with enhanced thermal stability were obtained.The main results are as follows:1.By comparing the crystal structures of XynⅡ collected at 100 K and 293 K,and with the help of molecular dynamics simulation technology,the instability of N-terminal region and loop 162～168 region of XynⅡ were identified.Disulfide bonds were introduced into these two regions and the mutants T2C/228C and T2C/T28C/R81C/T168C were constructed.Their genes were cloned and expressed,and their proteins were purified and characterized.The results showed that the optimal temperature of the two mutants increased from 55℃to 65℃and the half-life of the two mutants increased by 20 times and 80 times,respectively.Their T_mincreased by 8.3℃and 12.1℃,and their enzyme activity increased by 111.5%and 57.3%,respectively.By comparing XynⅡ WT-apo structure with WT-X6 complex structure,it was proved that the kinetics of thumb region is closely related to catalytic activity.The mutant Q125A/I129S was designed to increase the kinetics of thumb region.The results showed its enzyme activity increased from 10 261 U·mg^-1to 13 355U·mg^-1,increasing by 30.2%.Through sequence alignment with GH11A,a psychrophilic cellulose hydrolase from the compost macrotranscriptome,the loop 57～63 was inserted into the corresponding region of XynⅡ and constructed the mutant XynⅡ-loop,whose optimal temperature decreased from 55℃to 35℃compared with that of XynⅡ.2.A semi-rational mutational strategy focused on the calculation of protein unfolding free energy（ΔΔG）,amino acid frequency analysis and B-factors analysis was established by computer-aided thermal stability mutants screening of XynⅡ.And the semi-rational mutational strategy was evaluated and optimized according to the experimental results.First,theΔΔG of 3591 mutants of XynⅡ was calculated using the Rosetta ddg＿monomer program.Then,47 mutants withΔΔG<-2 kcal·mol^-1were screened with respect to“Frequency”,“B-factors”and“RMSF”,and 12 mutants were retained to conduct enzymatic characterization.Ultimately,4 mutants were obtained with improved thermal stability without causing significant decrease in activity.Then,a mutant library was constructed and the results shows:（ⅰ）there were 5 mutants with improved thermal stability among the 21 mutants with-1.5 kcal·mol^-1<ΔΔG<-2 kcal·mol^-1,accounting for 28.6%,which was lower than mutants withΔΔG<-2 kcal·mol^-1（33%）.This indicated that the lowerΔΔG value the mutants have,the higher probability obtaining mutants with improved thermotolerance.Among the 25 mutants with ΔΔG>0 kcal·mol^-1,19 mutants significantly become unstable,indicating that “ΔΔG>0”could be used to exclude unstable mutations.（ⅱ）Of all the 12mutants with Frequency=0,the activity decreased in different degrees,while among 12mutants with Frequency>0,only 3 mutants had a greater than 10%reduction in activity.This indicated that mutations affecting the activity can be excluded according to“Frequency=0”.（ⅲ）4 mutants had improved thermal stability,and their B-factor values were all higher than the average B-factors.Among the 17 mutants with B-factor values lower than the average B-factors,14 mutants had an increase in stability,accounting for 82.4%.The results showed that mutating site with higher B-factor values could increase the probability of obtaining thermal stability mutants.Finally,a semi-rational mutational strategy with high success rates was summed up:selecting residues with higher B-factor values and selecting mutants with lowerΔΔG value and higher Frequency could largely exclude adverse mutations and increase the probability of obtaining thermal stability mutants.3.According to the strategies summarized above,a rapid screening for thermotolerance mutants of the commercial GH11 family xylanase XynM1 was carried out,and the combination of multiple point mutations was performed.Firstly,B-factors value and sequence conservation of XynM1 were analyzed,and 8 sites with high B-factors value and low conservation were selected to construct a single-site saturation mutagenesis libraries and perform high-throughput screening.Meanwhile,theΔΔG of 3686 mutants in the whole sequence of XynM1 was calculated.Mutants withΔΔG<-2 kcal·mol^-1and Frequency>4%were selected to examine their activity and thermotolerance.Six mutants with improved thermal stability were obtained by above mentioned methods.Finally,by calculating theΔΔG of combined mutants,two mutants with lowΔΔG were obtained.After examining the thermal stability of mutants XynM1-2M and XynM1-6M,their t_1/2at 80℃enhanced by 1.25-fold and 2.6-fold respectively,and their T_mincreased by 4.41℃and4.87℃,respectively.In conclusion,this study shed light on the relationship between structure and function of GH11 family xylanase,providing a new idea for structure-based protein design.At the same time,the rapid and accurate screening strategy of thermotolerance xylanase also provides reference for the effective screening of other enzymes.