Improving Catalytic Efficiency Of Thermophilic And Alkalophilic Xylanase Xyn10A By Engineering

The stringent environmental policies have prompted the pulp and paper manufacturing to adopt biocatalysts-based green processes to reduce pollution and save energy.Thermostable and alkaline xylanase has attracted much attention for its potential application in pulp bio-bleaching,which can significantly reduce the usage of toxic chemical bleaching agents like chloride,increase whiteness,and improve paper quality.However,in enzyme-based pulp bio-bleaching,xylanase with higher activity and stability under conditions prevailing in the industrial applications(>60?,pH 10-11)is still needed.Currently,there are not many thermophilic alkalophilic xylanases isolated from basophilic or thermophilic bacteria or archaea.In our studies here,a thermophilic alkaline xylanase Xyn10A was characterized,which was from the Bacillus halodurans S7,the GH10 family.Its optimum pH and temperature were identified as pH10 and 70?,respectively.In addition,the specific activity of the heterologously expressed xylanase from E.coli was evaluated as 110 U/mg.Its optimum pH and temperature can fully meet the production conditions of industrial papermaking process,but the specific enzymatic activity of this enzyme is much lower than many acidic xylanases and difficult to meet the requirements of industrial papermaking.In this study,the specific enzyme activity of xylanase Xyn10A was greatly improved through all means of protein engineering methods.The specific strategies used in our study are as follows:1 Rational design.By analyzing previously published studies on the engineering of GH 10 families xylanases,computer-based structure simulation was performed to design a mutant D175S with higher specific activity.The D175S mutant presented a 1.42 times higher specific activity than that of the wild-type xylanase Xyn1OA.2 Random mutation.A random mutant library was constructed by using the Error-prone PCR.Through screening this random mutant libraries,13 variants from approximately 80,000 transformants were selected,which presented enhanced enzyme activity.Among them,the mutant with the highest specific activity was S17A9(N151I/E236V/A305T),and the specific activity increased to 2.15 times of the wild-type xylanase Xyn10A.3 Semi-rational design.Through the structure docking analysis of proteins and substrates,and the comparison of their amino acid sequences,a semi-rational mutant library focusing on the residues of the entrance of protein substrate channel was constructed.From the this mutant library,6 mutants from approximately 21600 transformants was identified to possess higher specific activity than that of the wild-type xylanase Xyn10A.Then the mutation sites of these 6 variants were selectively combined that one variant,V220N/Q251S/W254R,was identified that its specific activity increased to 3.41 times of the wild-type xylanase Xyn1OA.The structural simulation of this variant also indicated that a significantly larger protein substrates entrance than that of the wild-type xylanase Xyn10A.Moreover,the variant also presented a structure that could capture the substrate in a better ability.Our finding of improving the enzyme specific activity by engineering its substrate entrance region for a better accommodation is an investing observation.To verify the generality of this strategy in xylanases,three other xylanases,including Xyl-gt,Xyl-NG27,and xyn10 were also evaluated.Their homology with Xyn10A are 57.1%,69.8%,and 87.0%,respectively.Based on the similar strategy with Xyn10A engineering,the substrate entrance region was also engineered in these three xylanases.Interestingly,the specific enzymatic activities of these three xylanases were all improved,with xylanase xyl-gt exhibiting the most significant increase in enzyme activity after mutation.In details,the specific activities of the Xyl-gt*,Xyl-NG27*,and Xyn10*increased 1.75,1.23,and 1.04 times,respectively,as compared with the wild-type Xyl-gt,Xyl-NG27,and Xyn10.It can be concluded that the protein substrate entrance engineering might be a universal strategy for xylanase engineering.Moreover,to further increase the specific enzyme activity of xylanase based on the optimal mutant at the protein substrate entrance.We have further studied in three aspects:1 The amino acid sequence of Xyn10A was aligned to Xyl10C,the xylanase possessing the reported highest-activity.Site-directed mutations was designed and evaluated based on the sequence difference of these two xylanases,but no mutant with higher specific activity was identified.However,H113 was unexpectedly identified to be critical to the pH stability of Xyn1OA.2 On the basis of the best mutant(V220N/Q251S/W254R),alanine scanning was performed against the non-conservative amino acids within 5 angstroms of the around the active center in the Xyn10A protein structure.The E71 site was identified to exhibit significant effect on the xylanase activity.However,no mutant with higher specific activity was identified after we mutated it to other 19 amino acids.3 At the same time,we also combined the dominant single point mutations obtained from random mutations and virtual amino mutations on the best mutant(V220N/Q251S/W254R).Finally,we obtained a mutant A262TL2(V220N/Q251S/W254R/A262T)presenting a 4.42-fold higher specific enzymatic activity than that of the wild-type xylanase Xyn10A,which resulted in a significant increase in the xylanase activity at 70?,pH10.This study laid a foundation for the application of the xylanase Xyn10A to industrial papermaking in the future,and also provided some useful methods and ideas for the engineering of the xylanase.