The Reaction Mechanism Of GH10 Family Xylanase XynAF1 From Thermophilic Composting Strain Aspergillus Fumigatus Z5

Xylan is a kind of renewable biomass resource,of which the effective utilization not only reduces the pollution caused by discarding of the agricultural solid wastes but also has huge economic benefits.According to different plant sources,xylan can be divided into several types,such as glucuronoxylan（GX）,neutral arabinoxylan（AX）,and glucuronoarabinoxylan（GAX）.All types of xylan have the same backbone composing of xylose units through β-1,4-glycosidic linkages,and the difference is mainly caused by the variable side chain residues.Endo-β-1,4-xylanase is a kind of high-efficiency biocatalyst1 which is widely used in industry to cut off the xylose backbone from the inside and release the xylo-oligosaccharides and xylose.At present,the research of xylanase mainly focused on GH10-family and GH11-family proteins.GH10 family enzymes are more suitable for the application environment with wider substrate range,higher optimum reaction temperature and stronger thermal stability when compared to GH11 family proteins.Aspergillus fumigatus Z5,a thermophilic and high efficient fiber-degrading filamentous fungus,was isolated from the compost by the previous research.It was subsequently found that a series of xylan degrading enzymes could be induced and secreted by Z5.This study focused on the GH10 family thermophilic xylanase,XynAF1,whose apo-structure and the enzyme-substrate complex structures were obtained by heterologous expression,crystallization,substrate soaking and crystal structure analysis.Based on the enzyme-substrate structures,the catalytic process of enzyme XynAF1 was derived.The enzyme mutations with improved enzymatic properties were subsequently obtained by rational design,directed evolution and iterative saturation mutation.The results were summarized as follows:1.By analyzing the amino acid sequence of the xylanase encoded by the gene Y699₀4481 in Z5,it was found that the xylanase has a catalytic domain connected by a carbohydrate binding domain with a flexible linker.The full-length DNA sequence recombinant protein was expressed after codon optimization and named by XynAF0;the catalytic domain was also expressed and named by XynAF1.Both expression systems of E.coli and Pichia pastoris were tried and results showed that expression system of Pichia pastoris was more advantage than E.coli no matter in protein production and enzyme properties.After that,XynAF0 and XynAFl were collected,purified,and screened for crystallization conditions.Due to the over-glycosylation of XynAF0,only the truncating enzyme XynAFl was able to obtain the protein crystal.The three-dimensional structure of XynAF1 was obtained by X-ray diffraction.There are two protein molecules in each asymmetric cell,the apo-structure resolution was 1.68 A,the protein exhibits the typical（β/α）8 TIM-barrel structure,and the catalytic residuals were the glutamic acids E135 and E242.2.As a high optimal reaction temperature of 90℃,recombinant xylanase XynAFl only has 1%of enzyme activity remained at room temperature.However,at this temperature,the protein-ligand complex structure in different catalytic process could be obtained by substrate soaking for 20-120 minutes.The diffraction data of each crystal was collected after X-ray diffraction,and the configuration of xylose sugar ring in the glycone region of enzyme active center presented in regular changes.Combining with the pKa analysis of the catalytic amino acid and analysis of sugar ring configuration,the catalytic process of xylanase XynAFl was derived.The snapshots of the complex structures indicated a conformational itinerary of 4C1→[2,5B]（?）→4C1 for the first step and a 4C1→5S1→4C1 conformational change for the second step of the retaining mechanism.Differ with the existing researches which were usually based on protein mutation and substrate substitutes,the catalytic process of enzyme XynAF1 was obtained by natural substrate soaking with the wild-type enzyme,and this provided a new approach for the glycosidic hydrolase catalytic mechanism study.3.The catalytic performance of xylanase XynAF1 was poor under the moderate temperature conditions.In this study,mutants with improving catalytic activity were designed based on the homologous protein structure analysis.The mutant XynAF1-CM with two amino acids site-directed mutagenesis in the catalytic center of XynAF1 showed better catalytic efficiency at room temperature.XynAFl-CM could exert more than 50%relative enzyme during the temperature range over 35℃ while the wild-type enzyme played a similar performance within the range of 25℃.Besides,through analysis of the protein-substrate complex structures,the leaving group in protein active center was found to influence the enzyme catalytic efficiency.By designing a series of mutants having the gradually reduced protein-substrate binding abilities,a specific balance between protein-substrate binding capacity and the enzyme catalytic activity was demonstrated,which also helped for improving the enzyme catalytic efficiency.4.Considering the weaker thermal stability of xylanase XynAF1 when compared to other homologous proteins,amino acid sites that might affect the enzyme heat resistance were found through the homologous 3D structure alignment.By constructing saturation mutation library and a disulfide bond at the N-and C-terminus of XynAFl,different positive mutants were obtained by high-throughput screening.Finally,combining the positive mutations at different amino acid sites and the disulfide bond mutant,a complex mutant was constructed and named by XynAF1-AC,which had a thermal stability for approximately 5-fold than XynAF1.What’s more,the mutant presented an improved hydrolysis ability in corn cob degrading than the wild-type enzyme,and exhibits superior utilization potential.In conclusion,this study constructed the recombinant proteins and obtained the crystal structure of the thermophilic xylanase XynAF1.Based on the enzyme-substrate complex crystals prepared by the soaking method,the enzyme catalytic process was preliminarily derived,which provided a theoretical basis for understanding the catalytic mechanism of GH10 family xylanase.Meanwhile,using the directed evolution method with rational design,the mutants having better catalytic ability and thermal stability were constructed,which improved the application potential in agricultural solid wastes degradation.