Xylanases From Fusarium Sp.21:Characterization,Directed Evolution,Immobilization And Their Application

Xylan is a major component of hemicellulose in agricultural residues,such as wheat bran,wheat straw,and corn stover,and constitutes one-third of the total renewable organic carbon on earth.Due to the large quantity of this biomass as an agricultural waste,it presents potential for application of the biorefinery concept which permits the production of xylooligosaccharides,xylitol and fuels that offer economic and environmental advantages.Endo-β-1-4-xylanase plays a key role in the degradation of hemicelluloses and can be used as feed additive,biological bleaching agent and food additive in the feed,paper and food industries.Therefore,developing novel xylanases is of great advantage for the biorefinery concept and the development of feed and food biotechnology industries.In this study,the production of the xylanase from Fusarium sp.21 was analyzed.Partial xylanase genes were heterologously expressed in E.coli and characterized.Their application potential of these xylanases were evaluated depending on the enzymatic properties.Finally,directed evolution and immobilization studies were carried out to improve the catalytic efficiency and thermalstability of xylanases.The main results obtained are as follows:(1)The extracellular enzymes produced by Fusarium sp.21 were mainly xylanase,as well as very small quantities of cellulase,xylosidase,glucosidase and arabinosidase.Corncob,sugarcane bagasse,and rice chaff could be hydrolyzed to liberate XOS(DP>2)by the extracellular xylanases of Fusarium sp.21 and the most XOS was obtained from sugarcane bagasse.Zymogram analysis showed that Fusarium sp.21 produced multiple extracellular xylanases.Seven genes were annotated as endo-β-1-4-xylanase(EC 3.2.1.8)in the genome of this strain.The xylanases sequences of Fusarium sp.21 shared low sequence identity(often below 50%)and their phylogenetic relationship was far away from each other.(2)Three new xylanases of the glycosyl hydrolase 11(GH11)family,named Xyn11A,Xyn11B,and Xyn11C,from the fungus Fusarium sp.21,were heterologously expressed in E.coli and characterized.Both Xyn11A and Xyn11B had same optimal pH of 6.0 and optimal temperature of 45℃.Xyn11C displayed the maximum activity at pH 5.0 and 45℃ and had an outsanding pH stability with a weak loss of activity in the pH 2.0-10.5.Xyn11A showed much a higher activity against corncob xylan,while Xyn 11B and Xyn11C presented higher activities against beechwood xylan.Beechwood xylan hydrolysates produced by these three xylanases contained xylobiose(DP2),xylotriose(DP3)and xylooligosaccharides(XOS).Three xylanases have different efficiency on the degration of agricultural residues and the clarification of orange juices.Xyn11A liberated more quantity of XOS from the agricultural residues than Xyn11B and Xyn11C.And Xyn11C displayed the best performance in increasing the clarity of orange juice.(3)For further study of the relationship between the structure and function of GH11 xylanases,we carried out the homology modeling,molecular docking and molecular dynamics(MD)simulation of Xyn11A,Xyn11B and Xyn11C,respectively.These three xylanases had similar spatial structures of "jelly roll",while the structures of substrate binding sites of three xylanases were slightly differen.The N-terminus of Xyn11B(Thr27Gln28 Pro29Thr30)layed over the docked xylan hexasaccharide and stabilized a straighter path for the backbone direction.The presence of the sequence in Xyn11B may increase the stability of the binding of the ligand to the enzyme and,therefore,lead to an increase in the rate of xylan degradation.There were more aromatic amino acids near the active sites of Xyn11B which could contribute to its thermal stability.The "thumb" region of Xyn11A comprising D149/G150/I151/Q152 was more flexible which tolerate a higher degree of substitutions in this active site.While the "thumb" region of Xyn11B comprising E151/G152/N153/K154 would cause a steric hindrance effect at the-2 position and could not accommodate the side chain group of corncob xylan.(3)In order to improve the hydrolytic activity of Xyn11B,a random mutation library was constructed a variant of Xyn11BQ125T/K182A with improved activity on beechwood xylan was obtained,and it presented 2.24-fold increase in kcat/Km comparative to the wild type and displayed the maxium activity at pH5.5.Site-directed mutagenesis was used to create single mutants Xyn11BQ125T,Xyn11BK182A and Xyn11BK182E.Xyn11BQ125T showed 1.63fold increase in Kcat/Km contrast to wild type.The optimum pH of the enzymes with a residue mutation(K182A or K182E)shifted to 5,5,The result indicated that mutation K182 was important in optimum pH shifting.The structural model analysis of Xyn11BQ125T/K182A showed that the Q125 and K182 were located on the surface area far from the active center of the protein.Q125 was near the+2 and+3 subsites,and K182 was located at the end of the alpha helix.(5)In order to improve thermal and reusability properties of Xyn11A,the immobilization of Xyn11A using a protein-inorganic hybrid nanoflower system and immobilization on aminoethyl-agarose supports were assessed.The stability and reusability of Xyn11A-Agarose was significantly improved contrast to Xyn11A-Cu3(PO4)2.Xyn11ACu3(PO4)2was used in six consecutive cycles,maintaining about 30%of its residual activity.After 12 cycles of reuse,Xyn11A-Agarose retained 90%residual activity.Xyn11ACu3(PO4)2 and XynllA-Agarose hydrolyzed corncob and sugarcane bagasse to produce XOS degree of polymerization(DP 2-6),especially xylotriose and xylotetraose.These results demonstrate that xylanase immobilization on aminoethyl-agarose supports was an effective approach for production of XOS in industrial applications.