Gene Cloning,Expression And Characterization Of Xylosidase Xyl21 And AX543 From Fungus

Xylan is a kind of heterogeneous polysaccharide.xylose unit was connected by?-1,4-glycosidic bond to form the xylan main chain.Due to the complexity of its structure,the degradation of xylan need a variety of glycoside hydrolase,Among them,?-xylosidase and a-L-arabinofuranosidases are the important accessory enzymes in xylan depolymerization and show a great potential in many biotechnological application especially in food,bioconversion,pulp and paper industries.Bifunctional xylosidase gene ax543 from Acremonium is composed of 975 nucleotides,which encodes 324 amino acids and a termination codon.AX543 shared the highest identity(86%)with the reported xylosidase/arabinofuranosidase like protein from Acremenium chrysogenum.After expressed in P.pastoris GS115,it was comfirmed to be a bifunctional xylosidase/arabinofuranosidase enzyme through the determination of enzyme activity and the substrate docking experiment.The specific activity of AX543 was 4.99 U/mg,and the Km?Vmax and Kcat of AX543 were 5.94 mmol/L,3.53 ?mol/min/mg and 2.46 s-1,when use pNPX as substract.The specific activity of AX543 was 1.16 U/mg,and the Km?Vmax and Kcat of AX543 were 14.29 mmol/L,3.23?mol/min/mg and 2.25 s-1,when use pNPAf as substract.The optimum temperature was 25?,the relative activity of AX543 was 53.61%,20.57%and 6.28%for pNPX and 38.44%,14.04%,4.15%for pNPAf at 15?,4? and 0?,respectively.Bifunctional enzyme AX543 show a poor thermal stability,after treated at 50? for 5min,the enzyme activity was completely loss.The optimal pH of AX543 was 6.0.In the range of pH5.5-7,AX543 show higher xylosidase activity than arabinofuranosidase activity.pH stability results show that AX543 was more stable within pH5.0-9.0 when use pNPAf as substrate.Besides,AX543 has a broader substrate specificity and showed a significant synergic effect with beechwood xylanase.AX543 also had xylose and arabinose tolerance with Ki 84.78 mM and 45.08 mM.Xylosidase gene xyl21 from Penicillium is composed of 1506 nucleotides,which encodes 301 amino acids and a termination codon.The comparison results show that xyl21 sharing the identity of 63%with the reported xylosidase from Alicyclobacillus ferrooxydans and it is a novel xylosidase gene.The recombinant plasmid was successfully expressed in Escherichia coli BL21(DE3).The specific activity was 5.154 U/mg,Km?Vmax and Kcat were 1.71 mmol/L,17.6 ?mol/min.mg and 19.04 s-1,respectively.Biological activity detection show that the optimal pH of Xyl21 was 5.5,and it remains more than 80%relative activity within pH5-6.5.The optimum temperature is 45 ?,thermal stability results show that the residue activity of Xyl21 was 80%after incubation at 45? for 1h,but at 50? and 55?,Xyl21 show the poor thermal stability.It was worth mentioning that Xyl21 displayed high xylose-tolerance with Ki value approximately 1100 mM.In addition,through the analysis of the hydrolysis products of Xyl2l found that the enzyme has a wide substrate specificity.In this study,AX543 was a bifunctional enzyme with low temperature,Xyl2l was a high xylose tolerance xylosidase with medium temperature.Thus,both AX543 and Xyl2l have great potential for industrial applications.