Identification, Purification, Characterization And Gene Cloning Of Xylanase From Trametes Versicolor HN0511

Xylan, a major structural component of plant cell walls and the most abundant renewable hemicellulose, constitutes 35% of total plant biomass, which also has a complex structure consisting of (3-1,4-linked xylose residues in the backbone. The core chain hydrolysis is accomplished by the action of xylanases (1,4-β-xylan xylanohydrolases, EC 3.2.1.8), which release xylo-oligosaccharides of different sizes.Xylanases, associated or not with other enzymes, can be used for industrial applications, such as the food industry in order to enhance the digestibility of animal feeding, as well as in the textile industry and paper industry for bleaching purposes, and so on.In this study one xylanase-producing fungi:HN0511 strains were isolated from soil sample No.l collected from bagasse laydown area, one glyc-oside factory in Hainan province, according to Martin's reference with medium xylan as the sole carbon.The research we done including strains isolated, cultured, purification, enzymatic properties analysis of this xylanase which from HN0511. HN0511 had been selected as a promising strain for xylanase production.18S rDNA sequences of HN0511 were compared in GenBank database using the BLAST program, consequently our strain was identified as Trametes vevsicolor and named Trametes vevsicolor HN0511. The enzymatic hydrolysis of corncob experiments showed this xylanase can transform hemicellulose.HN0511was induced with oat spelt xylan as single carbon source to produce xylanase and the special activity was 177.36 U/mL which obtained after 144 h inducing. The enzyme was purified about 20.42 fold with recovery of 23.44% from the culture supernatant by cation exchange chromatography and size exclusion chromatography. Zymogram analysis of the purified enzyme confirmed the enzyme was xylanase. Hydrolysis products were analyzed by thin-layer chromatography (TLC), suggesting it is an endo-xylanase. The optimal temperature and pH for the action of the enzyme were at 60℃and 7.0, respectively. The purified enzyme was stable from pH 6.0 to 10.0 and up to 60℃. The activity of the enzyme was significantly stimulated by Fe2+,Mn2+,β-mercaptoethanol and inhibited by K+,Mg2+,Co2+,SDS,EDTA. The Km values of the xylanase for oat spelt xylan and birchwood xylan were 8.67 mg/mL and 3.79 mg/mL.Quantity, activity and cost of producing the xylanase in HN0511 are the factors limited large-scale produce in industry. Throughing MS/MS mass spectrometry analysis of its peptide amino acid sequence, we design degenerate primers to amplify DNA fragment of this xylanase. Gene specific primers derived from this partial sequence were used for the amplication of the 3'and 5'ends of this cDNA by RACE method.