Gene Cloning, Expression And Characterization Of Two Xylanase From Phialophora Sp. G5

Hemicellulose is the second most abundant renewable biomass after cellulose in nature, xylan is one of the major components of hemicellulose. But complete degradation requires the synergistic action of several glycoside hydrolytic enzymes, xylanase is the crucial enzyme to catalyze the hydrolysis of the main backbone of xylan, which can be degrade into xylose and oligosaccharides. Xylanase can find in most microbes, plants and lower animals and have a widely application in animal feed, papermaking, energy and other industrial areas.The research is using molecular biology techniques to obtaining xylanase with superior enzyme propertiesStrain Phialophora G5 was isolated from an acidic wastewater sample which was collected from a tin mine in Yunan province. Use bioinformatics approaches, blast the amino acid sequence of family 10 and family 11 xylanase from microbial, and combination with filamentous fungi published sequence, so as to determine the conservative amino acid sequence of family 10 and family 11 xylanase. Then Touchdown-PCR was used to cloning partial segment of xylanase, the full-length were obtained using thermal asymmetric interlaced (TAIL)-PCR technique. Family 10 and famliy 11 xylanase gene cloning from Phialophora G5 were designed as xyn10G5 and xyn11G5,they exhibited the highest identity with known xylanase amino acid sequence, 53% and 63%, respectively.Xylanase gene xyn10G5 and xyn11G5 were expression in Pichia pastoris GS115 and purification, the specific activity of purified recombinant XYN10G5 and XYN11G5 was 207.2 U/mg and 857.1U/mg. Enzyme characterization show that the optimal pH for the xylanase activity of purified recombinant XYN10G5 was pH 4.0, and more than 30% of the maximal activity was retained at pH 3.0. retaining more than 70% of the activity after incubation at pH 3.0–9.0 for 1 h at 37oC. The optimal temperature for the enzyme activity was 70°C, the enzyme was stable at 70°C. Substrate specificity of the purified recombinant XYN10G5 exhibited the highest relative activity for soluble wheat arabinoxylan, moderate activity on oat spelt xylan, birchwood xylan, beechwood xylan, and weak activity on cellulose. The hydrolysis products of xylan were xylose and xylobiose. Under simulated conditions in vitro, addition of XYN10G5 alone reduced the viscosity of barley-soybean feed. The optimum pH and optimum temperature for the XYN11G5 was pH 5.0 and 50°C, respectively. And show a pH stability under neutral condition, then have a resistance to pepsin and trypsin, the hydrolysis products of xylan were xylose, xylobiose and xylotriose.The research results will provide a methods for more superior enzyme characterization xylanase and develop widely applied prospect in application, as well as to have a theoretical foundation about the further research on structure and function relations of xylanase.