| Hemicellulose is the second largest renewable resource of nature. The xylan, its most abundant component, is ascendant to research the degradation and biotransformation tech-nology. The endo-xylanase and beta-xylosidase are very important glycoside hydrolase of the xylan degradation. Beta-xylosidase has been used in traditional industrial processes. Beta-xylosidase can improve the nutritional value of the feed in the breeding industry, to improve the quality of the meat. Xylosidase is generally used as a dipping enzyme in the food industry, mainly used in the extraction and clarification of fruit juice, and to improve its’quality. In the brewing industry, beta-xylosidase can reduce the viscosity and turbidity of the beer and grape juice to improve the clarity filter, and to improve the the liquor stab-ility and mellow. In the paper industry, beta-xylosidase can reduce the consumption of chemicals. Xylosidase, together with other hemicellulases, has many potential applicatio-ns including coffee process, immersed vegetables, high fiber baked material with the pig-ment preparation, spices, starch extraction, detergents industry as well as antibacterial and anti-oxidation polysaccharide active drug.Beta-xylosidases gene from the deep sea thermophilic bacteria Geobacillus stearoth-ermophilus and fungi Aspergillus fumigatus were cloned and expressed in E. coli DE3, purification and study its enzymatic properties. The study results are as follows.1. The beta-xylosidase (gsxyn) gene from Geobacillus stearothermophilus consists of2118bp, the initiation codon ATG, the terminator for the TAA and GC content of51.37%. GSxyn encodes a705-residue polypeptide with a calculated mass of79.82kDa, and no signal peptide predicted by the SignalP4.1Server. The isoelectric point of GSxyn is5.03. Through the BLAST alignment, GSxyn belongs to the family52glycoside hydro-lase with the catalytic amino acid residues of Glu-335and Asp-495. The xylosidasee gene was cloned in E. coli DE3with the vector pGEX-6p-1, and then expression, purification and analysis enzymatic properties. The purified GSxyn displayed the maximum activity at pH5.5and70℃; Using the p-nitrophenyl-beta-D-xylopyranoside (pNPX) as substrate, the Km, Vmax,kcat and kcat/Km of GSxyn was0.48mM,27.54μmol/mg-min,26.63s-1and76.99L/s-mmol, respectively.In order to explore the substrate specificity of the xylosidase and research the multi-functional enzyme structural characterisation, site-directed mutagenesis introduces xylan-ase activity, whereas the resultant enzyme variant, Y509E, retains xylosidase activity. Determination of the xylanase is endo-hydrolase by high performance liquid anion exch-ange chromatography (HPAEC). The xylosidase mutation and xylanase enzymatic prope-rties were studied in this process. The xylosidase activity of Y509E mutant (Y509Exyn) as follow. The purified Y509Exyn displayed the maximum activity at pH5.5and60℃; Using the p-nitrophenyl-beta-D-xylopyranoside (pNPX) as substrate, the Km, Vmax, kcat and kcat/Kmof GSxyn was0.51mM,15.52μmol/mg-min,20.64s-1and40.75L/s-mmol, respectively. The xylanase activity of Y509E mutant (Y509Exyl) was as follow. The purified Y509Exyl displayed the maximum activity at pH6.5and50℃; Using the beech-wood xylan as substrate, the Km, Vmax,kcat and kcat/Km of GSxyn was5.10mg/mL,16.94μmol/mg-min,22.53s-1and4.42mL/s-mg, respectively. Therefore, this part of the study results demonstrated that GH52xylosidase provides a platform for generating custom-made multifunctional enzymes that target industrially sigificant complex substrates, such as the plant cell wall.2. The beta-xylosidase (afc6xyn) gene from Aspergillus fumigatus consists of1595bp with an intron in the sixth chromosome. The afc6xyn gene ORF is1542bp, the initiation codon ATG, the terminator for the TAA and GC content of51.37%. Afc6xyn encodes a513-residue polypeptide with a calculated mass of57.36kDa, and no signal peptide predicted by the SignalP4.1Server. The isoelectric point of Afc6xyn is5.03. Through the BLAST alignment, Afc6xyn belongs to the family43glycoside hydrolase with the catalytic amino acid residues of Asp-140and Glu-194. The xylosidasee gene was cloned in E. coli DE3with the vector pGEX-6p-1, and then expression, purification and analysis enzymatic properties. The purified Afc6xyn displayed the maximum activity at pH6.0and50℃; Using the p-nitrophenyl-beta-D-xylopyranoside (pNPX) as substrate, the Km, Vmax, kcat and kcat/Kmof Afc6xyn was0.56mM,0.25μmol/mg-min,0.24s"1and0.42L/s-mmol, respectively. This is the first report on the cloning and expression of a GH43xylosidase gene from Aspergillus fumigatus. |
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