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摘要
A novel beta-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both beta-xylosidase and alpha-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60 degrees C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0-9.0 (incubated at 37 degrees C for 1 h) and at temperatures of 60 degrees C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 beta-xylosidases with a high K-I value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the samemicrobial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX(2). All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.
- 出版日期2015-2-6
- 单位浙江大学; 中国农业科学院
