Cloning and expression of a novel α-1,3-arabinofuranosidase from Penicillium oxalicum sp. 68

摘要

The discovery and creation of biocatalysts for plant biomass conversion are essential for industrial demand and scientific research of the plant cell wall. α-1,2 and α-1,3- l -arabinofuranosidases are debranching enzymes that catalyzing hydrolytic release of α- l -arabinofuranosyl residues in plant cell wall. Gene database analyses shows that GH62 family only contains specific α- l -arabinofuranosidases that play an important role in the degradation and structure of the plant cell wall. At present, there are only 22 enzymes in this group has been characterized. In this study, we cloned a novel α-1,3-arabinofuranosidase gene ( poabf62a ) belonging to glycoside hydrolase family 62 from Penicillium oxalicum sp. 68 and expressed it in Pichia pastoris . The molecular mass of recombinant PoAbf62A was estimated to be 32.9?kDa. Using p -nitrophenyl-α-l-arabinofuranoside ( p NPαAb f ) as substrate, purified PoAbf62A exhibited an optimal pH of 4.5 and temperature of 35?°C. Results of methylation and~(13)C NMR analyses showed that PoAbf62A was exclusively α-1,3-arabinofuranosidase, specific for cleavage of α-1,3-arabinofuranosyl residues, and with the absence of activity towards α-1,2-arabinofuranose and α-1,5-arabinofuranose. Therefore, PoAbf62A exhibits high activity on sugar beet arabinan and wheat arabinoxylan, because their branched side chain are decorated with α-1,3-arabinofuranose. On the other hand, there is a lack of activity with linear-α- l -1,5-arabinan and xylan that only contained α- l -1,5-arabinofuranose or β-1,4-xylose. The α-1,3-arabinofuranosidase activity identified here provides a new biocatalytic tool to degrade hemicellulose and analyze the structure of plant cell walls.