Cloning and characterization of a thermostable endo-arabinanase from Phanerochaete chrysosporium and its synergistic action with endo-xylanase

Putative arabinanase (PcARA) was cloned from cDNA of Phanerochaete chrysosporium. The gene sequencing indicated that PcARA consisted of 939 nucleotides that encodes for 312 amino acid arabinanase-polypeptide chain, including a signal peptide of 19 amino acids. Three-dimensional homology indicated that this enzyme is a five-bladed beta-propeller, belonging to glycosidase family 43 and its secondary structure is consisted of 24 beta-sheets. The PcARA-cDNA was expressed in Pichia pastoris using pPICZ alpha C. SDS-PAGE of purified arabinanase showed a single band of 33 kDa that is very close to theoretical molecular mass of 33.9 kDa calculated by its amino acid content. Recombinant arabinanase (rPcARA) exhibited maximum activity at pH and temperature of 5.0 and 60 degrees C, respectively. End-product analysis of debranched arabinan hydrolysis by thin-layer chromatography indicated that rPcARA acted as endo-type. The synergistic action of rPcARA with recombinant xylanase resulted in 72 and 9.3% release of total soluble sugar of arabinoxylan and NaOH-pretreated barley straw, respectively.