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 β-propeller, belonging to glycosidase family 43 and its secondary structure is consisted of 24 β-sheets. The PcARA-cDNA was expressed in Pichia pastoris using pPICZαC. 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 °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.
Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk, 570-752, Korea.
Nguyen Duc
ND
Huy
Saravanakumar
S
Thiyagarajan
Yoon-E
YE
Choi
Dae-Hyuk
DH
Kim
Seung-Moon
SM
Park
D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D020075:Phanerochaete / Q000201:enzymology / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D005656:Fungal Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C030080:araban; D043364:Endo-1,4-beta Xylanases