title: Heterologous expression and characterization of α-L-arabinofuranosidase 4 from Penicillium purpurogenum and comparison with the other isoenzymes produced by the fungus.
abstract: Penicillium purpurogenum secretes at least four arabinofuranosidases. In this work, the gene of α-L-arabinofuranosidase 4 (ABF4) has been sequenced and expressed in Pichia pastoris. The gene is 1521 pb long, has no introns and codes for a protein of 506 amino acid residues including a signal peptide of 26 residues. Mature protein has a calculated molecular mass of 55.4 kDa, shows 77% identity with α-L-arabinofuranosidase 1 from P. purpurogenum and belongs to family 54 of the glycosyl hydrolases. Purified enzyme has a molecular mass near 68 kDa, is active on p-nitrophenyl α-L-arabinofuranoside and p-nitrophenyl-β-D-galactofuranoside, and follows Michaelis-Menten kinetics with KM of 1.58 ± 0.13 mM and 5.3 ± 1.18 mM, respectively. The pH optimum is 4.6 and optimal temperature is 50 °C. The enzyme is active on sugar beet arabinan and wheat flour arabinoxylan but does not act on short arabinooligosaccharides or debranched arabinan. It shows synergistic effect on arabinose liberation from wheat arabinoxylan when combined with endoxylanase from P. purpurogenum. The properties of ABF4 have been compared with those of the other arabinofuranosidases produced by the fungus. P. purpurogenum is the first fungus possessing four biochemically characterized arabinofuranosidases. The availability of four different ABFs may be valuable for biotechnological applications.
authors.affiliation: Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago 8320000, Chile. Electronic address: mravanal@ing.uchile.cl.
authors.forename: María Cristina
authors.initials: MC
authors.lastname: Ravanal
authors.affiliation: Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago 8320000, Chile. Electronic address: jeyzaguirre@unab.cl.
authors.forename: Jaime
authors.initials: J
authors.lastname: Eyzaguirre
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005658:Fungi / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: Arabinoxylan; GH family 54; Lignocellulose biodegradation; Pichia pastoris; Recombinat enzyme