title: Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization.
abstract: Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60-80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4-5.5 and optimal temperature of 40 °C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions.
authors.affiliation: Facultad de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
authors.forename: Wladimir
authors.initials: W
authors.lastname: Mardones
authors.affiliation: BRIN-USDSSOM Proteomics Facility, University of South Dakota, Vermillion, SD, USA.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Callegari
authors.affiliation: Facultad de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile. Electronic address: jeyzaguirre@unab.cl.
authors.forename: Jaime
authors.initials: J
authors.lastname: Eyzaguirre
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; 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; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C434938:exo-alpha-L-arabinanase
keywords: Arabinan degradation; GH family 93; Lignocellulose; Pectin