title: A family GH51 α-L-arabinofuranosidase from Pleurotus ostreatus: identification, recombinant expression and characterization.
abstract: An α-L-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-L-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L(-1)) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-L-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-L-arabinofuranoside it follows Michaelis-Menten kinetics with a K (M) of 0.64 mM and a k (cat) of 3,010 min(-1). The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-L-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.
authors.affiliation: Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, Naples, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Amore
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
authors.forename: Leila
authors.initials: L
authors.lastname: Birolo
authors.forename: Bernard
authors.initials: B
authors.lastname: Henrissat
authors.forename: Gabriella
authors.initials: G
authors.lastname: Leo
authors.forename: Angelo
authors.initials: A
authors.lastname: Palmese
authors.forename: Vincenza
authors.initials: V
authors.lastname: Faraco
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D005091:Exons; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007438:Introns; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics; D018551:Solanum lycopersicum / Q000382:microbiology; D008970:Molecular Weight; D016366:Open Reading Frames; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase