title: Molecular and biochemical characterization of three GH62 α-l-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum.
abstract: Penicillium funiculosum is an industrial fungus exploited for its capacity to secrete a wide array of glycosyl hydrolases (GHs) and glycosyl transferases (GTs). These enzymes are part of an enzymatic cocktail that is commercialized under the name RovabioExcel(®), which is used as feed additive in animal nutrition. The genome sequence of this filamentous fungus has revealed a remarkable richness in several accessory enzymes, and notably in α-l-arabinofuranosidases (α-l-AFases) that participate in the hydrolysis of arabinoxylans (AX) in corn/wheat fibers used in poultry feed. Here, we report on the molecular and biochemical characterization of three GH62 family α-l-AFases encoding genes in this filamentous fungus. Amino acids sequences showed strong similarities (>65%) between them, as well with GH62 enzymes from other filamentous fungi. Interestingly, one of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1) of 37 amino acids length, which was shown to help the protein to bind to microcrystalline cellulose. Also, this PfABF62c showed optimal pH and temperature of 2.8 and 50°C, respectively, whereas optimal activity for PfABF62a and PfABF62b were measured at 40°C and at pH ranging between 2.6 and 4.5. Arabinan and arabinoxylan, but no other sugars or polymers were found to augment the thermal transition of the three enzymes by 3-5°C as measured by differential scanning fluorimetry. Finally, enzymatic hydrolysis fingerprints of heteroxylans allowed concluding that the mode of action of the GH62 enzymes from this fungal species was to remove arabinofuranosyl residues linked in position O-2 and O-3 of substituted xylose units in arabinoxylan chains.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; Cinabio-Adisseo France S.A.S., 135 Avenue de Rangueil, 31077 Toulouse, France. Electronic address: delamare@insa-toulouse.fr.
authors.forename: Marion
authors.initials: M
authors.lastname: De La Mare
authors.forename: Olivier
authors.initials: O
authors.lastname: Guais
authors.forename: Estelle
authors.initials: E
authors.lastname: Bonnin
authors.forename: Jérôme
authors.initials: J
authors.lastname: Weber
authors.forename: Jean M
authors.initials: JM
authors.lastname: Francois
mesh_terms: D000595:Amino Acid Sequence; D000821:Animal Feed; D000818:Animals; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: Arabinofuranohydrolase; Arabinoxylans; Carbohydrate module; Filamentous fungi; GH62 family