Molecular and biochemical characterization of three GH62 α-l-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum

• The genome sequence of Penicilium funiculosum 8/403 has revealed the presence of there GH62 family α-l-arabinofuranosidase encoding genes, termed PfABF62a, b and c.
• One of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1), which was shown to help the protein to bind to microcrystalline cellulose.
• Optimal pH and temperature, as well as thermostability were investigated for all three enzymes, showing disparity between pfABF62c from the two others.
• Mode of action of the three enzymes has been determined and was found to remove arabinofuranosyl residues linked in position O-2 and O-3 of substituted xylose units in arabinoxylan chains.

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.