Characterization of the family GH54 alpha-L-arabinofuranosidases in Penicillium funiculosum, including a novel protein bearing a cellulose-binding domain.

The soil deuteromycete Penicillium funiculosum is characterized by its remarkable capacity to produce a wide variety of cellulolytic and hemicellulolytic enzymes. In the course of the genome sequencing of this industrial fungus, four different genes encoding glycosyl hydrolase family 54 (GH54)22 alpha-L-arabinofuranosidases were identified. Three of them termed PfabfB1, PfabfB3, and PfabfB4 were highly similar, encoding proteins of 507, 508, and 505 amino acids, respectively. They exhibited structural features typical of GH54 enzymes, including an N-terminal catalytic domain connected to a C-terminal arabinose-binding domain (ABD). The fourth gene termed PfafbB2 codes for an unusual 400 amino acid length GH54 alpha-L: -arabinofuranosidase, in which the ABD was replaced by a fungal cellulose-binding domain (fCBD). This domain was shown to be functional since it allowed this protein to be retained onto microcrystalline cellulose, and the fusion of this CBD to the C-terminal end of PfAbfB1 allowed this protein to bind to cellulose. Expression analysis of the four PfabfB genes during an industrial-like process fermentation on complex carbohydrates revealed that PfafB2 was expressed more than 20,000-fold, while PfabfB3 and PfabfB4 were increased moderately at the end of the fermentation. In contrast, the transcript levels of PfabfB1 remained unchanged throughout the process. This new type of GH54 alpha-arabinofuranosidase encoded by PfabfB2 showed enzymatic properties slightly different to those of other GH54 enzymes characterized so far, including a higher thermostability, an optimum pH, and temperature of 2.6 and 50 degrees C, instead of 3.5 and 60 degrees C as found for PfAbfB1. Nonetheless, like other GH54 alpha-arabinofuranosidases, PfAbfB2 was able to release arabinose from various sources of branched arabinoxylan and arabinan.