Combined in situ Physical and ex-situ Biochemical Approaches to Investigate in vitro Deconstruction of Destarched Wheat Bran by Enzymes Cocktail Used in Animal Nutrition.

Wheat bran is a foodstuff containing more than 40% of non-starch polysaccharides (NSPs) that are hardly digestible by monogastric animals. Therefore, cocktails enriched of hydrolytic enzymes (termed NSPases) are commonly provided as feed additives in animal nutrition. However, how these enzymes cocktails contribute to NSPs deconstruction remains largely unknown. This question was addressed by employing an original methodology that makes use of a multi-instrumented bioreactor that allows to dynamically monitor enzymes in action and to extract in-situ physical and ex-situ biochemical data from this monitoring. We report here that the deconstruction of destarched wheat bran by an industrial enzymes cocktail termed Rovabio® was entailed by two concurrent events: a particles fragmentation that caused in <2 h a 70% drop of the suspension viscosity and a solubilization that released <30 % of the wheat bran NSPs. Upon longer exposure, the fragmentation of particles continued at a very slow rate without any further solubilization. Contrary to this cocktail, xylanase C alone caused a moderate 25% drop of viscosity and a very weak fragmentation. However, the amount of xylose and arabinose from solubilized sugars after 6 h treatment with this enzyme was similar to that obtained after 2 h with Rovabio®. Altogether, this multi-scale analysis supported the synergistic action of enzymes mixture to readily solubilize complex polysaccharides, and revealed that in spite of the richness and diversity of hydrolytic enzymes in the cocktail, the deconstruction of NSPs in wheat bran was largely incomplete.

Four GH11 xylanases from the xylanolytic fungus Talaromyces versatilis act differently on (arabino)xylans.

The filamentous fungus Talaromyces versatilis produces a wide range of cellulolytic and hemicellulolytic enzymes such as xylanases. The recent accessibility to the T. versatilis genome allows identifying two new genes, xynE and xynF, encoding glycoside-hydrolases from family GH11. Both genes were cloned and expressed in the methylotrophic yeast Pichia pastoris in order to compare these new xylanases with two other GH11 xylanases from T. versatilis (XynB and XynC) that were previously reported. High-level expression of recombinant enzymes was obtained for the four enzymes that were purified to homogeneity. The XynB, XynC, XynE and XynF enzymes have molecular masses of 34, 22, 45 and 23 kDa, an optimal pH between 3.5 and 4.5 and an optimal temperature between 50 °C and 60 °C. Interestingly, XynF has shown the best thermal stability at 50 °C for at least 180 min with a weak loss of activity. The four xylanases catalysed hydrolysis of low viscosity arabinoxylan (LVAX) with K m(app) between 11.5 and 23.0 mg.mL(-1) and k cat/K m(app) 170 and 3,963 s(-1) mg(-1).mL. Further investigations on the rate and pattern of hydrolysis of the four enzymes on LVAX showed the predominant production of xylose, xylobiose and some (arabino)xylo-oligosaccharides as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing degree of polymerisation of oligomer up to 6, suggesting that the specificity region of XynE and XynF spans at least six xylose residues. Because of their attractive properties, T. versatilis xylanases might be considered for biotechnological applications.

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

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.

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.

Proteomics analysis of "Rovabiot Excel", a secreted protein cocktail from the filamentous fungus Penicillium funiculosum grown under industrial process fermentation.

MS/MS techniques are well customized now for proteomic analysis, even for non-sequenced organisms, since peptide sequences obtained by these methods can be matched with those found in databases from closely related sequenced organisms. We used this approach to characterize the protein content of the "Rovabio Excel", an enzymatic cocktail produced by Penicillium funiculosum that is used as feed additive in animal nutrition. Protein separation by bi-dimensional electrophoresis yielded more than 100 spots, from which 37 proteins were unambiguously assigned from peptide sequences. By one-dimensional SDS-gel electrophoresis, 34 proteins were identified among which 8 were not found in the 2-DE analysis. A third method, termed 'peptidic shotgun', which consists in a direct treatment of the cocktail by trypsin followed by separation of the peptides on two-dimensional liquid chromatography, resulted in the identification of two additional proteins not found by the two other methods. Altogether, more than 50 proteins, among which several glycosylhydrolytic, hemicellulolytic and proteolytic enzymes, were identified by combining three separation methods in this enzymatic cocktail. This work confirmed the power of proteome analysis to explore the genome expression of a non-sequenced fungus by taking advantage of sequences from phylogenetically related filamentous fungi and pave the way for further functional analysis of P. funiculosum.