Proteomics analysis of “Rovabio™ Excel”, a secreted protein cocktail from the filamentous fungus Penicillium funiculosum grown under industrial process fermentation

Abstract

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.

Evaluation of Glycosyl Hydrolases in the Secretome of Aspergillus fumigatus and Saccharification of Alkali-Treated Rice Straw

“…The protein spots E and G (Fig. 1) were identified as polygalactouronase (GH 28) and spot 4 as N-acetyl hexaminidase (GH 20) in the secretome also indicated the ability of A. fumigatus strain to utilize pectin [45] and chitin [46] in addition to cellulose. N-Acetyl hexaminidase (GH 20) catalyzes the specific exohydrolysis of chitooligosaccharides from the non-reducing end generating monomers of N-acetylglucosamine [46].…”

Hunting down fungal secretomes using liquid‐phase IEF prior to high resolution 2‐DE

“…The separation of basic secreted proteins along 7-10 and 6.3-8.3 pH gradients resulted in low-quality 2-D profiles. The vast majority of fungal secretome results based on 2-DE published to date only reported acidic proteins [12,15,[18][19][20][21][22][44][45][46]. Oda et al [16] mentioned that pIs of proteins in the Aspergillus oryzae secretome fell exclusively within 4-7 range.…”

“…The objective of this study was to optimize 2-D separation of fungal secreted proteins, which was achieved using an initial liquid-phase IEF step. Yet, combining several proteomic techniques greatly increased protein identification coverage, as evidenced by Guais et al [46].…”

“…3B). Using phenol/ammonium acetate method to extract secreted proteins from Penicillium funiculosum liquid cultures, Guais et al [46] were able to separate about 100 spots along 3-6 pH range. Precipitation using ammonium sulfate instead of ammonium acetate was also performed but secreted proteins were completely lost (data not shown).…”

“…These observations suggest that L. maculans secreted proteins were more reliably identified using 2-DE than 1-DE. More proteins were identified in Penicillum funiculosum using 2-DE than shotgun proteomics [46]. Although 2-DE is more time-consuming than 1-DE, its higher resolving power unravels some PTMs such as glycosylations, which massively affect extracellular proteins and in particular GPI-anchored proteins.…”