Proteomic analysis of Viscozyme L and its major enzyme components for pectic substrate degradation.

ABSTRACT

Enzymatic degradation of plant biomass requires the coordinated action of various enzymes. In this study, the production of reducing sugars from pectic substrates and sugar beet pulp (SBP) was investigated and compared using commercial enzyme preparations, including M2, pectinase (E1), Viscozyme L (V-L) and L-40. V-L, a cellulolytic enzyme mix produced by Aspergillus sp. was further evaluated as the most robust enzyme cocktail with the strongest SBP degradation ability in terms of the release of monosaccharides, methanol, and acetate from SBP. Mass-spectrometry-based proteomics analysis of V-L revealed 156 individual proteins. Of these, 101 proteins were annotated as containing a carbohydrate-active enzyme module. Notably, of the 50 most abundant proteins, ca. 44 % were predicted to be involved in pectin degradation. To reveal the role of individual putative key enzymes in pectic substrate decomposition, two abundant galacturonases (PglA and PglB), were heterologously expressed in Pichia pastoris and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 68 °C, respectively, and exhibited endo-type cleavage patterns towards polygalacturonic acid. Further studies along this line may lead to a better understanding of efficient SBP degradation and may help to design improved artificial enzyme mixtures with lower complexity for future application in biotechnology.