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) from Aspergillus niger, 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 dynamic release of monosaccharides, methanol, and acetate from substrate SBP. Glucose, the building block of cellulose, was the first product to reach its maximum concentration within 2.5 hours. In contrast, the major monosaccharides in pectin, including arabinose and galacturonic acid, were continuously released from the residue until reaching a peak of 2,092 mg L-1 and 7,069 mg L-1 after 19 hours of fermentation. Label-free proteomics analysis (MS/MS) of V-L revealed 151 individual proteins. Of these, 137 proteins were annotated as containing a carbohydrate-active enzymes (CAZyme) module. Notably, of the 50 most abundant proteins, which accounted for over 90% of the protein amount in V-L, ca. 40% were predicted to be involved in pectin degradation (belonging mainly to CAZyme families GH28, CE8, CE16, PL1 and PL3). To reveal the role of individual putative key enzymes of pectic substrate decomposition, two galacturonases (PglA and PglB), which are core of pectin-degrading enzymes of the GH28 family, were heterologously expressed in Pichia pastoris strain GB10 and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 67 °C, respectively. Both enzymes exhibited endo-cleavage patterns towards polygalacturonic acid (PGA) and released oligosaccharides with different degrees of polymerization (DP). In conclusion, our study identified two pectin-cleaving enzymes present in major amounts in a highly efficient SBP-saccharifying enzyme mix and characterized some of their properties. Further studies along this line may facilitate the understanding of SBP degradation and may help to design improved artificial enzyme mixtures with a lower complexity than V-L for future application in biotechnology.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Aspergillus Aculeatus Atcc 16872

SUBMITTER: Christina Ludwig  

LAB HEAD: Christina Ludwig

PROVIDER: PXD045553 | Pride | 2024-04-09

REPOSITORIES: Pride