Analysis of Post-translational Modifications of endo-Glucanases using High Accuracy Mass Spectrometry
EMSL Project ID
48931
Abstract
A critical step in the lignocellulosic biofuels pipeline is the extraction of fermentable sugars from plant biomass. Extracellular fungal enzymes are currently the most effective way of deconstructing biomass, but their efficiency needs to be improved. The Walton lab has shown that an endo-?1,4-glucanase (EG) from the fungus Sporotrichum (StCel5A) outperforms the ortholog from Trichoderma (TrCel5A). The two enzymes define two phylogenetic sub-branches of subfamily 5 of Glycosyl Hydrolase (GH) family 5 of endo-glucanases. To test the correlation between sub-branch, superiority, and substrate range, an additional four members of the two sub-branches have been expressed in T. reesei. Although all six enzymes have similar predicted molecular weights, they differ significantly in apparent molecular weights by SDS-PAGE. We hypothesize that this is due to dramatically different post-translational modifications such as N- and O-glycosylation. The goal of this proposal is to take advantage of the advanced mass spectrometric capacity at EMSL to analyze the glycosylation and other posttranslational modifications (PTMs) that the heterologous enzymes undergo in T. reesei. In particular, the proposed research will use “top down” proteomics to characterize in detail the proteoforms (isoforms) of heterologous EG enzymes. A major ultimate goal is to understand what effects the observed PTMs have on critical enzymatic properties such as specific activity, pH and temperature optima, cooperativity with other enzymes, and substrate range. The expected results will not only be relevant to the discovery of superior forms of EG for biomass applications but also more generally to a better understanding of the limitations of expressing heterologous proteins in filamentous fungi.Project Details
Project type
Large-Scale EMSL Research
Start Date
2015-10-01
End Date
2016-09-30
Status
Closed
Released Data Link
Team
Principal Investigator