Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase
Guusje van Schaick1, Nadi El Hajjouti1, Simone Nicolardi1
1Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
International Journal of Molecular Sciences
|February 15, 2022
Summary
This study characterizes endo-1,4-β-xylanase (ENDO-I) proteoforms using a novel workflow. Thermal stress induced glycation, reducing enzyme activity, highlighting the importance of understanding post-translational modifications for enzyme optimization.
Area of Science:
- Biotechnology
- Enzymology
- Protein Chemistry
Background:
- Xylanases are crucial industrial enzymes with biotechnological production.
- Post-translational modifications (PTMs) significantly impact enzyme function.
- Understanding PTMs guides the development of high-performance enzymes.
Purpose of the Study:
- To develop and validate a workflow for structural and functional characterization of endo-1,4-β-xylanase (ENDO-I) proteoforms.
- To investigate the impact of thermal stress on ENDO-I PTMs and subsequent function.
- To establish structure-function relationships for ENDO-I proteoforms.
Main Methods:
- Orthogonal native separation techniques including size exclusion chromatography (SEC), ion exchange chromatography (IEX), and boronate affinity chromatography (BAC).
- Mass spectrometry for proteoform identification and characterization.
- Enzyme activity assays to quantify functional differences between proteoforms.
Main Results:
- A workflow was established for resolving and characterizing ENDO-I proteoforms.
- Thermal stress induced low-abundance glycated variants in ENDO-I.
- Glycated ENDO-I variants exhibited reduced enzymatic activity compared to non-modified enzyme.
Conclusions:
- The developed workflow enables in-depth structural and functional analysis of enzyme proteoforms.
- Glycation, induced by thermal stress, negatively affects ENDO-I activity.
- This research provides insights for optimizing enzyme production and application by controlling PTMs.
