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RSS FeedsIJMS, Vol. 23, Pages 1307: Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-xylanase Proteoforms (International Journal of Molecular Sciences)

 
 

24 january 2022 16:39:29

 
IJMS, Vol. 23, Pages 1307: Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-xylanase Proteoforms (International Journal of Molecular Sciences)
 


Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.


 
71 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
IJMS, Vol. 23, Pages 1305: Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants (International Journal of Molecular Sciences)
IJMS, Vol. 23, Pages 1311: Recent Development in NKT-Based Immunotherapy of Glioblastoma: From Bench to Bedside (International Journal of Molecular Sciences)
 
 
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