Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms.

Guusje Van Schaick,Manfred Wuhrer,Maurien M A Olsthoorn,Romana Jansen,Simone Nicolardi,Wim Bijleveld,Nadi El Hajjouti,Rob Van Der Hoeven,Joost Den Hartog,Elena Domínguez-Vega,Nicolas Abello

doi:10.3390/ijms23031307

Abstract

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.

Highlights

Xylanases are industrially produced enzymes with a broad application in the paper and pulp, textile, food, and biorefinery industries [1]
To investigate in detail the changes of the product, we developed size exclusion chromatography (SEC), ion exchange chromatography (IEX), and boronate affinity chromatography (BAC) methods to resolve proteoforms based on size, charge, and presence of glycation
An industrially produced ENDO-I granulated enzyme product expressed in Aspergillus niger was employed as a model sample

Summary

Introduction

Xylanases are industrially produced enzymes with a broad application in the paper and pulp, textile, food, and biorefinery industries [1]. The filamentous fungi Aspergillus and Trichoderma are known to produce high xylanase levels, making them suitable for commercial production [3]. Xylanases exist in many sizes varying from 6 to 80 kDa, with optimal performance between pH 3.0 and 6.5 and at temperatures of between 25 and 60 ◦ C [4]. These enzymes often contain a plethora of post-translational modifications (PTMs), including glycosylation, disulfide bond formation, and proteolytic truncation [5]. These modifications might play a decisive role in the functionality of the enzyme [11], potentially influencing product performance and, they should be carefully monitored

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Conclusion