Evaluating the effect of glycation on lipase activity using boronate affinity chromatography and mass spectrometry

Abstract

Protein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme performance (e.g., stability or function) is often not predictable, highlighting the importance of having appropriate analytical methodologies to monitor the influence of glycation on performance. Here, a boronate affinity chromatography method was developed to enrich glycated species followed by mass spectrometry for structural characterization and activity assays for functional assessment. This approach was applied to a (temperature-stressed) lipase used for food applications revealing that storage at -20 °C and 4 °C resulted in minor glycation (below 9%), whereas storage at 25 °C led to a higher glycation level with up to four sugars per lipase molecule. Remarkably, activity measurements revealed that glycation did not reduce lipase activity or stability. Altogether, this novel strategy is a helpful extension to the current analytical toolbox supporting development of enzyme products.

Keywords: Boronate affinity chromatography; Glycation; Lipase; Mass spectrometry; Structure–function relationship.

Conflict of interest statement

Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Co-authors Olaf Schouten, Joost den Hartog, Michiel Akeroyd, Rob van der Hoeven, Wim Bijleveld, Nicolas Abello, and Maurien Olsthoorn are affiliated with Royal DSM, a global company active in health, nutrition, and bioscience.

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