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| Auteurs principaux: | , , , , , |
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| Format: | Artículo Open Access |
| Publié: |
Wiley
2025
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| Accès en ligne: | https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/bit.70003 |
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- The P134Q Sucrose Phosphorylase: Subtle Changes in the Catalytic Properties Benefit the Production of 2‐O‐α‐Glucosyl Glycerol Alexander Sigg Mario Klimacek Martin Pfeiffer Jorick Franceus Tom Desmet Bernd Nidetzky Biotechnology and Bioengineering ABSTRACTProtein engineering of Bifidobacterium adolescentis sucrose phosphorylase (BaSucP) has previously identified the P134Q enzyme variant for site‐selective glycosylation at the 2‐OH of glycerol. Besides improvement in selectivity, the P134Q‐BaSucP additionally involves enhanced affinity for glycerol as a biochemical property potentially important for the production of 2‐O‐α‐glucosyl glycerol (2GG), a commercialized skincare ingredient for cosmetic applications. Here, we performed a detailed kinetic model‐based evaluation of P134Q‐BaSucP in initial‐rate and full reaction time course analyses to obtain a mechanistic interpretation and a comprehensive assessment of the process improvements achievable by the P134Q variant compared to the native enzyme. We show that P134Q‐BaSucP involves ∼50‐fold lowered reactivity with phosphate compared to native enzyme. The effect likely arises from decreased conformational flexibility of the substrate binding pocket in the P134Q variant that may also serve to constrain the positioning of glycerol for glycosylation. Glycerol reactivity is decreased ∼1.3‐fold in P134Q‐BaSucP; yet because the hydrolytic reactivity is lowered even more (threefold), the transfer efficiency to glycerol of the variant is enhanced ∼twofold compared to the native enzyme. Product inhibition by 2GG is decreased ∼threefold in P134Q‐BaSucP. These properties of P134Q‐BaSucP combine into major benefits for 2GG synthesis in terms of productivity and product yield. Model‐based window‐of‐operation analysis for 2GG production from sucrose and glycerol further reveals the significant potential for saving on the excess glycerol used in the process that results from replacing the wild‐type BaSucP with the P134Q variant. Collectively, this study shows the important interplay of enzyme and reaction engineering in the optimization of glycoside production through biocatalytic transglycosylation. 10.1002/bit.70003 http://creativecommons.org/licenses/by/4.0/