{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T23:01:42Z","timestamp":1792710102814,"version":"4.5.0"},"reference-count":48,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2025,6,20]],"date-time":"2025-06-20T00:00:00Z","timestamp":1750377600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2025,9]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>Protein engineering of <jats:italic>Bifidobacterium adolescentis<\/jats:italic> sucrose phosphorylase (<jats:italic>Ba<\/jats:italic>SucP) has previously identified the P134Q enzyme variant for site\u2010selective glycosylation at the 2\u2010OH of glycerol. Besides improvement in selectivity, the P134Q\u2010<jats:italic>Ba<\/jats:italic>SucP additionally involves enhanced affinity for glycerol as a biochemical property potentially important for the production of 2\u2010<jats:italic>O<\/jats:italic>\u2010\u03b1\u2010glucosyl glycerol (2GG), a commercialized skincare ingredient for cosmetic applications. Here, we performed a detailed kinetic model\u2010based evaluation of P134Q\u2010<jats:italic>Ba<\/jats:italic>SucP in initial\u2010rate 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\u2010<jats:italic>Ba<\/jats:italic>SucP involves \u223c50\u2010fold 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 \u223c1.3\u2010fold in P134Q\u2010<jats:italic>Ba<\/jats:italic>SucP; yet because the hydrolytic reactivity is lowered even more (threefold), the transfer efficiency to glycerol of the variant is enhanced \u223ctwofold compared to the native enzyme. Product inhibition by 2GG is decreased \u223cthreefold in P134Q\u2010<jats:italic>Ba<\/jats:italic>SucP. These properties of P134Q\u2010<jats:italic>Ba<\/jats:italic>SucP combine into major benefits for 2GG synthesis in terms of productivity and product yield. Model\u2010based window\u2010of\u2010operation 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\u2010type <jats:italic>Ba<\/jats:italic>SucP 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.<\/jats:p>","DOI":"10.1002\/bit.70003","type":"journal-article","created":{"date-parts":[[2025,6,20]],"date-time":"2025-06-20T05:43:30Z","timestamp":1750398210000},"page":"2465-2477","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["The P134Q Sucrose Phosphorylase: Subtle Changes in the Catalytic Properties Benefit the Production of 2\u2010<i>O<\/i>\u2010\u03b1\u2010Glucosyl Glycerol"],"prefix":"10.1002","volume":"122","author":[{"given":"Alexander","family":"Sigg","sequence":"first","affiliation":[{"name":"Institute of Biotechnology and Biochemical Engineering Graz University of Technology, NAWI Graz Graz Austria"},{"name":"Austrian Centre of Industrial Biotechnology (acib) Graz 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