Three‐level hybrid modeling for systematic optimization of biocatalytic synthesis: α‐glucosyl glycerol production by enzymatic trans‐glycosylation from sucrose

Abstract

Mechanism‐based kinetic models are rigorous tools to analyze enzymatic reactions, but their extension to actual conditions of the biocatalytic synthesis can be difficult. Here, we demonstrate (mechanistic‐empirical) hybrid modeling for systematic optimization of the sucrose phosphorylase‐catalyzed glycosylation of glycerol from sucrose, to synthesize the cosmetic ingredient α‐glucosyl glycerol (GG). The empirical model part was developed to capture nonspecific effects of high sucrose concentrations (up to 1.5 M) on microscopic steps of the enzymatic trans‐glycosylation mechanism. Based on verified predictions of the enzyme performance under initial rate conditions (Level 1), the hybrid model was expanded by microscopic terms of the reverse reaction to account for the full‐time course of GG synthesis (Level 2). Lastly (Level 3), the application of the hybrid model for comprehensive window‐of‐operation analysis and constrained optimization of the GG production (~250 g/L) was demonstrated. Using two candidate sucrose phosphorylases (from Leuconostoc mesenteroides and Bifidobacterium adolescentis), we reveal the hybrid model as a powerful tool of “process decision making” to guide rational selection of the best‐suited enzyme catalyst. Our study exemplifies a closing of the gap between enzyme kinetic models considered for mechanistic research and applicable in technologically relevant reaction conditions; and it highlights the important benefit thus realizable for biocatalytic process development.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production II: Model‐based multiobjective optimization

“…The apparent violation of mass balance is explainable by enzymatic hydrolysis of Suc, G1P or both happening in small degree. ScP is known to possess low hydrolase activity towards Suc conditions in which the [Pi] is strongly limiting in the reaction (Klimacek et al, 2020; Sigg et al, 2021). However, as it did not affect the Cb formation, the side reaction leading to excess Glc in the process was not further investigated here.…”

“…A more robust approach uses design of experiments which can be performed in a purely data-driven fashion based on statistical models or under support from kinetic models of the synthetic reactions (Siedentop et al, 2021;Taylor, Pomberger, et al, 2023). Mechanism-based kinetic models achieve the most comprehensive and in-depth description of the reactions analyzed (Almquist et al, 2014;Gernaey et al, 2010;Harris et al, 2022;Sigg et al, 2021Sigg et al, , 2023. These models are powerful engineering tools of optimization in their own right.…”

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development

“…Given this fundamental relevance of modeling, surprisingly, there seems to be a certain vagueness on its systematic use for practical effect in reaction development and optimization (for reviews, see Kara & Rudroff 2021; Siedentop et al, 2021). Analogous to experiment, kinetic models differ widely in the degree and detail of system description reached (Berendsen et al, 2006; Klimacek et al, 2020; Sigg et al, 2021; Sun et al, 2021). Central questions, like that of minimum model requirements for reliable application in cascade optimization, appear to lack authoritative answers.…”

Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

“…Broad specificity for the nucleophile and low hydrolysis represent a combination of properties that is rare among GHs and makes sucrose phosphorylase a promising transglycosylase candidate, for synthetic applications . The enzyme reaction with glycerol is used industrially to produce the cosmetic ingredient 2-O-α- d -glucosyl glycerol from sucrose (Figure S2 , ). Reactions with l -ascorbic acid and glucose , provide other commercially relevant products, namely, l -ascorbic acid 2-α- d -glucoside and kojibiose, respectively (Figure S2).…”

“…The current study was performed to obtain a better understanding of the energetics underlying the versatile catalysis of sucrose phosphorylase. The approach used was a detailed study of temperature effects on steady-state rate parameters for the main types of enzymatic reaction: , phosphorolysis, transglycosylation (to glycerol), and hydrolysis (Figure a).…”