Kinetic modeling of phosphorylase-catalyzed iterative β-1,4-glycosylation for degree of polymerization-controlled synthesis of soluble cello-oligosaccharides

Abstract

Background Cellodextrin phosphorylase (CdP; EC 2.4.1.49) catalyzes the iterative β-1,4-glycosylation of cellobiose using α-d-glucose 1-phosphate as the donor substrate. Cello-oligosaccharides (COS) with a degree of polymerization (DP) of up to 6 are soluble while those of larger DP self-assemble into solid cellulose material. The soluble COS have attracted considerable attention for their use as dietary fibers that offer a selective prebiotic function. An efficient synthesis of soluble COS requires good control over the DP of the products formed. A mathematical model of the iterative enzymatic glycosylation would be important to facilitate target-oriented process development. Results A detailed time-course analysis of the formation of COS products from cellobiose (25 mM, 50 mM) and α-d-glucose 1-phosphate (10–100 mM) was performed using the CdP from Clostridium cellulosi. A mechanism-based, Michaelis–Menten type mathematical model was developed to describe the kinetics of the iterative enzymatic glycosylation of cellobiose. The mechanistic model was combined with an empirical description of the DP-dependent self-assembly of the COS into insoluble cellulose. The hybrid model thus obtained was used for kinetic parameter determination from time-course fits performed with constraints derived from initial rate data. The fitted hybrid model provided excellent description of the experimental dynamics of the COS in the DP range 3–6 and also accounted for the insoluble product formation. The hybrid model was suitable to disentangle the complex relationship between the process conditions used (i.e., substrate concentration, donor/acceptor ratio, reaction time) and the reaction output obtained (i.e., yield and composition of soluble COS). Model application to a window-of-operation analysis for the synthesis of soluble COS was demonstrated on the example of a COS mixture enriched in DP 4. Conclusions The hybrid model of CdP-catalyzed iterative glycosylation is an important engineering tool to study and optimize the biocatalytic synthesis of soluble COS. The kinetic modeling approach used here can be of a general interest to be applied to other iteratively catalyzed enzymatic reactions of synthetic importance.

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

“…Enzyme (0.17 U/mL) was incubated with 50 mM or 8.0 mM G1P in the presence of Glc varied at 80, 300, and 600 mM. Initial rates were determined from the phosphate release measured in five samples withdrawn at appropriate times up to 40 min (Klimacek et al, 2021).…”

Bottom‐Up Synthesized Glucan Materials: Opportunities from Applied Biocatalysis

“…[ 46a ] Reactions from d ‐glucose 1‐phosphate have equilibrium with K eq typically below 4. [ 61 ] Transglucosidase reactions are kinetically controlled. [ 46b ] The d ‐glucan product can be hydrolyzed as it accumulates.…”

Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production

“…DP control in the oligosaccharide product is the crucial element of successful COS production. In the absence of such control, the CdP continues the β-1,4-chain oligomerization to a point (DP ≥ 6) at which self-assembly driven aggregation of the oligosaccharides from solution can no longer be suppressed adequately [ 17 , 23 , 29 , 30 ]. While useful for the preparation of insoluble cellulose with property-tunable characteristics of material [ 13 , 25 , 29 , 31 – 35 ], product precipitation lowers the efficiency of the COS production.…”