Abstract
BackgroundCellodextrin 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.ResultsA 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.ConclusionsThe 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.
Highlights
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
Time‐course analysis of the enzymatic COS synthesis The molar substrate ratio of α-d-glucose 1-phosphate (αGlc1-P) and cellobiose determines the degree of polymerization (DP) distribution in the COS products released from the CdP reaction [6, 8, 15]
The larger this ratio, the greater is the abundance of high-DP products (DP ≥ 5) and so the portion of total product going into insoluble material
Summary
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. Cellodextrin phosphorylase (CdP; EC 2.4.1.49) catalyzes the consecutive (non-processive) depolymerization of cello-oligosaccharides (COS) in the presence of phosphate, forming α-d-glucose 1-phosphate (αGlc1-P) as the cleavage product [1, 2]. The CdP reaction in reverse direction under ex vivo conditions can provide an interesting route for the bottom-up synthesis of COS. Iterative β-1,4-glycosylation of cellobiose from αGlc1-P was shown in several studies [6,7,8, 11, 12] and it was recently demonstrated for the enzymatic production of COS at ~ 100 g/L final concentration [13]. Based on the emerging evidence on possible applications of COS for food and feed use [14], there is considerable interest in the intensification of the CdP-catalyzed conversion for the development of an efficient biocatalytic production
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