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

Mario Klimacek, Chao Zhong, Bernd Nidetzky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Background: Cellodextrin phosphorylase (CdP; EC 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.

Original languageEnglish
Article number134
Number of pages15
JournalBiotechnology for Biofuels
Issue number134
Publication statusPublished - 10 Jun 2021


  • Bottom-up oligosaccharide synthesis
  • Cello-oligosaccharides
  • Cellodextrin phosphorylase (EC
  • Cellulose
  • Iterative glycosylation
  • Kinetic analysis and modeling

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Energy(all)
  • Biotechnology
  • Management, Monitoring, Policy and Law
  • Renewable Energy, Sustainability and the Environment

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