Letter to the Editor

A Simple Individual-based Model of Insoluble Polysaccharide Hydrolysis: the Potential for Autosynergism with Dual-activity Glycosidases

Processes involving the enzymatic hydrolysis of polysaccharides in biorefineries are receiving increased attention. Mathematical models of these processes can guide process design and operation. For complex reaction networks, stochastic models are preferable to deterministic models. To be useful for guiding process design and operation, stochastic models cannot advance in steps of degree of reaction, they must advance in steps of time. Several current stochastic models for the hydrolysis of polysaccharides do advance in steps of time. However, some use parameters that have no recognized biological meaning; in others, the time axis is fitted to experimentally determined profiles. In the current work, we propose stochastic models that advance in time in a manner that is consistent with classical enzyme kinetic theory: the parameters of these models are specificity constants and saturation constants. We present three case studies. (1) We use the hydrolysis of thiophenyl N-acetyl-β-(1,6)-pentaglucosamine by DispersinB to demonstrate that the predictions of the stochastic model are equal to those of a deterministic model based on differential equations with classical kinetic expressions. (2) We model the hydrolysis of a mixture of oligomers of D-galacturonic acid by an endopolygalacturonase, a system for which it is not convenient to use a deterministic model. (3) We extend the method to a system with two enzymes and demonstrate how the model can be used to optimize the ratio of the enzymes. Our approach can lay the basis for the development of a new family of stochastic models describing the hydrolysis of colloidal suspensions of polysaccharides.

The recovery of fermentable sugars from lignocellulosic biomass is very challenging and requires multiple process steps. Numerous parameters are affecting this process and increase in the process steps increases the final cost of the product. Chemical and enzymatic methods are the most prevalent techniques in this conversion process and each of these methods has its own advantages and disadvantages. Chemical methods are toxic, corrosive, not environmentally friendly, and hazardous. Moreover it requires reactors and equipment that are resistant to corrosion which make the capital investment expensive. Enzymatic conversion is more environmentally friendly method but the efficiency and process costs are major concerns. The current chapter discusses the various chemical and biological methods for the hydrolysis of lignocellulosic biomass with more emphasis on enzymatic hydrolysis. The various factors which influence the process efficiency are discussed.

Solvent water is an essential factor for high solids enzymatic hydrolysis. To investigate its effect on substrate conversion efficiency in high solids hydrolysis of sugarcane bagasse (SCB), oleyl alcohol was used to partially substitute the solvent water. The results in batch hydrolysis tests in which diverse ratio of solvent water was replaced found that the majority of the substrate was insoluble. Then high solids fed-batch hydrolysis with the reaction solution mixed with solvent water and oleyl alcohol in the ratio of 3:1 (solids concentration correspond to 24% (w/v)) was carried out at the final real solids loading of 18% (w/v). The produced sugars were found to be less than pure water system, which indicated that water played a significant role in high solids hydrolysis process, and solids effect was related to the solvent water content.

Cellulose hydrolysis during bioethanol production from lignocellulosic biomass requires the use of strong acids or synergistic action of several cellulase enzymes. The high cost of cellulase enzymes is considered as the big hurdle in the commercialization success of the process. This chapter provides the basics of cellulose structure, the mechanism of the synergistic action of enzymatic hydrolysis, and the factors affecting enzymatic hydrolysis. Synthetic enzyme mixtures, a novel approach to reduce the overall enzyme cost, is discussed in the chapter. The chapter also provides an overview of the various approaches used to model enzymatic hydrolysis, along with their major assumptions and limitations.

Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and “steady state” conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the “steady state” rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on ¹⁴C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios.

Substrate channeling is a process of transferring the product of one enzyme to an adjacent cascade enzyme or cell without complete mixing with the bulk phase. Such phenomena can occur in vivo, in vitro, or ex vivo. Enzyme–enzyme or enzyme–cell complexes may be static or transient. In addition to enhanced reaction rates through substrate channeling in complexes, numerous potential benefits of such complexes are protection of unstable substrates, circumvention of unfavorable equilibrium and kinetics imposed, forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of toxic metabolite inhibition, and so on. Here we review numerous examples of natural and synthetic complexes featuring substrate channeling. Constructing synthetic in vivo, in vitro or ex vivo complexes for substrate channeling would have great biotechnological potentials in metabolic engineering, multi-enzyme-mediated biocatalysis, and cell-free synthetic pathway biotransformation (SyPaB).