Elsevier

Biotechnology Advances

Volume 27, Issue 6, November–December 2009, Pages 833-848
Biotechnology Advances

Research review paper
Modeling cellulase kinetics on lignocellulosic substrates

https://doi.org/10.1016/j.biotechadv.2009.06.005Get rights and content

Abstract

The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps involved in the conversion of lignocellulosic biomass to yield biofuel. This hydrolysis by cellulases, a heterogeneous reaction, currently suffers from some major limitations, most importantly a dramatic rate slowdown at high degrees of conversion. To render the process economically viable, increases in hydrolysis rates and yields are necessary and require improvement both in enzymes (via protein engineering) and processing, i.e. optimization of reaction conditions, reactor design, enzyme and substrate cocktail compositions, enzyme recycling and recovery strategies. Advances in both areas in turn strongly depend on the progress in the accurate quantification of substrate–enzyme interactions and causes for the rate slowdown. The past five years have seen a significant increase in the number of studies on the kinetics of the enzymatic hydrolysis of cellulose. This review provides an overview of the models published thus far, classifies and tabulates these models, and presents an analysis of their basic assumptions. While the exact mechanism of cellulases on lignocellulosic biomass is not completely understood yet, models in the literature have elucidated various factors affecting the enzymatic rates and activities. Different assumptions regarding rate-limiting factors and basic substrate–enzyme interactions were employed to develop and validate these models. However, the models need to be further tested against additional experimental data to validate or disprove any underlying hypothesis. It should also provide better insight on additional parameters required in the case that more substrate and enzyme properties are to be included in a model.

Introduction

The possibility of deriving fuel from the largest carbon source on Earth — lignocellulose in various forms, such as grass, wood, trees or husks — has resulted in large investments in the biofuel industry in recent past (Schubert, 2006, Sheridan, 2008, Waltz, 2007). Ethanol derived from lignocellulose is produced via four major consecutive steps: pretreatment, hydrolysis, fermentation, and separation. It has been recognized by experts that major improvements have to be made in the enzymatic hydrolysis of cellulosic biomass for cellulosic ethanol to compete economically with corn ethanol and petroleum-derived gasoline (Galbe and Zacchi, 2002, Lynd et al., 2008, Sun and Cheng, 2002). Main challenges include decreasing rates, high cellulase costs, and little understanding of cellulase kinetics on lignocellulosic substrates. The advantages of enzymatic hydrolysis of cellulose over other hydrolysis methods such as acid hydrolysis are lower utility (cooling water, gas, electricity) and disposal costs and no corrosion issues for equipment (Sun and Cheng, 2002). The huge investments are mainly driven by the potential reduction in the cost of cellulosic ethanol as projected by advances in cellulase-based technology (Lynd et al., 2008). Cost-competitive technology can be developed by improving the cellulase machinery as well as by rendering the cellulosic substrates more susceptible to hydrolysis (Himmel et al., 2007). To do so, it is first necessary to understand the enzyme–substrate interactions and both identify and quantify the contribution of various system properties to the hydrolysis process. Cellulose is degraded synergistically into glucose by three types of cellulases: endoglucanases (EC 3.2.1.4), that randomly cleave β-1,4-glycosidic bonds on cellulose chains away from chain ends, cellobiohydrolases (EC 3.2.1.91), that produce cellobiose by attacking cellulose from chain ends (Cel7A (cellobiohydrolase I), acts from the reducing ends, and Cel6A (cellobiohydrolase II) acts from the non-reducing ends of the cellulose chains) as well as β-glucosidases (EC 3.2.1.21) that convert cellobiose to glucose (Henrissat, 1994, Lynd et al., 2002, Rabinovich et al., 2002, Teeri, 1997, Zhang and Lynd, 2004).

Experimental data on cellulose hydrolysis by cellulases point to various bottlenecks that contribute to decreasing rates with conversion (see Section 3). To deconvolute the data, mathematical modeling of the hydrolysis process is an important tool. A robust model is also needed to develop rate expressions that can be incorporated into process models required for large-scale biofuels production. Recent works on simultaneous saccharification and fermentation (SSF) (Shao et al., 2009a,b) have shown how kinetic models can be used for modeling staged reactor configurations with different feeding frequencies of the reaction mixture. Fed-batch strategies have also been developed for the enzymatic hydrolysis of cellulose (Hodge et al., 2009). Further improvement of cellulase kinetics will be guided by the relative importance of physical parameters of the model, such as those associated with adsorption or surface accessibility. To find and alleviate bottlenecks, the kinetic and the physical parameters in the model have to be estimated correctly.

The current paper reviews the various published models of enzymatic hydrolysis of both pure cellulosic and lignocellulosic materials, and gives an analysis of their key aspects as well as their shortcomings to highlight their role in advancing our understanding of this field. The experimental data present in the literature are discussed, with the aim of understanding the kinetics and rate-limiting causes. We also discuss the experimental data that could be generated to distinguish between the hypotheses regarding the decreasing rates. Lee et al. (1980) in 1980 reviewed the models published up to that point. Zhang and Lynd (2004) discussed the potential use of various models in literature, based on the number of substrate and enzyme variables considered. Both these articles concluded that to achieve a more detailed and phenomenological understanding of the hydrolysis process, more substrate and enzyme properties have to be considered in the kinetic models. While models which do so would be more robust, they would require more experimental data for validation due to the increase in the number of variables and parameters. In any case, the two main challenges of modeling the cellulose hydrolysis process are i) to gain a more fundamental understanding of the relevant enzyme and substrate variables (substrate-concentration, degree of polymerization, accessibility, adsorption capacity, size distribution of chains, crystallinity; enzyme-concentration, cellulase composition, adsorbed cellulase concentration, synergism), and ii) to identify rate-limiting factors.

Since the last review in 2004 (Zhang and Lynd, 2004), about thirty more works have been published on kinetic modeling of cellulose bioconversion. This is more than one third of the number of works in the literature on kinetic modeling of cellulose hydrolysis by cellulases. Given the recent enthusiasm in biofuels, we believe that the time has arrived for another review on the subject.

Product inhibition of cellulases (by cellobiose) is a phenomenon that can be quantified by independent experiments and can be alleviated with an excess of β-glucosidase (Bommarius et al., 2008). The overall structure of the kinetic models of enzymatic hydrolysis of cellulose and lignocellulose is not affected by the inclusion of product inhibition parameters. The phenomenon has been previously reviewed in 2002 (Lynd et al., 2002) and 2004 (Zhang and Lynd, 2004), and the state of the art in modeling product inhibition has not advanced since then. Therefore, in this article we do not discuss the various expressions used for product inhibition. However, we also discuss the incorporation of adsorption of cellulases on cellulosic substrates into the various models and the interchangeability of models for pure cellulosic vs. lignocellulosic substrates.

Section snippets

Model classes and classification

Biohydrolysis of cellulose, due its heterogeneous nature, involves more steps than classical enzyme kinetics. The major steps are (Fig. 1):

  • 1.

    Adsorption of cellulases onto the substrate via the binding domain (Ståhlberg et al., 1991),

  • 2.

    Location of a bond susceptible to hydrolysis on the substrate surface (Jervis et al., 1997) (chain end if cellobiohydrolase, cleavable bond if endoglucanase),

  • 3.

    Formation of enzyme–substrate complex (by threading of the chain end into the catalytic tunnel if

Rate limitations and decreasing rates with increasing conversion

Most of the experimental studies showed that the rate of hydrolysis drops by two to three orders of magnitude at high degrees of conversions (Fig. 2, from Bommarius et al. (2008)). Even after alleviating product inhibition from cellobiose, cellulase activities and hydrolysis rates fall precipitously as the reaction proceeds (Bommarius et al., 2008). To be able to increase the rates, the various bottlenecks in cellulose hydrolysis need to be elucidated.

The contributing factors to decreasing

Modeling synergism of cellulase components

A mixture of cellulase components, cellobiohydrolases and endoglucanases, has higher activity than the individual components alone (Beldman et al., 1988, Fujii and Shimizu, 1986, Gusakov et al., 2007, Henrissat et al., 1985, Kleman-Leyer et al., 1996, Nidetzky et al., 1994b, Schell et al., 1999, Wood and McCrae, 1978, Woodward et al., 1988a, Woodward et al., 1988b). Modeling synergistic kinetics of the cellulases requires separate mathematical expressions for the individual components and the

Models of pure cellulosic substrates and lignocellulosic substrates

Lignin reduces the accessibility of cellulose to cellulases and also adsorbs cellulases, resulting in lower hydrolysis rates (Mansfield et al., 1999). The effect of lignin content is also evident from numerous empirical models (see Table 1A). Since the presence of lignin can significantly affect the hydrolysis rates, models developed for pure cellulosic substrates cannot be extended to substrates having high lignin content. For example, in the presence of lignin, a two-phase model might be

Conclusions and outlook

Cellulase hydrolysis of cellulose is a reaction in heterogeneous medium. Classical homogenous enzyme catalysis is modeled by Michaelis–Menten kinetics and heterogeneous catalysis on a catalyst support, by Langmuir–Hinshelwood kinetics. Cellulase kinetics on insoluble lignocellulosic substrates is a combination of the above two kinds of reactions and also involves other factors (product inhibition, enzyme deactivation, substrate crystallinity, substrate accessibility changes, substrate

Acknowledgment

The authors thank the Chevron Corporation for the financial support.

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