Edward A. Bayer, Bernard Henrissat, and Raphael Lamed

13.1 Introduction

Plant cell wall polysaccharides provide an exceptional source of carbon and energy that can be potentially utilized as a low-cost renewable source of mixed sugars for fermentation to biofuels like ethanol (1-3). Perhaps the major bottleneck for conversion of biomass to ethanol is the combined high cost and low efficiency of the enzymes — the cellulases and other glycoside hydrolases — that are capable of degrading cellulose and myriads of complex plant cell wall polysaccharides to simple sugars. Efficient hydrolysis is impeded by limited accessibility of the enzymes and the recalcitrance of cellulose, owing to the extremely stable “paracrystalline” arrangement of the cellulose chains in the microfibrils (4).

The rate-limiting step in the hydrolysis of cellulose is not the catalytic cleavage of the (3 -1,4- glucosidic bond, but the disruption of a single chain of the substrate from its native crystalline matrix, thereby rendering it accessible to the active site of the enzyme. Thus, the processes and interactions that are most significant are those that facilitate the disruption of strong interchain hydrogen-bonding network that characterizes the microcrystalline arrangement of the insoluble cellulose structure (5). Moreover, single cellulolytic enzymes alone are generally incapable of efficient cellulose hydrolysis (6). The mode of action of the various cellulases is different, and they act synergistically, such that the combined extent of hydrolysis is much more than the sum of the individual parts (6-8). A simplistic example of this phenomenon is apparent when comparing the action of endoglucanases versus that of the exoglucanases: Endoglucanases cleave at random points along the cellulose chain, whereas exoglucanases cleave successive units (e. g., cellobiose or cellotetraose) from the chain ends in a “processive” (sequential) manner (9). Processive endoglucanases have also been described (10,11), whereby an initial endo cleavage is followed by more systematic, successive cleavage along the chain, owing to either an appropriate change in the active-site architecture or the effects of an associated ancillary module(s) that might confer processivity on an otherwise endo-acting enzyme. In any case, the secret to potent enzymatic degradation of a recalcitrant cellulosic substrate is how these different types of enzymes work together, which is difficult to approach experimentally but is a key conceptual component for improving the overall degradation of cellulosic biomass.

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel © 2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6

In nature, degradation of cellulosic substrates is accomplished by various microorgan­isms, thus contributing a central component to the carbon cycle. In some cases, free-living microorganisms exploit such polysaccharides from decaying plant matter, found, for ex­ample, in compost piles and sewage sludge; in others, the microbes assist higher animals (e. g., ruminants, termites, etc.) in converting the polysaccharides to digestible components. Microbial degradation of cellulosic materials is one of the most important processes in na­ture, and different bacteria and fungi approach the task in different ways (12-16). Whereas aerobic microbes commonly produce copious quantities of the relevant enzymes (e. g., cel — lulases and hemicellulases), the biosynthetic apparatuses of anaerobes are much more frugal in their output of such enzymes. In this context, it is believed that the anaerobic environ­ment presents a greater selective pressure for the evolution of highly efficient machinery for the extracellular degradation of polymeric substrates, such as the recalcitrant crystalline components of the plant cell wall. The energy yield of aerobes per hydrolyzed sugar unit is much greater than that of anaerobes, which have evolved extensive energy-conserving mechanisms for physiological adaptation to environmental stresses such as novel enzyme activities (17). Consequently, the anaerobes tend to adopt alternative strategies for degrad­ing plant matter, and of these the organization of the enzymes into cellulosomes appears to be the most remarkable.

Much of the pioneering work on cellulases was first performed on “free” enzyme systems, commonly produced by fungi and some, aerobic bacterial species. The free cellulases are relatively simple macromolecules consisting generally of a catalytic module and cellulose­binding module (CBM) on a single polypeptide chain. The CBM targets the catalytic module to the cellulose surface, whereupon it begins to disrupt and degrade the cellulose chains. The different types of cellulase enzymes are thus distributed and interact freely in a synergistic manner (8).