Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method. In this study, four different techniques incorporating X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) were compared using eight different cellulose preparations. We found that the simplest method, which is also the most widely used, and which involves measurement of just two heights in the X-ray diffractogram, produced significantly higher crystallinity values than did the other methods. Data in the literature for the cellulose preparation used (Avicel PH-101) support this observation. We believe that the alternative X-ray diffraction (XRD) and NMR methods presented here, which consider the contributions from amorphous and crystalline cellulose to the entire XRD and NMR spectra, provide a more accurate measure of the crystallinity of cellulose. Although celluloses having a high amorphous content are usually more easily digested by enzymes, it is unclear, based on studies published in the literature, whether CI actually provides a clear indication of the digestibility of a cellulose sample. Cellulose accessibility should be affected by crystallinity, but is also likely to be affected by several other parameters, such as lignin/hemicellulose contents and distribution, porosity, and particle size. Given the methodological dependency of cellulose CI values and the complex nature of cellulase interactions with amorphous and crystalline celluloses, we caution against trying to correlate relatively small changes in CI with changes in cellulose digestibility. In addition, the prediction of cellulase performance based on low levels of cellulose conversion may not include sufficient digestion of the crystalline component to be meaningful.

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Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance

Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.20282

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

Outlook for cellulase improvement: screening and selection strategies.

Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the β-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.

The biological degradation of cellulose

Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review.

binding and hydr~lysis.~~",' ~~~~ There also have been severa1 st~dies~~~~~~~,~ ~ investigating how various cellulases act synergistically to yield higher rates and extents of hydrolysis than would be predicted from the sum of the hydrolytic activities of the individual enzymes. In most of these studies only cellulase binding and reducing sugar formation were measured. Very little is known about the fragmentation activities of pure cellulases and their mixtures. It has been known for some time that cellulose undergoes fragmentation during enzymatic hydrolysis.6,28 Several investigators have observed transverse cracks and complete tranverse breakage of cellul~se.~~'~~~ ~~~~ Others have observed an increase in the number of cellulose and a shift in the volume fraction distrib~tion.~',~' The rate and extent of particle formation particles 10,18,19,23,26 * To whom all correspondence should be addressed. during hydrolysis increase linearly with increasing enzyme c~ncentration.'~,'~,~~,~'"~ Also, the rate and extent of fragmentation, as measured by changes in mean particle volume with time, decrease with decreasing initial particle size.31 It has been reported that both endoglucanases (E) and exoglucanases (CBH) are capable of fragmenting cell~lose,'~~~~~ ~~ but that the bulk of the fragmentation activity is associated with endoglucana~es.~ ~~~~ Wood et al.43 concluded that a mixture of endoglucanases and exoglucanases is necessary to fragment cotton fiber. In this study, the fragmentation activity of Thermomonospora fusca E , E , E s , Trichoderma reesei CBHI, and mixtures of these cellulases was measured. T. fusca E2 and Es were selected because of their high CMC activity, which is characteristic of highly active endoglucana~es,~' and the availability of clones for the production of these pure enzymes. T. fusca E3 was selected because it gives synergism when mixed with the other T. fusca cellulases, while i? reesei CBHI was selected to determine whether cross mixing a fungal cellulase and a bacterial cellulase would yield synergism.

C. McQuire

Papers

Fragmentation of cellulose by the major Thermomonospora fusca cellulases, Trichoderma reesei CBHI, and their mixtures.