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Evidence for a novel mechanism of microbial cellulose degradation
Abstract and Figures
There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant
cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use cellulases
to hydrolyze the β-1,4 linkages present in cellulose, however the way in which cellulases are presented to the environment
are quite different for each mechanism. Most aerobic microorganisms secrete a set of cellulases outside the cell (free cellulase
mechanism) while most anaerobic microorganisms produce large multi enzyme complexes on their outer surface (cellulosomal mechanism).
Their genomic sequences suggest that the aerobic bacterium, Cytophaga hutchinsonii and the anaerobic bacterium, Fibrobacter succinogenes, do not use either of these mechanisms for degrading cellulose, as these organisms only code for normal endocellulases not
for processive cellulases like exocellulases and processive endocellulases which are used in both of the well studied mechanisms.
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... Lignin slows cellulose decomposition due to its association with cellulose in the cell wall. In addition, the addition of readily decomposable organic matter initially enhances cellulose decomposition by facilitating microbial growth, but the rate may decrease as the readily available organic matter is depleted (Chmolowska et al., 2017;Fujii et al., 2023;Haddad et al., 2019;Shen et al., 2013;Wilson, 2009). Frank et al. (2021)showed that the degradation rate of nanocellulose mainly depends on the degree of surface replacement and not on the bulk. ...
This review investigates the potential of nanocellulose in agriculture, encompassing its structure, synthesis, modification, and applications. Our investigation of the characteristics of nanocellulose includes a comprehensive classification of its structure. Various mechanical, chemical and enzymatic synthesis techniques are evaluated, each offering distinct possibilities. The central role of surface functionalization is thoroughly examined. In particular, we are evaluating the conventional production of nanocellulose, thus contributing to the novelty. This review is a pioneering effort to comprehensively explore the use of nanocellulose in slow and controlled release fertilizers, revolutionizing nutrient management and improving crop productivity with reduced environmental impact. Additionally, our work uniquely integrates diverse applications of nanocellulose in agriculture, ranging from slow-release fertilizers, superabsorbent cellulose hydrogels for drought stress mitigation, and long-lasting crop protection via nanocellulose-based seed coatings. The study ends by identifying challenges and unexplored opportunities in the use of nanocellulose in agriculture. This review makes an innovative contribution by being the first comprehensive study to examine the multiple applications of nanocellulose in agriculture, including slow-release and controlled-release fertilizers.
... Most aerobic cellulolytic microorganisms degrade cellulose by secreting a series of free cellulases, while most anaerobic cellulolytic microorganisms perform the degradation process via cellulosome, a large multienzyme complex which bounds to the outer surface of the microorganisms (Wilson 2009;Bayer et al. 2004). In both strategies, synergistic action of endoglucanases (EC 3.2.1.4), ...
Cellulose is the most abundant renewable bioresources on earth, and the biodegradation and utilization of cellulose would contribute to the sustainable development of global environment. Sporocytophaga species are common aerobic cellulose-degrading bacteria in soil, which can adhere to the surface of cellulose matrix and motile by gliding. In this study, a differential transcriptome analysis of Sporocytophaga sp. CX11 was performed and a total of 4,217 differentially expressed genes (DEGs) were identified. Gene Ontology enrichment results showed that there are three GO categories related to cellulose degradation function among the annotated DEGs. A total of 177 DEGs were identified as genes encoding carbohydrate-active enzymes (CAZymes), among which 54 significantly upregulated CAZymes were mainly cellulases, hemicellulases, pectinases, etc. 39 DEGs were screened to associate with gliding function. In order to explore unannotated genes potentially related to cellulose metabolism, cluster analysis was performed using the Short-Time Series Expression Miner algorithm (STEM). 281 unannotated genes were predicted to be associated with the initial-middle stage of cellulose degradation and 289 unannotated genes might function in the middle-last stage of cellulose degradation. Sporocytophaga sp. CX11 could produce extracellular endo-xylanase, endo-glucanase, FPase and β-glucosidase, respectively, according to different carbon source conditions. Altogether, this study provides valuable insights into the transcriptome information of Sporocytophaga sp. CX11, which would be useful to explore its application in biodegradation and utilization of cellulose resources.
Graphical Abstract
... Both play an important role in cellulose degradation in some bacteria, but are not universally found in known lignocellulose degraders. They were not identified in Fibrobacter succinogenes, Cytophaga hutchinsonii or several gut and rumen metagenomes with lignocellulose-degrading capabilities [17,20,24,57]. In line with these findings, we found GH6 and GH48 to be annotated in less than 5% of the samples of our input collection, and only a single GH6 annotation (no GH48) in the metagenome bins. ...
Background: Efficient industrial processes for converting plant lignocellulosic materials into biofuels are a key challenge in global efforts to use alternative energy sources to fossil fuels. Novel cellulolytic enzymes have been discovered from microbial genomes and metagenomes of microbial communities. However, the identification of relevant genes without known homologs, and elucidation of the lignocellulolytic pathways and protein complexes for different microorganisms remain a challenge. Results: We describe a new computational method for the targeted discovery of functional modules of plant biomass-degrading protein families based on their co-occurrence patterns across genomes and metagenome datasets, and the strength of association of these modules with the genomes of known degraders. From more than 6.4 million family annotations for 2884 microbial genomes and 332 taxonomic bins from 18 metagenomes, we identified five functional modules that are distinctive for plant biomass degraders, which we call plant biomass degradation modules (PDMs). These modules incorporated protein families involved in the degradation of cellulose, hemicelluloses and pectins, structural components of the cellulosome and additional families with potential functions in plant biomass degradation. The PDMs could be linked to 81 gene clusters in genomes of known lignocellulose degraders, including previously described clusters of lignocellulolytic genes. On average, 70% of the families of each PDM mapped to gene clusters in known degraders, which served as an additional confirmation of their functional relationships. The presence of a PDM in a genome or taxonomic metagenome bin allowed us to predict an organism's ability for plant biomass degradation accurately. For 15 draft genomes of a cow rumen metagenome, we validated by cross-linking with confirmed cellulolytic enzymes that the PDMs identified plant biomass degraders within a complex microbial community. Conclusions: Functional modules of protein families that realize different aspects of plant cell wall degradation can be inferred from co-occurrence patterns across (meta-)genomes with a probabilistic topic model. The PDMs represent a new resource of protein families and candidate genes implicated in microbial plant biomass degradation. They can be used to predict the ability to degrade plant biomass for a genome or taxonomic bin. The method would also be suitable for characterizing other microbial phenotypes.
... Pour les Fibrobacteres, la dépolymérisation de la cellulose implique une adhérence au substrat suivie par son clivage par plusieurs types de cellulases (Seon Park et al., 2007;Wilson, 2009;Suen et al., 2011;Ransom-Jones et al., 2014;Neumann and Suen, 2018). Fibrobacter succinogenes est une bactérie connue pour s'adhérer à la cellulose et produire du succinate à partir de cette fraction. ...
La lignocellulose, principal composant des parois végétales et sous-produit agricole très abondant, est une des sources de carbone renouvelable les plus prometteuses pour la production d'énergie, de produits chimiques ou de biocarburants. L’utilisation de la lignocellulose pour la production de carboxylates repose sur l'utilisation de consortia microbiens. Pour améliorer les rendements de production de carboxylates, ces travaux de thèse se sont intéressés à i) caractériser le fonctionnement des consortia sélectionnés pour leur potentiel lignocellulolytique à partir de différents écosystèmes naturels et ii) à mieux comprendre leurs réponses fonctionnelles face à des changements dans la composition et la structure physicochimique du substrat. Pour cela, des approches d’écologie microbienne et des outils multi-omiques ont été appliquées.Ainsi, des consortia dérivés de rumen bovin ou d'intestin de termites ont été étudiés par des approches de métabarcoding 16S et de métaprotéomique comparative label-free. Leurs réponses fonctionnelles, enzymatiques et taxonomiques ont été évaluées en utilisant la paille de blée prétraitée par des méthodes mécaniques et/ou chimiques. Ces recherches ont permis de mettre en lumière le rôle et la complémentarité des différentes populations composant les consortia microbiens, ainsi que l’effet des modifications induites par le prétraitement du substrat sur la régulation de l’expression des enzymes impliquées dans la bioconversion des carbohydrates (CAZymes) en carboxylates. De plus, un consortium issu de rumen bovin et enrichi sur des résidus de maïs a également été étudié. L’étude de la dynamique des populations bactériennes au cours de l’enrichissement et de la bioconversion de la lignocellulose a permis de mettre en lumière le rôle des microorganismes libres et attachées au substrat, et de leurs enzymes, fournissant un aperçu inédit du fonctionnement des communautés lignocellulolytiques en bioréacteur.
... Apart from the two mechanisms of cellulase degradation discussed above, Cytophaga hutchinsonii and Fibrobacter succinogenes degrade cellulose through an entirely different mechanism [51][52][53]. In C. hutchinsonii, the genes encoding dockerin domains are not present, while very few CBM encoding genes are found. ...
Lignocellulose, the main component of plant cell walls, comprises polyaromatic lignin and fermentable materials, cellulose and hemicellulose. It is a plentiful and renewable feedstock for
chemicals and energy. It can serve as a raw material for the production of various value-added products, including cellulase and xylanase. Cellulase is essentially required in lignocellulose-based biorefineries and is applied in many commercial processes. Likewise, xylanases are industrially important enzymes applied in papermaking and in the manufacture of prebiotics and pharmaceuticals. Owing to the widespread application of these enzymes, many prokaryotes and eukaryotes have been exploited to produce cellulase and xylanases in good yields, yet yeasts have rarely been explored for their plant-cell-wall-degrading activities. This review is focused on summarizing reports about cellulolytic and xylanolytic yeasts, their properties, and their biotechnological applications.
... In contrast, solids retention times (SRT) is, practically the only controllable parameter affecting cellulose degradation in a municipal WWTP. Long SRT increase the level and diversity of cellulolytic bacteria, and the cellulose (as a particulate) is retained in the reactor for a longer time, which may result in higher biodegradability (Wilson, 2009). Although SRT change may shift hydrolytic populations, with possibly different optimal pH and temperature, the linked effect of SRT, temperature, and pH has not been identified due to the aggregate nature of a complex microbial community. ...
Cellulose contributes approximately one third of the influent suspended solids to wastewater treatment plants and is a key target for resource recovery. This study investigated the temperature impact on biological aerobic degradation of cellulose in laboratory-scale sequencing batch reactors (SBR) at four different temperatures (10–33 °C) and two different solids retention times (SRT) of 15 days and 3 days. The degradation efficiency of cellulose was observed to increase with temperature and was slightly dependent on SRT (80%–90% at an SRT of 15 days, and 78%–85% at an SRT of 3 days). Hydrolysis followed 1st order kinetics, rather than the biomass dependent Contois kinetics (default in the activated sludge models), with a hydrolysis coefficient at 20 °C of 1.14 ± 0.01 day⁻¹.
... Direct contact of C. hutchinsonii cells with cellulose is needed in the process of cellulose degradation, and regular arrangement of C. hutchinsonii cells was detected along the cellulose fiber (Wilson, 2009;Zhu and McBride, 2017). The unique cellulose degradation mechanism of C. hutchinsonii suggested that the cellulose adhesion ability is crucial for cellulose degradation. ...
The type IX secretion system (T9SS) is a novel protein secretion system, which is found in and confined to the phylum Bacteroidetes. T9SS is involved in the secretion of virulence factors, cell surface adhesins, and complex biopolymer degrading enzymes to the cell surface or extracellular medium. Cytophaga hutchinsonii is a widely distributed bacterium, which is able to efficiently digest cellulose and rapidly glide along the solid surfaces. C. hutchinsonii has a full set of orthologs of T9SS components. However, the functions of most homologous proteins have not been verified. In C. hutchinsonii, CHU_0029 and CHU_2709 are similar in sequence to Flavobacterium johnsoniae T9SS components SprA and SprT, respectively. In this study, the single deletion mutants of chu_0029 (sprA) and chu_2709 (sprT) were obtained using a complex medium with the addition of Ca2+ and Mg2+. Single deletion of sprA or sprT resulted in defects in cellulose utilization and gliding motility. Moreover, the ΔsprA and ΔsprT mutants showed growth defects in Ca2+- and Mg2+-deficient media. The results of ICP-MS test showed that both the whole cell and intracellular concentrations of Ca2+ were dramatically reduced in the ΔsprA and ΔsprT mutants, indicating that SprA and SprT are both important for the assimilation of trace amount of Ca2+. While the assimilation of Mg2+ was not obviously influenced in the ΔsprA and ΔsprT mutants. Through proteomics analysis of the cell surface proteins of the wild type and mutants, we found that the ΔsprA and ΔsprT mutants were defective in secretion of the majority of T9SS substrates. Together, these results indicate that SprA and SprT are both essential components of C. hutchinsonii T9SS, which is required for protein secretion, Ca2+ acquisition, cellulose degradation, and gliding motility in C. hutchinsonii. Our study shed more light on the functions of SprA and SprT in T9SS, and further proved the link between the T9SS and Ca2+ uptake system.
The recently discovered type IX secretion system (T9SS) is limited to the Bacteroidetes phylum. Cytophaga hutchinsonii, a member of the Bacteroidetes phylum widely spread in soil, has complete orthologs of T9SS components and many T9SS substrates. C. hutchinsonii can efficiently degrade crystalline cellulose using a novel strategy, in which bacterial cells must be in direct contact with cellulose. It can rapidly glide over surfaces via unclear mechanisms. Studies have shown that T9SS plays an important role in cellulose degradation, gliding motility, and ion assimilation in C. hutchinsonii. As reported recently, T9SS substrates are N- or O-glycosylated at their C-terminal domains (CTDs), with N-glycosylation being related to the translocation and outer membrane anchoring of these proteins. These findings have deepened our understanding of T9SS in C. hutchinsonii. In this review, we focused on the research progress on diverse substrates and functions of T9SS in C. hutchinsonii and the glycosylation of its substrates. A model of T9SS functions and the glycosylation of its substrates was proposed.
Microbes in the rumen of herbivores are responsible for effective plant biomass decomposition and digestion. Recent efforts to transform cellulosic biomass into biofuels have heightened interest in the bacterial fibrinolytic processes used by these bacteria. In ecology, plant cell wall material is used to transmit energy between the host and parasitic organisms. Herbivores eat plant material and digest it via symbiotic stomach microbiota (protozoa, fungi, and bacteria). Much anaerobic lignocellulose and hemicellulose-digesting bacteria populate the rumen. Cellulosome is a plant cell wall destroying bacteria’s strategic arsenal. Raphael Lamed identified this complex protein in 1983 in an extremophile Clostridium thermocellum. The cellulosome complex and its actions were also being studied as “Swiss knife” shape and protein complex, the cellulosome protein complex (carbohydrate-binding modules (CBM), cohesin, dockerin, enzymes, and scaffoldings. Scientists discovered these compounds in rumen microbes. A constant study has helped us learn more about rumen bacteria and how their cellulosomes break down plant cell walls. The rumen community depends on cellulolytic Ruminococcus spp. Cohesin-dockerin molecules combine to form cellulosome complexes. Designer cellulosomes are chimeric-tailored cellulosomes that function in a cell-free system. They improve the hydrolysis of cellulosic substrates to create value-added products. For this purpose, recombinant constructions and artificial self-assembling chimeric proteins are produced. The capacity of rumen microbes to digest refractory cellulose is of great industrial importance, and metagenomics research is helping to understand and determine the quantities and kinds of cellulolytic bacteria found in the bovine rumen complex ecosystem. This chapter explains the cellulosomal machinery’s extensive function in lignocellulose-degrading bacteria.
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures.
Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying
genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular
glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes,
are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a
single putative β-1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles
were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated
during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially
involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More
broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with
the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin
was lost.
The complete DNA sequence of the aerobic cellulolytic soil bacterium Cytophaga hutchinsonii, which belongs to the phylum Bacteroidetes, is presented. The genome consists of a single, circular, 4.43-Mb chromosome containing 3,790 open reading frames, 1,986
of which have been assigned a tentative function. Two of the most striking characteristics of C. hutchinsonii are its rapid gliding motility over surfaces and its contact-dependent digestion of crystalline cellulose. The mechanism
of C. hutchinsonii motility is not known, but its genome contains homologs for each of the gld genes that are required for gliding of the distantly related bacteroidete Flavobacterium johnsoniae. Cytophaga-Flavobacterium gliding appears to be novel and does not involve well-studied motility organelles such as flagella or type IV pili. Many
genes thought to encode proteins involved in cellulose utilization were identified. These include candidate endo-β-1,4-glucanases
and β-glucosidases. Surprisingly, obvious homologs of known cellobiohydrolases were not detected. Since such enzymes are needed
for efficient cellulose digestion by well-studied cellulolytic bacteria, C. hutchinsonii either has novel cellobiohydrolases or has an unusual method of cellulose utilization. Genes encoding proteins with cohesin
domains, which are characteristic of cellulosomes, were absent, but many proteins predicted to be involved in polysaccharide
utilization had putative D5 domains, which are thought to be involved in anchoring proteins to the cell surface.
From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during ligno-cellulose degradation. Globally, termites are an extremely success-ful group of wood-degrading organisms1 and are therefore important both for their roles in carbon turnover in the envir-onment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis2. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding ‘higher’ Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial com-munity specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-ml environment can be.
The hydrolysis of Whatman no. 1 filter paper by purified cellulolytic components from Trichoderma reesei and the synergistic action of binary combinations of these enzymes on the same substrate were investigated. At 20 milligrams filter paper, enzyme concentrations needed to obtain half-maximal hydrolysis rates (KE values) were in the 3-4 microM range for the cellobiohydrolases (CBHs) and 0.05-0.10 microM for the endoglucanases (EGs). Catalytic-core proteins of CBH I and EG III, lacking the cellulose-binding domain, exhibit KE values 2.3 and 5.1 times higher than those of the intact enzymes. In synergistic combinations of two cellulases, the KE value of at least one enzyme was 3-10-fold reduced. CBH I/CBH II and CBH I/EG III combinations showed the most powerful synergism, and optimal ratios were a function of the total protein concentration. Results obtained in activity and adsorption assays using filter paper pretreated with one component, followed by inactivation and subsequent hydrolysis with the same or another cellulase component, point to a sequential enzymic attack of the cellulose and seems consistent with the mathematical model presented.
The activities of six purified Thermomonospora fusca cellulases and Trichoderma reesei CBHI and CBHII were determined on filter paper, swollen cellulose, and CMC. A simple method to measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found to effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% to 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases to digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 μmol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 to 7.8. T. reesei CBHII and T. fusca E3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) of T. fusca endocellulases E2 and E5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a β-glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHI to T. fusca crude cellulase increased its activity on filter paper 1.7-fold. © 1993 John Wiley & Sons, Inc.
Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley β-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose.
Fibrobacter succinogenes is a major cellulolytic species in the rumen. On the basis of molecular data, this species can be classified into four phylogenetic groups. Recently gathered ecological and physiological data have revealed the importance of this species, particularly phylogenetic group 1, in rumen fiber digestion. These data indicate that group 1 should be the focus of future efforts to maximize the fibrolytic function of the rumen. © 2008 Institute of Microbiology, v.v.i, Academy of Sciences of the Czech Republic.
The chapter focuses on the recent advances in understanding the structural and functional organization of individual cellulases, their regulation, and the ways in which the multiple enzyme components of cellulolytic systems cooperate. It overviews the cellulose structures because cellulose is more than a homopolymer of β-1 ,4 linked glucose units. An appreciation of its complex physical organization and its interactions with other plant cell wall components is central for understanding of the mechanisms of cellulase action. Cellulose nearly always occurs in close association with plant cell wall matrix polysaccharides so that enzymes such as xylanases are intimately involved in the attack of cellulose in vivo. Cellulases effect important changes to their substrate before releasing soluble products and the key to understanding cellulase action rest in the examination of these events. Progress in this area is limited by the availability of appropriate analytical tools although new techniques, such as atomic force microscopy are promising. The properties of cellulases are profoundly altered by the presence of trace enzyme contaminants. Future studies in vitro are proposed to be restricted to enzymes from recombinant sources. The reasons for the individual cellulolytic bacteria and fungi requiring many related cellulases with specificities that overlap is still not clear, but perhaps this is because the complexity of the substrates and of the task these microorganisms face is underestimated.
The celF gene from the predominant cellulolytic ruminal bacterium Fibrobacter succinogenes encodes a 118.3-kDa cellulose-binding endoglucanase, endoglucanase F (EGF). This enzyme possesses an N-terminal cellulose-binding domain and a C-terminal catalytic domain. The purified catalytic domain displayed an activity profile typical of an endoglucanase, with high catalytic activity on carboxymethyl cellulose and barley beta-glucan. Immunoblotting of EGF and the formerly characterized endoglucanase 2 (EG2) from F. succinogenes with antibodies prepared against each of the enzymes demonstrated that EGF and EG2 contain cross-reactive epitopes. This data in conjunction with evidence that the proteins are the same size, share a 19-residue internal amino acid sequence, possess similar catalytic properties, and both bind to cellulose allows the conclusion that celF codes for EG2.