Cellulosomal Cellulases Research Articles

In vitro assembly and cellulolytic activity of a β-glucosidase-integrated cellulosome complex.

The cellulosome is a supramolecular multi-enzyme complex formed by protein interactions between the cohesin modules of scaffoldin proteins and the dockerin module of various polysaccharide-degrading enzymes. In general, the cellulosome exhibits no detectable β-glucosidase activity to catalyze the conversion of cellobiose to glucose. Because β-glucosidase prevents product inhibition of cellobiohydrolase by cellobiose, addition of β-glucosidase to the cellulosome greatly enhances the saccharification of crystalline cellulose and plant biomass. Here, we report the in vitro assembly and cellulolytic activity of a β-glucosidase-coupled cellulosome complex comprising the three major cellulosomal cellulases and full-length scaffoldin protein of Clostridium (Ruminiclostridium) thermocellum, and Thermoanaerobacter brockii β-glucosidase fused to the type-I dockerin module of C. thermocellum. We show that the cellulosome complex composed of nearly equal numbers of cellulase and β-glucosidase molecules exhibits maximum activity toward crystalline cellulose, and saccharification activity decreases as the enzymatic ratio of β-glucosidase increases. Moreover, β-glucosidase-coupled and β-glucosidase-supplemented cellulosome complexes similarly exhibit maximum activity toward crystalline cellulose (i.e. 1.7-fold higher than that of the β-glucosidase-free cellulosome complex). These results suggest that the enzymatic ratio of cellulase and β-glucosidase in the assembled complex is crucial for the efficient saccharification of crystalline cellulose by the β-glucosidase-integrated cellulosome complex.

Synergy between recombinant endoglucanase a and exoglucanase s secreted by bacillus subtilis WB800N

An approach for dertemining the synergistic activity of cellulosomal catalytic units in culture media is among essential steps in the research of artificial cellulosomes. Endoglucanase A (CelA) and exoglucanase S (CelS) are two abundant cellulosomal cellulases secreted by anaerobic thermophilic bacterium Clostridium thermocellum and mostly responsible for the cellulolytic activities. They are the well-known candidates of catalytic units for artificial mini-cellulosome. Their synergistic activities play important roles in cellulolytic degradation. CelA cleaves inside the cellulose chains and produces more chain ends for the next step controlling by CelS. While endoglucanase demonstrates distinct activity on carboxymethyl cellulose, it still lacks the specific substrate for quantifying exoglucanase activity. In this research, we introduced an approach for measuring the synergistic activity for these endo and exoglucanase. We designed two recombinant proteins CelA and CelS secreted by the host-cell Bacillus subtilis WB800N in order to investigate their synergistic activity in culture media. The secretory expression was confirmed by tandem mass spectrometry. A modified dinitrosalicylic acid assay was performed on 96 well-plate for quantifying cellulolytic activities of the secreted cellulases in culture media. When adding CelA into CelS, CMCase activities were enhanced and higher than the total of their individual CMCase activities at some cases. When mixing 3 of CelA with 5 of CelS, the CMCase activity was enhanced about 35.5% of the total activities from individual ones. This indicated the synergistic activity of the endo and exoglucanase could degrade cellulose more efficiently than their individual activities. The research also provides the essential materials and methods for further research on designing mini-cellulosome secreted by B. subtilis.

Methods for Discovery of Novel Cellulosomal Cellulases Using Genomics and Biochemical Tools.

Cell wall degradation by cellulases is extensively explored owing to its potential contribution to biofuel production. The cellulosome is an extracellular multienzyme complex that can degrade the plant cell wall very efficiently, and cellulosomal enzymes are therefore of great interest. The cellulosomal cellulases are defined as enzymes that contain a dockerin module, which can interact with a cohesin module contained in multiple copies in a noncatalytic protein, termed scaffoldin. The assembly of the cellulosomal cellulases into the cellulosomal complex occurs via specific protein-protein interactions. Cellulosome systems have been described initially only in several anaerobic cellulolytic bacteria. However, owing to ongoing genome sequencing and metagenomic projects, the discovery of novel cellulosome-producing bacteria and the description of their cellulosomal genes have dramatically increased in the recent years. In this chapter, methods for discovery of novel cellulosomal cellulases from a DNA sequence by bioinformatics and biochemical tools are described. Their biochemical characterization is also described, including both the enzymatic activity of the putative cellulases and their assembly into mature designer cellulosomes.

Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity

BackgroundClostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA.ResultsAlthough all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity.ConclusionsThe identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum

BackgroundClostridium thermocellum utilizes a wide variety of free and cellulosomal cellulases and accessory enzymes to hydrolyze polysaccharides present in complex substrates. To date only a few studies have unveiled the details by which the expression of these cellulases are regulated. Recent studies have described the auto regulation of the celC operon and determined that the celC–glyR3–licA gene cluster and nearby manB–celT gene cluster are co-transcribed as polycistronic mRNA.ResultsIn this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivo transcription of the celC–glyR3–licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription.ConclusionsTogether these results reveal a mechanism by which manB is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, C. thermocellum is able to co-regulate both the celC and manB gene clusters in response to the availability of β-1,3-polysaccharides in its environment.

Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells

Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes.

Cellulosomal expansin: functionality and incorporation into the complex.

BackgroundExpansins are relatively small proteins that lack enzymatic activity and are found in plants and microorganisms. The function of these proteins is to disrupt the plant cell walls by interfering with the non-covalent interchain bonding of the polysaccharides. Expansins were found to be important for plant growth, but they are also expressed by various bacteria known to have interactions with plants. Clostridium clariflavum is a plant cell wall-degrading bacterium with a highly elaborate cellulosomal system. Among its numerous dockerin-containing genes, two expansin-like proteins, Clocl_1862 and Clocl_1298 (termed herein CclEXL1 and CclEXL2) were identified, and CclEXL1 was found to be expressed as part of the cellulosome system. This is the first time that an expansin-like protein is identified in a cellulosome complex, which implicates its possible role in biomass deconstruction.ResultsIn the present article, we analyzed the functionality of CclEXL1. Its dockerin was characterized and shown to bind selectively to type-I cohesins of C. clariflavum, with preferential binding to the cohesin of ScaG, and additionally to a type-I cohesin of C. cellulolyticum. We demonstrated experimentally that the expansin-like protein binds preferentially to microcrystalline cellulose, but it also binds to acid-swollen cellulose, xylan, and wheat straw. CclEXL1 exhibited a pronounced loosening effect on filter paper, which resulted in substantial decrease in tensile stress. The C. clariflavum expansin-like protein thus enhances significantly enzymatic hydrolysis of cellulose, both by C. clariflavum cellulosomes and two major cellulosomal cellulases from this bacterium: GH48 (exoglucanase) and GH9 (endoglucanase). Finally, we demonstrated CclEXL1-mediated enhancement of microcrystalline cellulose degradation by different cellulosome fractions and the two enzymes.ConclusionsThe results of this study confirm that the C. clariflavum expansin-like protein is part of the elaborate cellulosome system of this bacterium with capabilities of cellulose creeping. The data suggest that pretreatment of cellulosic materials with CclEXL1 can bring about substantial improvement of hydrolysis by cellulases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0474-5) contains supplementary material, which is available to authorized users.

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome.

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display seven distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G, and Cel9M). In this study, the 10 uncharacterized GH9 enzymes were overproduced in Escherichia coli and purified, and their activity pattern was investigated in the free state or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in mini-cellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole noncellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous mini-cellulosomes that exhibit more activity on crystalline cellulose than the best homogeneous tri-functional complex. Altogether, our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerization.

Dockerin-containing protease inhibitor protects key cellulosomal cellulases from proteolysis inClostridium cellulolyticum

Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin-containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome-localized protease inhibitors in Clostridia.

Scaffoldin Modules Serving as “Cargo” Domains To Promote the Secretion of Heterologous Cellulosomal Cellulases by Clostridium acetobutylicum

The secretion of large heterologous cellulases by Clostridium acetobutylicum was formerly shown to be deleterious. To circumvent this issue, various scaffoldins' modules were grafted at their N termini. Family 3a cellulose binding module combined with an X2 module(s) was found to trigger the secretion of Clostridium cellulolyticum cellulases by the solventogenic bacterium.

The processive endoglucanase EngZ is active in crystalline cellulose degradation as a cellulosomal subunit of Clostridium cellulovorans

Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZ's specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans.

Demonstration of the importance for cellulose hydrolysis of CelS, the most abundant cellulosomal cellulase in Clostridium thermocellum

Anaerobic and aerobic bacteria use different mechanisms to degrade cellulose, with many aerobic microorganisms secreting a synergistic set of individual enzymes that includes multiple cellulases, most of which contain a carbohydrate-binding module (CBM) in addition to their catalytic domain (free enzyme mechanism). Anaerobic microorganisms usually produce a similar set of enzymes, but they are present in large multienzyme complexes (>1 million molecular weight) called cellulosomes (cellulosomal mechanism) (1). Most of the cellulosomal enzymes do not contain a CBM, but the scaffoldin protein in the cellulosome does contain a CBM. At this time, there are still a number of unanswered questions about how cellulosomes are able to degrade cellulose so well, despite their large size, which should limit their access to the cellulose surface, much of which is present in narrow pores (2). One suggestion is that enzyme proximity on the cellulosomes increases the rate of cellulose degradation, but it would seem that enzyme proximity would be most effective if enzymes were incorporated in a specific order in cellulosomes. All of the different cohesin domains in the scaffoldin protein have equal affinity for all of the docerin domains present on the cellulosomal enzymes so that it is thought that the enzymes are randomly incorporated into cellulosomes, but this has not been proven. Synergism between cellulases that have different sites of attack (i.e., endocellulases and exocellulases) is important for crystalline cellulose degradation by both mechanisms, because the specific activity of a synergistic mixture can be up to 15 times that of the most active individual cellulase (3).

The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 Å resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature—a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose “whiskers” of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.

Three Microbial Strategies for Plant Cell Wall Degradation

Cellulolytic bacteria and fungi have been shown to use two different approaches to degrade cellulose. Most aerobic microbes secrete sets of individual cellulases, many of which contain a carbohydrate binding molecule (CBM), which act synergistically on native cellulose. Most anaerobic microorganisms produce large multienzyme complexes called cellulosomes, which are usually attached to the outer surface of the microorganism. Most of the cellulosomal enzymes lack a CBM, but the cohesin subunit, to which they are bound, does contain a CBM. The cellulases present in each class show considerable overlap in their catalytic domains, and processive cellulases (exocellulases and processive endocellulases) are the most abundant components of both the sets of free enzymes and of the cellulosomal cellulases. Analysis of the genomic sequences of two cellulolytic bacteria, Cytophaga hutchinsonii, an aerobe, and Fibrobacter succinogenes, an anaerobe, suggest that these organisms must use a third mechanism. This is because neither of these organisms, encodes processive cellulases and most of their many endocellulase genes do not encode CBMs. Furthermore, neither organism appears to encode the dockerin and cohesin domains that are key components of cellulosomes.

Synergistic Interaction of Clostridium cellulovorans Cellulosomal Cellulases and HbpA

Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum.

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.

Effect of sulfur-containing compounds onBacilluscellulosome-associated ‘CMCase’ and ‘Avicelase’ activities

The isolation of cellulosomes from clostridial sources has been extensively studied; however, the isolation of cellulosomes from facultative soil anaerobes of the family Bacillaceae is not as well characterized. The Bacillus cellulosome (celluloxylanosome) essentially consists of two complex components: C-I and C-II. This multi-component complex enables Bacillus to degrade a variety of carbonaceous compounds as it is composed of several enzymes, such as cellulases, xylanases and other degradative enzymes. The cellulosomal cellulases from Bacillus megaterium were purified using cellulose affinity chromatography, followed by Sepharose 4B gel filtration chromatography. The objective of this investigation was to establish the effect of sulfate and sulfide on cellulosomal 'cellulase' activity. An increase in sulfide concentration led to a general enhancement of cellulosomal-associated cellulolytic activity, whereas an increase in sulfate concentration resulted in an inhibition of the cellulosome-associated cellulolytic activity.

Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHDeltaCBM and EngYDeltaCBM devoid of the CBM lost activity toward all substrates including CMC. EngKDeltaCBM and EngMDeltaCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.