RESUMO
Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), ß-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.
Assuntos
Membrana Celular/metabolismo , Celulossomas/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Celulossomas/enzimologia , Celulossomas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Clostridium thermocellum/genética , Etanol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Kluyveromyces/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , CoesinasRESUMO
Cellulosomes are elaborate multienzyme complexes capable of efficiently deconstructing lignocellulosic substrates, produced by cellulolytic anaerobic microorganisms, colonizing a large variety of ecological niches. These macromolecular structures have a modular architecture and are composed of two main elements: the cohesin-bearing scaffoldins, which are non-catalytic structural proteins, and the various dockerin-bearing enzymes that tenaciously bind to the scaffoldins. Cellulosome assembly is mediated by strong and highly specific interactions between the cohesin modules, present in the scaffoldins, and the dockerin modules, present in the catalytic units. Cellulosomal architecture and composition varies between species and can even change within the same organism. These differences seem to be largely influenced by external factors, including the nature of the available carbon-source. Even though cellulosome producing organisms are relatively few, the development of new genomic and proteomic technologies has allowed the identification of cellulosomal components in many archea, bacteria and even some primitive eukaryotes. This reflects the importance of this cellulolytic strategy and suggests that cohesin-dockerin interactions could be involved in other non-cellulolytic processes. Due to their building-block nature and highly cellulolytic capabilities, cellulosomes hold many potential biotechnological applications, such as the conversion of lignocellulosic biomass in the production of biofuels or the development of affinity based technologies.
Assuntos
Celulose/metabolismo , Celulossomas/enzimologia , Celulossomas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteômica , CoesinasRESUMO
The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism's fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.
Assuntos
Celulossomas/enzimologia , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/enzimologia , Animais , Evolução Biológica , Biopolímeros/metabolismo , Celulossomas/genética , Celulossomas/metabolismo , Proteínas Fúngicas/genética , Fungos/genética , Fungos/metabolismo , Genoma Fúngico/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rúmen/microbiologiaRESUMO
OBJECTIVES: To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular ß-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights. RESULTS: Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular ß-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit. CONCLUSION: This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular ß-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.
Assuntos
Actinobacteria/enzimologia , Celulossomas/enzimologia , Complexos Multienzimáticos/metabolismo , Actinobacteria/metabolismo , Biotecnologia , Celulossomas/metabolismo , Paclitaxel/análogos & derivados , Paclitaxel/metabolismo , Xilosidases/metabolismoRESUMO
Anaerobic fungi (phylum Neocallimastigomycota), an early branching family of fungi, are commonly encountered in the digestive tract of mammalian herbivores. To date, isolates from ten described genera have been reported, and several novel taxonomic groupings are detected using culture-independent molecular methods. Anaerobic fungi are recognized as playing key roles in the decomposition of lignocellulose (up to 50% of the ingested and untreated lignocellulose), with their physical penetration and extracellular enzymatical secretion of an unbiased diverse repertoire of cell-wall-degrading enzymes. The secreted cell-wall-degrading enzymes of anaerobic fungi include both free enzymes and extracellular multi-enzyme complexes called cellulosomes, both of which have potential as fiber degraders in industries. In addition, anaerobic fungi can provide large amounts of substrates such as hydrogen, formate, and acetate for their co-cultured methanogens. Consequently, large amounts of methane can be produced. And thus, it is promising to use the co-culture of anaerobic fungi and methanogens in the biogas process to intensify the biogas yield owing to the efficient and robust degradation of recalcitrant biomass by anaerobic fungi and improved methane production from co-cultures of anaerobic fungi and methanogens.
Assuntos
Biodegradação Ambiental , Biotecnologia , Fermentação , Fungos/metabolismo , Metano/metabolismo , Neocallimastigomycota/metabolismo , Ácido Acético/metabolismo , Anaerobiose/fisiologia , Biocombustíveis , Biomassa , Celulase/genética , Celulase/metabolismo , Celulossomas/enzimologia , Técnicas de Cocultura , Fibra de Algodão , Euryarchaeota/metabolismo , Formiatos/metabolismo , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Hidrogênio/metabolismo , Lignina/metabolismo , Neocallimastigomycota/classificação , Neocallimastigomycota/enzimologia , Neocallimastigomycota/genética , Polissacarídeos/metabolismo , Especificidade por SubstratoRESUMO
The cellulosome is an extracellular multi-enzyme complex that is considered one of the most efficient plant cell wall-degrading strategies devised by nature. Its unique modular architecture, achieved by high affinity and specific interaction between protein modules (cohesins and dockerins) enables formation of various enzyme combinations. Extensive research has been dedicated to the mechanistic nature of the cellulosome complex. Nevertheless, little is known regarding its distribution and abundance among microbes in natural plant fibre-rich environments. Here, we explored these questions in bovine rumen microbial communities, specialized in efficient degradation of lignocellulosic plant material. We bioinformatically screened for cellulosomal modules in this complex environment using a previously published ultra-deep fibre-adherent rumen metagenome. Intriguingly, a large portion of the functions of the dockerin-containing proteins were related to alternative biological processes, and not necessarily to the classic fibre degradation function. Our analysis was experimentally validated by characterizing specific interactions between selected cohesins and dockerins and revealed that cellulosome is a more generalized strategy used by diverse bacteria, some of which were not previously associated with cellulosome production. Remarkably, our results provide additional proof of similarity among rumen microbial communities worldwide. This study suggests a broader and widespread role for the cellulosomal machinery in nature.
Assuntos
Bactérias/isolamento & purificação , Celulossomas/enzimologia , Microbioma Gastrointestinal , Filogenia , Rúmen/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Celulossomas/genética , Metagenoma , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismoRESUMO
Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/química , Celulase/química , Celulose/química , Clostridiales/química , Thermotoga maritima/química , Sequência de Aminoácidos , Repetição de Anquirina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocombustíveis , Biomassa , Celulase/genética , Celulase/metabolismo , Celulose/metabolismo , Celulossomas/química , Celulossomas/enzimologia , Clonagem Molecular , Clostridiales/enzimologia , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Thermotoga maritima/enzimologiaRESUMO
Ruminococcus champanellensis is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production. The basic elements of its cellulosome architecture, mainly cohesin and dockerin modules from scaffoldins and enzyme-borne dockerins, have been characterized recently. In this study, we cloned, expressed and characterized all of the glycoside hydrolases that contain a dockerin module. Among the 25 enzymes, 10 cellulases, 4 xylanases, 3 mannanases, 2 xyloglucanases, 2 arabinofuranosidases, 2 arabinanases and one ß-glucanase were assessed for their comparative enzymatic activity on their respective substrates. The dockerin specificities of the enzymes were examined by ELISA, and 80 positives out of 525 possible interactions were detected. Our analysis reveals a fine-tuned system for cohesin-dockerin specificity and the importance of diversity among the cohesin-dockerin sequences. Our results imply that cohesin-dockerin pairs are not necessarily assembled at random among the same specificity types, as generally believed for other cellulosome-producing bacteria, but reveal a more organized cellulosome architecture. Moreover, our results highlight the importance of the cellulosome paradigm for cellulose and hemicellulose degradation by R. champanellensis in the human gut.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Celulose/metabolismo , Celulossomas/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Microbioma Gastrointestinal/fisiologia , Ruminococcus/enzimologia , Glicosídeo Hidrolases/metabolismo , Humanos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Ruminococcus/genética , CoesinasRESUMO
Ruminococcus champanellensis is a keystone species in the human gut that produces an intricate cellulosome system of various architectures. A variety of cellulosomal enzymes have been identified, which exhibit a range of hydrolytic activities on lignocellulosic substrates. We describe herein a unique R. champanellensis scaffoldin, ScaK, which is expressed during growth on cellobiose and comprises a cohesin module and a family 25 glycoside hydrolase (GH25). The GH25 is non-autolytic and exhibits lysozyme-mediated lytic activity against several bacterial species. Despite the narrow acidic pH curve, the enzyme is active along a temperature range from 2 to 85°C and is stable at very high temperatures for extended incubation periods. The ScaK cohesin was shown to bind selectively to the dockerin of a monovalent scaffoldin (ScaG), thus enabling formation of a cell-free cellulosome, whereby ScaG interacts with a divalent scaffodin (ScaA) that bears the enzymes either directly or through additional monovalent scaffoldins (ScaC and ScaD). The ScaK cohesin also interacts with the dockerin of a protein comprising multiple Fn3 domains that can potentially promote adhesion to carbohydrates and the bacterial cell surface. A cell-free cellulosomal GH25 lysozyme may provide a bacterial strategy to both hydrolyze lignocellulose and repel eventual food competitors and/or cheaters.
Assuntos
Proteínas de Bactérias/metabolismo , Celulossomas/enzimologia , Muramidase/metabolismo , Ruminococcus/enzimologia , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Celulose/metabolismo , Celulossomas/genética , Celulossomas/metabolismo , Humanos , Muramidase/genética , Ruminococcus/genética , Ruminococcus/metabolismoRESUMO
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 â¼2.4-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.
Assuntos
Celulose/metabolismo , Celulossomas/metabolismo , Clostridium thermocellum/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulases/genética , Celulases/metabolismo , Celulose/química , Celulossomas/enzimologia , Celulossomas/genética , Clostridium thermocellum/enzimologia , Clostridium thermocellum/genética , CristalizaçãoRESUMO
The process of bioethanol production from biomass comprises pretreatments and enzyme-mediated hydrolysis to convert lignocellulose into fermentable sugars. Because of the recalcitrant character of cellulose, the enzymatic hydrolysis is considered the major challenge in this process to be economically competitive. These technical difficulties highlight the need for the discovery of new enzymes to optimize and lower the cost of current technologies. Microorganisms have developed efficient systems for cellulose degradation. Among cellulolytic microbes, Thermobifida fusca possesses great physiological and cellulolytic characteristics (thermostability, high activity and tolerance to a broad pH range) making it an interesting organism to be studied from an applied perspective. In this review we describe the main enzymes/proteins produced by T.fusca (cellulases, xylanases, mannanase, manosidase, CBM33 and CelR), the effect of substrate on T. fusca proteome, enzyme improvement approaches, synergism between enzymes/proteins and artificial cellulosomes.
Assuntos
Actinomycetales/metabolismo , Celulossomas/metabolismo , Enzimas/metabolismo , Etanol/metabolismo , Lignina/metabolismo , Actinomycetales/efeitos dos fármacos , Actinomycetales/enzimologia , Actinomycetales/efeitos da radiação , Celulossomas/efeitos dos fármacos , Celulossomas/enzimologia , Celulossomas/efeitos da radiação , Estabilidade Enzimática , Enzimas/química , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design.
Assuntos
Redes e Vias Metabólicas , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Animais , Celulossomas/enzimologia , Celulossomas/metabolismo , Cilióforos/enzimologia , Cilióforos/metabolismo , Evolução Molecular Direcionada , Transporte de Elétrons , Euryarchaeota/enzimologia , Euryarchaeota/metabolismo , Humanos , Modelos Biológicos , Complexos Multienzimáticos/metabolismo , Periplaneta/metabolismo , Esgotos/microbiologia , Simbiose , Leveduras/enzimologia , Leveduras/metabolismoRESUMO
The efficient deconstruction of lignocellulosic biomass remains a significant barrier to the commercialization of biofuels. Whereas most commercial plant cell-wall-degrading enzyme preparations used today are derived from fungi, the cellulosomal enzyme system from Clostridium thermocellum is an equally effective catalyst, yet of considerably different structure. A key difference between fungal enzyme systems and cellulosomal enzyme systems is that cellulosomal enzyme systems utilize self-assembled scaffolded multimodule enzymes to deconstruct biomass. Here, the possible function of the X1 modules in the complex multimodular enzyme system cellobiohydrolase A (CbhA) from C. thermocellum is explored. The crystal structures of the two X1 modules from C. thermocellum CbhA have been solved individually and together as one construct. The role that calcium may play in the stability of the X1 modules has also been investigated, as well as the possibility that they interact with each other. Furthermore, the results show that whereas the X1 modules do not seem to act as cellulose disruptors, they do aid in the thermostability of the CbhA complex, effectively allowing it to deconstruct cellulose at a higher temperature.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose/química , Celulossomas/enzimologia , Clostridium thermocellum/enzimologia , Complexos Multienzimáticos/química , Sítios de Ligação , Biomassa , Celulose 1,4-beta-Celobiosidase/metabolismo , Cristalografia por Raios X , Estrutura Terciária de ProteínaRESUMO
Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1â Å resolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed ß-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.
Assuntos
Celulase/química , Celulossomas/enzimologia , Clostridium thermocellum/enzimologia , Glicosídeo Hidrolases/química , Sítios de Ligação , Domínio Catalítico , Celulase/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Glicosídeo Hidrolases/metabolismo , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.
Assuntos
Celulossomas/enzimologia , Clostridium thermocellum/enzimologia , Glicosídeo Hidrolases/metabolismo , Lignina/metabolismo , Bactérias Anaeróbias/enzimologia , Bactérias Anaeróbias/genética , Celulose/metabolismo , Celulossomas/ultraestrutura , Cromatografia em Gel , Clonagem Molecular , Clostridium thermocellum/genética , Escherichia coli , Glicosídeo Hidrolases/genética , Hidrólise , Microscopia Eletrônica de Varredura , Oryza/metabolismo , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Ribossômico 16S/biossíntese , RNA Ribossômico 16S/genética , Esgotos/microbiologia , Tailândia , ResíduosRESUMO
Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.
Assuntos
Proteínas de Bactérias/metabolismo , Celulossomas/metabolismo , Clostridium cellulolyticum/metabolismo , Polissacarídeos/metabolismo , Proteômica/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Metabolismo dos Carboidratos/genética , Celulossomas/enzimologia , Celulossomas/genética , Clostridium cellulolyticum/enzimologia , Clostridium cellulolyticum/genética , Hidrólise , Polissacarídeos/genética , Especificidade por SubstratoRESUMO
The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.
Assuntos
Celulases/genética , Celulases/metabolismo , Celulossomas/enzimologia , Clostridium thermocellum/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Regulação Bacteriana da Expressão Gênica , Especificidade por Substrato , CoesinasRESUMO
Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to colocalize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. In this chapter, we describe several DNA- and protein-based scaffold approaches to assemble artificial enzyme cascades for a wide range of applications. We highlight the key benefits and drawbacks of these approaches to provide insights on how to choose the appropriate scaffold for different cascade systems.
Assuntos
Bactérias/enzimologia , DNA/química , Enzimas Imobilizadas/química , Bactérias/química , Biocatálise , Materiais Biocompatíveis/química , Celulossomas/química , Celulossomas/enzimologia , Modelos MolecularesRESUMO
The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.
Assuntos
Celulase/metabolismo , Ruminococcus/enzimologia , beta-Manosidase/metabolismo , Sequência de Aminoácidos/genética , Celulase/biossíntese , Celulase/química , Celulase/genética , Celulossomas/enzimologia , Quelantes , Cromatografia em Camada Fina , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Galactanos/química , Galactanos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Mananas/química , Mananas/metabolismo , Oligossacarídeos/química , Gomas Vegetais/química , Gomas Vegetais/metabolismo , Ruminococcus/genética , Especificidade por Substrato , Temperatura , beta-Manosidase/química , beta-Manosidase/genéticaRESUMO
Cellulosomes are intricate multienzyme systems produced by several cellulolytic bacteria, the first example of which was discovered in the anaerobic thermophilic bacterium, Clostridium thermocellum. Cellulosomes are designed for efficient degradation of plant cell wall polysaccharides, notably cellulose--the most abundant renewable polymer on earth. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture. A full generation of research has elapsed since the original publications that documented the cellulosome concept. In this review, we provide a personal account on the discovery process, while describing how divergent cellulosome systems were identified and investigated, culminating in the collaboration of several labs worldwide to tackle together the challenging field of cellulosome genomics and metagenomics.