Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum

Bagnara-Tardif, Chantal; Gaudin, Christian; Belaı̈ch, Anne; Hoest, Philippe; Citard, Thierry; Bélaı̈ch, Jean-Pierre

doi:10.1016/0378-1119(92)90062-t

Cited by 80 publications

(60 citation statements)

References 38 publications

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“…0, 1, 2, etc.). This premise is consistent with the tandem position of the cipC and cel48F genes on the C. cellulolyticum genome; the two genes form part of an operon and lack an intergenic terminator (31,32). With respect to the relatively low levels of Cel9G, the native cellulosome includes two other family-9 enzymes of identical modular architecture (Cel9H and Cel9J) (18), and these three enzymes together would presumably work in concert with Cel48F in a similar manner, thus enhancing the overall synergy displayed by the system.…”

Section: Discussionsupporting

confidence: 81%

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

Fierobe

Mingardon

Mechaly

et al. 2005

Journal of Biological Chemistry

217

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(2002) J. Biol. Chem. 277, 49621-49630), we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.

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Section: Discussionsupporting

confidence: 81%

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

Fierobe

Mingardon

Mechaly

et al. 2005

Journal of Biological Chemistry

217

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“…These reiterated domains have been found to be essential for CelD and XynZ to bind to the assembly factor CipA in C. therrnocellum [ 5 ] . The high degree of similarity existing between the reiterated zones of C. tlzerrnocellum and C. cellulolyticum [2], suggests that they may mediate interaction of the same kind in C. cellulolyticum. The catalytic effects observed with CelCCA and CelCCC when this particular domain is lacking are not incompatible with these data ; the most probable explanation is that its absence, in addition to the incapacity to bind the assembly factor, may induce small conformational changes in the catalytic cores which are responsible for the catalytic differences observed between the two forms of CelCCA or the two forms of CelCCC.…”

Section: Discussionmentioning

confidence: 99%

“…Although protease inhibitors (phenylmethylsulphonyl fluoride and EDTA) were present in the early steps of the purification, two distinct forms of the enzyme were found to exist from the first chromatography on phenyl-Sepharose, a large one called CL, the molecular mass of which (48 kDa) corresponds to the theoretical estimate from the gene sequence (47.2 kDa) [2] and a smaller one, CS (41 kDa). From this step, two purifications were carried out in parallel to isolate each form (the general pathway is summarized in Table 1).…”

Section: Purification Of Celcccmentioning

confidence: 99%

Purification and characterization of endoglucanase C from Clostridium cellulolyticum

Fierobe

Bagnara-Tardif

Gaudin

et al. 1993

European Journal of Biochemistry

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An Escherichia colz clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. Nterminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity. The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy ; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.Clostridiurn cellulolyticum is a mesophilic Clostridium which is able to completely degrade crystalline cellulose. The four known cellulase sequences of this organism [l-31 exhibit reiterated domains. These particular sequences are thought to be characteristic of enzymes integrated into the cellulosome structure [4], and yet they are observed with cellulolytic systems of Clostridium thermocellum [4, 51 and Clostridium cellulovorans [6]. These organisms secrete highmolecular-mass particles named cellulosomes which are active against cellulose, and they possess an assembly factor, CbpA in C. cellulovorans [7] and CipA in C. thermocellum [8, 91. Four genes, celCCA, celCCC, coding for enzymes of the cellulolytic system of C. cellulolyticum have been completely sequenced and the four corresponding proteins are members of three different fami-Correspondence to H

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“…The sequence of the 10-gene, 20-kb cluster is cipC-celF-celCcelG-celE-orfX p -celH-celJ-manK-celM. Results of Northern blot analysis revealed that celC and celG are expressed as a polycistronic transcriptional unit which possibly includes a third gene (12).…”

Section: Cellulase Gene Clustersmentioning

confidence: 99%

Cellulase, Clostridia, and Ethanol

Demain

Newcomb

2005

Microbiol Mol Biol Rev

799

579

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SUMMARY Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.

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Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum

Cited by 80 publications

References 38 publications

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates

Purification and characterization of endoglucanase C from Clostridium cellulolyticum

Cellulase, Clostridia, and Ethanol

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