Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans

Doi, Roy H.; Park, Jae-Seon; Liu, Chichi; Malburg, Laercio M.; Tamaru, Yutaka; Ichiishi, Akihiko; Ibrahim, Atef M.

doi:10.1007/s007920050042

Cited by 77 publications

(51 citation statements)

References 20 publications

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“…Although this discussion focuses primarily on the action of hydrolytic enzyme systems on cellulose, it should be realized that such systems are also active on hemicellulose, and enzymes active specifically on hemicellulose are commonly coproduced by cellulolytic microorganisms. Prior reviews consider the complexed cellulases of anaerobic bacteria (31,33,36,37,38,39,40,41,43,165,166,182,383,565,606,621), noncomplexed fungal and bacterial cellulases (122,349,579,653,672,673,756), cellulase structure and catalytic mechanisms (58,136,470,624,683,722,757,758), cellulase (hydrolase) families (254,255,256,258,260), and biotechnological applications (52,214,501,604,605).…”

Section: Cellulase Enzyme Systemsmentioning

confidence: 99%

“…ExgS is the major exoglucanase in the cellulosome of C. cellulovorans (166). ExgS is homologous to CelS of C. thermocellum and CelF of C. cellulolyticum (family 48 cellulases) and probably fulfills a similar function in the cellulosome.…”

Section: Cellulase Enzyme Systemsmentioning

confidence: 99%

“…However, C. cellulovorans grown on cellobiose and CMC does exhibit a high cellulase activity and transcription of cellulase genes. This suggests that cellulases may be produced on certain soluble carbohydrates, such as cellobiose, but that (poly)cellulosome assembly and detachment from the cell wall need some "triggering" from the presence of insoluble microcrystalline cellulose (60,166,665). Analysis of mRNA transcripts in the ruminal bacterium R. flavefaciens FD-1 has revealed somewhat contradictory results.…”

Section: Molecular Biology Of Cellulase Enzymesmentioning

confidence: 99%

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Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,005

2,941

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Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts

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Section: Cellulase Enzyme Systemsmentioning

confidence: 99%

Section: Cellulase Enzyme Systemsmentioning

confidence: 99%

Section: Molecular Biology Of Cellulase Enzymesmentioning

confidence: 99%

See 1 more Smart Citation

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,005

2,941

View full text Add to dashboard Cite

show abstract

“…The C. cellulovorans cellulosome consists of three major subunits-CbpA, P100, and P70-and several minor subunits (20,27,31). Recent work in our laboratory has contributed to better understanding of the molecular biology of degradation of crystalline cellulose by C. cellulovorans cellulosome (7,8,33). Furthermore, we have also found that C. cellulovorans utilizes not only cellulose but also xylan, pectin, and several other carbon sources (28, 32).…”

mentioning

confidence: 83%

Characterization of Xylanolytic Enzymes in Clostridium cellulovorans : Expression of Xylanase Activity Dependent on Growth Substrates

2001

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Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High ␣-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose-and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that C. cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate.Plant cell walls, the major reservoir of fixed carbon in nature, have three major polymers: cellulose, hemicellulose, and lignin (29). In anaerobic environments and decaying plant materials, complex communities of interacting microorganisms carry out the decomposition of lignocellulose. Among the lignocellulolytic bacteria, cellulolytic clostridia play important roles in plant biomass turnover (19). Clostridium cellulovorans (ATCC 35296) (28), an anaerobic, mesophilic, and spore-forming bacterium, produces a large extracellular polysaccharolytic multicomponent complex (with a molecular weight of about one million) called the cellulosome (7), in which several cellulases are tightly bound to a scaffolding protein called CbpA (26). The C. cellulovorans cellulosome consists of three major subunits-CbpA, P100, and P70-and several minor subunits (20,27,31). Recent work in our laboratory has contributed to better understanding of the molecular biology of degradation of crystalline cellulose by C. cellulovorans cellulosome (7,8,33). Furthermore, we have also found that C. cellulovorans utilizes not only cellulose but also xylan, pectin, and several other carbon sources (28, 32). Among these carbon sources, xylan is mainly found in secondary walls of plants, the major component of woody tissue (35) and, after cellulose, xylan is the most abundant renewable polysaccharide in plants (34).Xylan, which has a backbone of ␤-1,4-linked xylopyranosyl residues contains various substituted side-groups such as acetyl, L-arabinofuranosyl, and o-met...

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“…This sophisticated complex of cellulolytic enzymes has been characterized most extensively in the thermophilic species Clostridium thermocellum (Bayer et al, 1998;Shoham et al, 1999). A considerable amount is also known about the cellulosomes of two mesophilic species, C. cellulovorans (Doi et al, 1998) and C. cellulolyticum Gal et al, 1997;Page' s et al, 1997Page' s et al, , 1999Reverbel-Leroy et al, 1997). This last species has also been quite extensively characterized physiologically (Giallo et al, 1985;Gelhaye et al, 1993a, b;Guedon et al, 1999a, b;Payot et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Gene transfer to Clostridium cellulolyticum ATCC 35319

et al. 2000

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Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electrotransformation was employed to establish plasmids containing the replication functions of pAMβ1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAMβ1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10 2 transformants per µg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10 5 and 10 7 transformants per µg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.

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Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans

Cited by 77 publications

References 20 publications

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Characterization of Xylanolytic Enzymes in Clostridium cellulovorans : Expression of Xylanase Activity Dependent on Growth Substrates

Gene transfer to Clostridium cellulolyticum ATCC 35319

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