Cellulomonas Fimi Research Articles

Streptomyces lividans glycosylates an exoglucanase (Cex) from Cellulomonas fimi

Exoglucanase Cex from Cellulomonas fimi is a glycoprotein [Langsford et al., J. Gen. Microbiol. 130 (1984) 1367–1376]. Cex produced by Streptomyces lividans from the cloned cex gene is also glycosylated. The extent and nature of glycosylation are similar for Cex from both organisms. The glycosylation affords protection against proteolysis for the enzymes from both organisms when they are bound to cellulose, but not in solution. The ability to glycosylate cloned gene products enhances the utility of Streptomyces as a host for the production of heterologous polypeptides.

Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of Cex, an exo-β-1,4-glucanase and β-1,4-xylanase from the bacterium Cellulomonas fimi

Single crystals of the catalytic domain of Cex, an exo-β-1,4-glucanase and β-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to better than 2.0 Å resolution, belong to space group P4 12 12 or P4 32 12 and have cell constants: a = b = 88.21 A ̊ , c = 81.10 A ̊ ; α = β = γ = 90 °.

Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum

The sequence of a 5633-bp EcoRI- PvuII DNA fragment from Clostridium cellulolyticum was determined. This fragment contains two complete endo-β-1,4-glucanase-encoding genes, designated celCCC and celCCG. These two genes are flanked by two other partial open reading frames (ORF1 and celCCE) that probably encode two cellulases or related enzymes. The celCCC and celCCG genes appear to be present in a polycistronic transcriptional unit. Northern blot hybridisations with intragenic probes derived from celCCC and celCCG gave similar patterns. Two transcripts of about 5 and 6 kb were identified. The celCCC and celCCG ORFs extend over 1380 bp and 2175 bp, respectively. They are separated by only 87 nt. A typical signal sequence is present at the N terminus of the deduced polypeptides. The mature CelCCC and CelCCG proteins have M r s 47 201 and 76 101, respectively. Comparisons between their amino acid (aa) sequences and other known cellulase sequences revealed that: first, they both contain the repeated 24-aa sequence characteristic of clostridial β-glycanases, secondly, the N-terminal catalytic domains of CelCCC and CelCCG can be classified into the D and E2 families, respectively, and thirdly, the largest CelCCG contains an additional internal domain which is very similar to that of the Bacillus-type cellulose-binding domain (CBD). The ORF1-C-terminal-encoded sequence also contains the clostridial 24-aa repeat. The CelCCE N-terminus consists of a typical signal sequence followed by a 168-aa domain homologous to the N-terminal repeated domain of Cellulomonas fimi CenC. This domain is connected to an incomplete catalytic domain of family E1 by a Pro-rich junction linker.

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant.

In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex). The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2). The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae. After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates. This step efficiently removed the majority of contaminating host proteins. While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments. Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e. the cellulose binding domain, stayed bound to the cellulose matrix.

The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats.

Endoglucanase C (CenC) from Cellulomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150-amino-acid repeats (N1 and N2) at its N-terminus and two unrelated contiguous 100-amino-acid repeats (C1 and C2) at its C-terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non-crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated ('amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose-binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose-binding domains of Cex and C. fimi endoglucanases A and B.

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.

The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose.

CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward). The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model. A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.

Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli

By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium cellulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl beta-1,4-cellobiosidase activity. The two proteins were very homologous (80%) up to the Pro-Thr-Thr region which divided the protein into -NH2- and -COOH-terminals. The -COOH- region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocellum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH- region of EngD, which had homology to the cellulose-binding domains of Cellulomonas fimi exo- and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.

Cellulose-binding domains: potential for purification of complex proteins.

The endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi each contain a discrete cellulose-binding domain (CBD), at the amino-terminus or carboxyl-terminus respectively. The gene fragment encoding the CBD can be fused to the gene of a protein of interest. Using this approach hybrid proteins can be engineered which bind reversibly to cellulose and exhibit the biological activity of the protein partner. Alkaline phosphatase (PhoA) from Escherichia coli, and a beta-glucosidase (Abg) from an Agrobacterium sp. are dimeric proteins. The fusion polypeptides CenA-PhoA and Abg-CBC(Cex) are sensitive to proteolysis at the junctions between the fusion partners. Proteolysis results in a mixture of homo- and heterodimers; these bind to cellulose if one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoA and CenA-PhoA/PhoA. CBD fusion polypeptides could be used in this way to purify polypeptides which associate with the fusion partner.

Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.

Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).

Immunological versatility and carbon regulation of Cellulomonas fimi endo-1,4-beta-glucanases.

More than 10 protein molecules with endo-1,4-beta-glucanase activity were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source. These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised. The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose. Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively. In the extracellular environment this group showed the largest versatility in protein molecules. The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group. The main extracellular protein of this group showed high affinity toward crystalline cellulose. One intracellular precursor was identified for the third group, which was subject to carbon catabolite control. Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion. It appears that the C. fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult.

Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.

Selection of surrogates for a genetically engineered microorganism with cellulolytic capability for ecological studies in streams

Aerobic cellulolytic bacteria were ranked according to ability to degrade cellulose azure and to clear cellulose agar. Cellulomonas uda NRRL B404 and Cellulomonas sp. NRC 2406 showed greater clearing of cellulose agar than other isolates, but differences in cellulose azure decomposition were not statistically significant. Isolates were tested for ability to accelerate decomposition of tulip poplar (Liriodendron tulipifera) leaves and Cladophora glomerata (Chlorophyta) detritus in stream water. There was significantly more cellulose lost from leaves exposed to Cellulomonas flavigena NRC 2403, Cellulomonas fimi NRRL B402, Cellulomonas sp. NRC 2406, and Cellvibrio gilvus ATCC 13127 and NRC 2407 than in the stream-water control, and the weight losses of leaves exposed to some isolates were significantly greater than in the control. There was significantly more cellulose lost from Cladophora glomerata detritus exposed to these and five other isolates, and there were greater weight losses than in the stream-water control. Cellulomonas uda NRRL B404 was the slowest growing isolate, although growth rates of isolates did not differ statistically. Cellulomonas uda NRRL B404, Cellulomonas sp. NRC 2406, Cellulomonas fimi NRRL B402, Cellulomonas flavigena NRC 2403, and Cellvibrio gilvus ATCC 13127 were selected as the best candidates for larger scale experiments. Persistence of Cellulomonas uda, Cellulomonas sp. NRC 2406, and Cellulomonas sp. CS1-1 in stream-bed sediments was studied in flowing-water microcosms, using fluorescent antibodies and epifluorescence microscopic counts to asses densities of target cells. Isolate densities declined from postinoculation maxima, but organisms were detected 2–4 weeks later in three different experiments. Key words: cellulolytic bacteria, detritus, decomposition, population dynamics, microcosms.

Multiple xylanases of Cellulomona fimi are encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.

Multiple xylanases ofCellulomona fimiare encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.

Glutamic acid 274 is the nucleophile in the active site of a “retaining” exoglucanase from Cellulomonas fimi

In addition to its known substrate activity with p-nitrophenyl beta-cellobioside, the exoglucanase from Cellulomonas fimi has been shown to utilize substituted phenyl beta-glucosides as substrates, of which the best is 2',4'-dinitrophenyl beta-D-glucopyranoside. The enzyme can be inactivated by treatment with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside, by trapping of the covalent intermediate in catalysis, as has been shown for a beta-glucosidase (Withers, S.G., and Street, I.P. (1988) J. Am. Chem. Soc. 110, 8551-8553). The intermediate formed is stable but can undergo turnover in the presence of cellobiose, reactivating the enzyme by transglycosylation. Using a tritium-labeled inactivator it has been possible to isolate and sequence a radiolabeled peptide from this enzyme, and the active site nucleophile has been identified as glutamic acid residue 274. This glutamic acid residue and its sequentially proximal amino acids are absolutely conserved in the homologous family F of cellulases.

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.

Deletion of the linker connecting the catalytic and cellulose-binding domains of endoglucanase A (CenA) of Cellulomonas fimi alters its conformation and catalytic activity

The Pro-Thr box is a linker of 23 amino acids ((PT)4T(PT)7) connecting the catalytic domain and the cellulose-binding domain (CBD) of endoglucanase A (CenA) from the bacterium Cellulomonas fimi. Deletion of the Pro-Thr box alters the conformation of CenA by changing the relative orientation of the catalytic domain and the CBD. The tertiary structures of the catalytic domain and the CBD appear to be unchanged. The change in conformation reduces the catalytic efficiency of the enzyme and masks one of two protease-sensitive sites between the domains. The deletion does not affect the adsorption of the enzyme to microcrystalline cellulose, but it does affect its desorption from cellulose. The results suggest that the Pro-Thr box in CenA has an extended, kinked, and rigid conformation.

Nucleotide sequence of the endoglucanase C gene (cenC) of Cellulomonas fimi, its high‐level expression in Escherichia coli, and characterization of its products

The cenC gene of Cellulomonas fimi, encoding endoglucanase CenC, has an open reading frame of 1101 codons closely followed by a 9 bp inverted repeat. The predicted amino acid sequence of mature CenC, which is 1069 amino acids long, is very unusual in that it has a 150-amino-acid tandem repeat at the N-terminus and an unrelated 100-amino-acid tandem repeat at the C-terminus. CenC belongs to subfamily E1 of the beta-1,4-glycanases. High-level expression in Escherichia coli of cenC from a 3.6 kbp fragment of C. fimi DNA leads to levels of CenC which exceed 10% of total cell protein. Most of the CenC is in the cytoplasm in an inactive form. About 60% of the active fraction of CenC is in the periplasm. The catalytic properties of the active CenC are indistinguishable from those of native CenC from C. fimi. The Mr of CenC from E. coli and C. fimi is approximately 130 kDa. E. coli and C. fimi also produce an endoglucanase, CenC', of approximate Mr 120kDa and with the same N-terminal amino acid sequence and catalytic properties as CenC. CenC' appears to be a proteolytic product of CenC. CenC and CenC' can bind to cellulose and to Sephadex. CenC is the most active component of the C. fimi cellulase system isolated to date.

Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46

Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.