Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.     

Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum.

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Isolation and characterization of a lichenan-degrading hydrophobic endoglucanase of Clostridium thermocellum

Endoglucanase 7 (EG7) of Clostridium thermocellum was isolated from a recombinant strain of Escherichia coli TG1 cells harbouring recombinant plasmid pCU110 containing the cel7 gene of C. thermocellum. The enzyme was purified to electrophoretic homogeneity and was presented as two components with a molecular mass of 49 and 47 kDa and a pI of 4.35 and 4.30, respectively. The enzyme was shown to have optimum pH of 5.5 and optimum temperature of 55–60° C with carboxymethylcellulose (CMC) as a substrate. EG7 displayed hyper-lichenase, high CMCase, low cellobiosidase and negligibly small activities towards Avicel, amorphous cellulose, laminarin and xylan. The enzyme was shown to be stable at 55° C and within a broad range of pH from 4.5 to 11.0. It is insensitive towards ethanol (up to 5%) and end-product (cellobiose or glucose) inhibition. The hydrophobic nature of the protein, as revealed by retarded elution on gel filtration columns, resulted in an unprecedentedly high yield (about 80%) of purified enzyme. Due to the above-mentioned characteristics, the enzyme should to be quite suitable for use in the mashing process of beer brewing. Correspondence to: N. P. Golovchenko     

Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.     

Various kinetic properties of beta-glucosidase cloned from Clostridium thermocellum in E. coli

The kinetics of pNPG, pNPX and cellobiose hydrolysis by beta-glucosidase cloned from C. thermocellum into E. coli was studied. The V values for these substrate hydrolysis are 102, 357 and 6.7 mumols/min/mg protein, respectively; Km are 0.44 mM, 50 mM and 100 mM, respectively, sigma-Gluconolactone inhibits the hydrolysis of all substrates according to a competitive mechanism with Ki of 0.032 mM, 6.0 mM and 0.25 mM, respectively. Glucose inhibits the hydrolysis of pNPG and pNPX also via a competitive mechanism with Ki of 10 mM and 37 mM, while cellobiose--via a mixed type mechanism with Ki of 110 mM and 350 mM. The existence of separate adsorption sites for each substrate and of a common catalytic site for pNPG and pNPX hydrolysis is supposed.     

A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells

The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.     

Purification and characterization of an endoglucanase (1,4-beta-D-glucan glucanohydrolase) from Clostridium thermocellum.

An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E. coli strain]

Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E. coli K12 C600 carrying plasmid pCU 304 with a C. thermocellum chromosomal DNA insertion. The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification. According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15. The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate. Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively). The activity optimum of the enzyme is at pH 5.6, 60 degrees C. The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively. The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases.     

Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B.

The nucleotide sequence of the CelB gene, encoding the extracellular endoglucanase B of Clostridium thermocellum, is reported. The putative start of the 1689 bp coding sequence was assigned to an ATG codon which is preceded by an AGGAGG sequence typical of ribosomal binding sites in Gram-positive bacteria. The amino-terminal end of the deduced protein sequence is similar to signal peptides described for other bacterial secretory proteins. The carboxy-terminal ends of endoglucanases A and B appear to be remarkably homologous. A striking feature of the conserved region is that both proteins contain two reiterated stretches of 23 aminoacids each, separated by 9 residues.     

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.     

Purification and characterization of an endoglucanase from the cellulosomes (multicomponent cellulase complexes) of Clostridium thermocellum.

A subunit with carboxymethyl cellulase (CMCase) activity was isolated from the cellulosomes of Clostridium thermocellum after dissociation of the cellulosomes by a mild sodium dodecyl sulfate (SDS) treatment. The subunit displayed only one protein band of 51 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but after boiling with SDS it had 3 bands of 60, 56, and 48 kDa. Prolonged incubation with SDS changed the subunit to display exclusively the 48-kDa band after boiling. The 51-kDa subunit was presumably a partially denatured form, and differentiated into 3 species with apparent M(r) of 60, 56, and 48 k through deglycosylation in SDS solution. Enzymatic properties of the 51-kDa subunit resembled those of the endoglucanase A which was purified from the culture fluid and from a E. coli clone with exceptions of temperature and pH optima.     

The use of a thermostable β-glucanase gene from Clostridium thermocellum as a reporter gene in plants

In order to take advantage of the high thermostability of its product, β-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine T_R-DNA 2′ gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.     

Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum.

The nucleotide sequence of the celD gene, encoding the previously crystallized endoglucanase D of Clostridium thermocellum, is reported. The enzyme shares a conserved, reiterated domain with the COOH-terminal end of endoglucanases A and B from the same organism. The overexpression in Escherichia coli of celD subcloned in pUC8 appears to result from a translational fusion of the NH2-terminal end of the endoglucanase with the NH2-terminal end of beta-galactosidase.     

Expression and secretion of Clostridium thermocellum endoglucanase A gene (celA) in different Saccharomyces cerevisiae strains

To develop effective and powerful probiotics, Saccharomyces cerevisiae strains producing cellulolytic enzymes were genetically engineered. Two plasmids were constructed in which the carboxymethyl cellulase (CMCase) gene of Clostridium thermocellum was connected in frame with GAPDH or ADH1 promoter. The resultant plasmids were transformed into various S. cerevisiae host cells, and the expression level and secretion efficiency of the CMCase were examined. The difference in genetic background of host strains did affect significantly the cell growth and expression and secretion levels of CMCase. Irrespective of the nature of the host cells, the ADH1 promoter-employing plasmid showed a greater expression level and plasmid stability than the GAPDH promoter-based plasmid. By the optimal host-vector system, S. cerevisiae SEY2102 harboring pVT-CT1 plasmid (ADH1 promoter), the highest expression level of 277 unit CMCase/L was obtained.     

Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum

The endoglucanase CelA from Clostridium thermocellum was strongly expressed in Bacillus subtilis. The enzyme was purified by Ni²⁺-affinity chromatography. Site-directed substitution of D278 with an asparagine or an alanine residue surprisingly did not decrease the apparent k _cat value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent k _cat value for Y215P and complete loss of activity for D152N.     

Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.

Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.     

Cloning and expression of Clostridium thermocellum genes coding for thermostable exoglucanases (cellobiohydrolases) in Escherichia coli cells

By special screening approach two independent Cl. thermocellum genes directing the synthesis of thermostable glucanases with an exo-mode of action have been isolated from pUC19-based gene bank in E. coli TG1. The genes are located on 3.4 and 11.3 kb DNA fragments showing no homology. E. coli-derived exoglucanases, presumably, cellobiohydrolases, are able to cleave lichenan, carboxymethyl cellulose, xylan and p-nitrophenyl derivatives of cellobioside and lactoside. Cellobiose is the main degradation product of carboxymethyl cellulose, treated with the identified exoglucanases. With p-nitrophenil-beta-D-cellobioside as substrate the enzymes had a pH optimum around 6.5 and a temperature optimum at 65 degrees C. The identified and expressed enzymes differ from all other Cl. thermocellum proteins known to date.     

Cloning and expression of Clostridium thermocellum F7 endoglucanase gene in gram-negative bacteria

The endoglucanase gene of Clostridium thermocellum F7 was cloned in Escherichia coli cells using pKM4 vector. Both the physical mapping and analysis of the gene products in the E. coli mini-cells system suggest cloning of a new cel gene different from those described earlier. The activity of endoglucanase in E. coli cells is localized in the periplasm, which correlates with secretion of enzymes of this type in C. thermicellum. Apart from 2 major components with Mr 42.5 and 43 kDa, corresponding to mature protein forms, we observed the formation of minor products of various electrophoretic motilities. Cloning of the endoglucanase gene on bhr vector pBS954 controlled by its own regulatory signal yielded high level of the endoglucanase activity in the recombinant strains of Klebsiella pneumoniae, Serratia marcescens and Erwinia carotovora comparable with the level of the gene expression in E. coli cells.