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.     

Expression of a celE gene from Clostridium thermocellum in Bacillus

The level of expression of the Clostridium thermocellum celE gene in the asporogenous Bacillus subtilis strain 1A718 did not exceed the endogenous background level. However, when transformed into sporogenous strains, celE-containing constructs allowed the cells to express a high level of thermostable carboxymethylcellulase (CMCase) activity which was detected exclusively in the culture supernatant. The sporulation efficiency was impaired in the celE-carrying strains. Most of the thermostable CMCase activity in the recombinant strains was attributed to the stationary phase of growth, and production of the enzyme could be further enhanced by increasing the cultivation temperature from 37°C to 42°C. Even when expressed in an extracellular proteases deficient mutant, the protein product was cleaved in the P-T-rich linker sequence (2 sites) and at a site downstream of the putative signal peptidase recognition site. As a consequence, the enzymatically active protein could be isolated only in a truncated form. Plasmid pHE9102, the celE-containing construct, undergoes significant structural rearrangements in Bacillus stearothermophilus strains, preventing any detectable expression.     

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.     

Expression of a Clostridium thermocellum xylanase gene in Bacillus subtilis

Summary The native promoter of a xylanase gene isolated from Clostridium thermocellum was replaced with a strong promoter screened from Bacillus subtilis chromosomes. A part of the C-terminal region of the gene which is not related to the xylanase activity was removed. With the modified xylanase gene, B. subtilis was transformed and grown in LB medium. The xylanase gene was expressed well in B. subtilis and extracellular xylanase was produced up to 30 units per ml when the growth reached OD₆₀₀ of 4.8.     

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.     

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.     

Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.     

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 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     

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.     

Characterization of two crystal forms of Clostridium thermocellum endoglucanase CelC

Endoglucanase CelC from Clostridium thermocellum expressed in Escherichia coli has been crystallized in two different crystal forms by the hanging drop method. Crystals of form I were grown with polyethylene glycol as a precipitant. They are orthorhombic, space group P2₁2₁2₁, with cell dimensions a =51.4 Å, b =84.3 Å, and c =87.5 Å. Crystals of form II, obtained in ammonium sulfate solutions, belong to the tetragonal space group P4₁2₁2 (or P4₃2₁2) with cell dimensions of a = b = 130.7 Å and c = 69.6 Å. Diffraction data to 2.8 Å resolution were observed for both crystal forms with a rotating anode generator. Preliminary oscillation images of the orthorhombic form I crystals using a synchrotron radiation source show diffraction to 2.2 Å resolution, indicating that these crystals are suitable for high resolution crystallographic analysis. © 1994 Wiley-Liss, Inc.     

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.     

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to β-1,4- and β-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.     

Site-directed mutagenesis of conserved residues of Clostridium thermocellum endoglucanase CelC.

Four conserved residues of Clostridium thermocellum endoglucanase CelC were replaced by site-directed mutagenesis. Proteins mutated in His-90, Asn-139 and Glu-140 showed strongly reduced activity, in agreement with predictions of sequence alignments. Mutations in Glu-140 did not result in any detectable change in Km, or apparent size, suggesting that Glu-140 is directly involved in catalysis. The pH optimum of the proteins carrying the Glu-140/Ala and Glu140/Gln mutations was lower than that of the wild type, whereas the activity vs. pH profile of Glu-140/Asp CelC was similar to that of the wild type, suggesting that Glu-140 may act as a proton donor.     

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.     

Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum

Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.     

Statistical optimization of one-step immobilization process for recombinant endoglucanase from Clostridium thermocellum

Endoglucanase CelA from Clostridium thermocellum (CtCelA) is a thermophilic endo-β-1,4-glucanase and has a low solubility when expressed in Escherichia coli. To make industrial application of CtCeA more appealing, artificial oil bodies (AOBs) was implemented for one-step renaturation and immobilization of recombinant CtCelA. CtCelA was first fused with oleosin (Ole-CtCelA), a structural protein of plant seed oils. Ole-CtCelA was overexpressed in E. coli, and its insoluble form was recovered and mixed with plant oils to assemble AOBs. Moreover, the Box–Behnken design and the central composite design were employed to optimize the condition for assembly of AOBs and the enzymatic reaction condition, respectively. Consequently, the approach led to the resumption of active CtCelA on AOBs. CtCelA-bound AOBs exhibited an optimum activity at 69 °C and pH 6.3 while the immobilized protein remained stable for several hours at 70 °C and after 5 repeated uses. Overall, it indicates a promise of this novel approach for direct processing and immobilization of recombinant CtCelA.