Search

In protein microarray performance, the choice of an appropriate surface is a crucial factor. Three‐dimensional substrates like nitrocellulose are known to have higher binding capacities than planar surfaces. Furthermore, they can enable the immobilization of proteins in a functional manner. One disadvantage of today's nitrocellulose‐based microarrays is the high background fluorescence, which can interfere with the detection of low‐abundance proteins. We have developed an innovative black nitrocellulose membrane‐based protein microarray that exhibits low autofluorescence in combination with increased sensitivity and improved LOD (limit of detection). The applicability of the novel material was demonstrated with main focus on reversed‐phase microarray experiments. In comparison to various commercially available microarrays, a higher sensitivity in regard to the spotted protein was achieved. In contrast to other porous nitrocellulose‐based microarrays, the black nitrocellulose provides a significant lower autofluorescence and background intensity.

A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Bélaı̈ch, A., Lamed, R., Shoham, Y., Bélaı̈ch, J.-P., and Bayer, E. A. (2001)J. Biol. Chem.276, 21257–21261), based on the high affinity species-specific cohesin-dockerin interaction. Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulose-binding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. The activities of the resultant ternary complexes were assayed using different types of cellulose substrates. Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates. On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate. For certain enzyme pairs, however, one effect appeared to predominate over the other. The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites.

alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant. The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.

A bacterial strain (TA13) capable of utilizing t-anethole as the sole carbon source was isolated from soil. The strain was identified as Arthrobacter aurescensbased on its 16 S rRNA gene sequence. Key steps of the degradation pathway of t-anethole were identified by the use of t-anethole-blocked mutants and specific inducible enzymatic activities. In addition to t-anethole, strain TA13 is capable of utilizing anisic acid, anisaldehyde, and anisic alcohol as the sole carbon source. t-Anethole-blocked mutants were obtained following mutagenesis and penicillin enrichment. Some of these blocked mutants, accumulated in the presence of t-anethole quantitative amounts of t-anethole-diol, anisic acid, and 4,6-dicarboxy-2-pyrone and traces of anisic alcohol and anisaldehyde. Enzymatic activities induced by t-anethole included: 4-methoxybenzoate O-demethylase, p-hydroxybenzoate 3-hydroxylase, and protocatechuate-4,5-dioxygenase. These findings indicate that t-anethole is metabolized to protocatechuic acid through t-anethole-diol, anisaldehyde, anisic acid, and p-hydroxybenzoic acid. The protocatechuic acid is then cleaved by protocatechuate-4,5-dioxygenase to yield 2-hydroxy-4-carboxy muconate-semialdehyde. Results from inducible uptake ability and enzymatic assays indicate that at least three regulatory units are involved in the t-anethole degradation pathway. These findings provide new routes for environmental friendly production processes of valuable aromatic chemicals via bioconversion of phenylpropenoids.

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellumand Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

A β‐xylosidase from Bacillus stearothermophilusT‐6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coliand purified. The enzyme showed maximum activity at 65°C and pH 5.6–6.3. The stereochemistry of the hydrolysis of p‐nitrophenyl β‐D‐xylopyranoside was followed by 1H‐nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site‐directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

A β‐xylosidase from Bacillus stearothermophilusT‐6 was cloned, overexpressed in Escherichia coliand purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH‐A clan. The conserved Glu160 was proposed as the acid‐base catalyst. An E160A mutant was constructed and subjected to steady state and pre‐steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid‐base catalytic residue.

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000)Proteins39, 170–177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellumand Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of theC. thermocellumdockerin differentiates between its nonrecognition and high affinity recognition (Ka∼ 109m−1) by a cohesin fromC. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.

Abstract: Previous work from our group [Morag (Morgenstern), E., Bayer, E. A., and Lamed, R. (1991), Appl. Biochem. Biotechnol. 30, 129–136] has demonstrated an anomalous electrophoretic mobility pattern for scaffoldin, the 210-kDa cellulosome-integrating subunit of Clostridium thermocellum. Subsequent evidence [Morag, E., Bayer, E. A., and Lamed, R. (1992), Appl. Biochem. Biotechnol. 33, 205–217] indicated that the effect could be attributed to a nonproteolytic fragmentation of the subunit into a defined series of lowermolecular-weight bands. In the present work, a recombinant segment of the scaffoldin subunit was employed to determine the site(s) of bond breakage. An Asp-Pro sequence within the cohesin domain was identified to be the sensitive peptide bond. This sequence appears quite frequently in the large cellulosomal proteins, and the labile bond may be related to an as yet undescribed physiological role in the hydrolysis of cellulose by cellulosomes.

A β-xylosidase from Bacillus stearothermophilusT-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coliand purified. The enzyme showed maximum activity at 65°C and pH 5.6–6.3. The stereochemistry of the hydrolysis of p-nitrophenyl β- D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

A β-xylosidase from Bacillus stearothermophilusT-6 was cloned, overexpressed in Escherichia coliand purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517–527) we predicted that four amino acid residues of the ~70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177. © 2000 Wiley-Liss, Inc.

The cohesin‐dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517–527) we predicted that four amino acid residues of the ∼70‐residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site‐directed mutagenesis of the suspected residues, whereby the species‐specificity of the cohesin‐dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177. © 2000 Wiley‐Liss, Inc.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517–527) we predicted that four amino acid residues of the ~70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177. © 2000 Wiley-Liss, Inc.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517–527) we predicted that four amino acid residues of the ~70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177. © 2000 Wiley-Liss, Inc.

The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valoniaventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Iα/Iβ composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its ‘commitment’ towards complete degradation.

α‐d‐Glucuronidases cleave the α‐1,2‐glycosidic bond of the 4‐O‐methyl‐α‐d‐glucuronic acid side chain in xylan. Of the xylan‐debranching hydrolases, these enzymes are the least studied and characterized. The α‐glucuronidase gene (aguA) from Bacillus stearothermophilusT‐6 has been cloned, sequenced and overproduced in Escherichia coli. The gene encodes for a protein of 679 amino acids with a calculated molecular weight of 78480 and a pIof 5.42. α‐Glucuronidase T‐6 shows high homology to the α‐gluc­uronidases of Thermotoga maritima(60% identity) and of Tri­choderma reesei(44% identity). Based on the amino‐acid sequence similarity, it is likely that these enzymes represent a new class of glycosyl hydrolases. Crystallographic studies of α‐glucuronidase T‐6 were initiated to study the mechanism of catalysis, as well as to provide a structural basis for rational introduction of enhanced thermostability by site‐specific mutagenesis. In this report, the crystallization and preliminary crystallographic characterization of the native α‐­glucuronidase T‐6 enzyme is described. Two crystal forms were found suitable for detailed crystal structure analysis. The T1 form was obtained by the vapour‐diffusion method using PEG 4000 as a precipitant and 2‐propanol as an organic additive. The crystals belong to a primitive tetragonal crystal system (space group P41212 or P43212) with unit‐cell dimensions a= b= 76.1 and c= 331.2 Å. These crystals are mechanically strong, are stable in the X‐­ray beam and diffract X‐rays to better than 2.4 Å resolution. A full 3.0 Å resolution diffraction data set (97.3% completeness, Rmerge9.8%) has recently been collected on one crystal at room temperature using a rotating‐anode X‐ray source and an R‐AXIS IIc imaging‐plate detector. The M1 form was obtained and characterized by similar techniques. The best crystallization occurred at a slightly lower pH and a lower concentration of 2‐propanol. The crystals belong to a primitive monoclinic crystal system (space group P21) with unit‐cell dimensions a= 65.8, b= 127.4, c= 96.6 Å and β = 97.9°. These crystals are also quite strong and stable, and diffract to better than 2.8 Å resolution. A full 2.8 Å resolution diffraction data set (96.2% completeness, Rmerge7.6%) has recently been collected on one crystal at room temperature using the same R‐AXIS IIc setup. Both forms are currently being used to obtain crystallographic phasing viaisomorphous heavy‐atom derivatives and selenomethionine MAD experiments.

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module—the cellulose-binding domain—is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelope via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dynamic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are then taken up and assimilated by normal cellular processes.

We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli.The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellumCBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein–CBD fusions in which the fusion partner is insoluble upon expression.

The cross-species specificity of the cohesin–dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin–dockerin interaction seems to be species-specific. Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Proteins 29:517–527, 1997. © 1997 Wiley-Liss, Inc.

Bacillus stearothermophilus L1 was isolated by enrichment culture using an alkaline extract of pulp as the carbon source at 65°C and pH 9.0. The bacterium produced extracellular xylanase and a-l-arabinofuranosidase (EC 3.2.1.55). The xylanase activity was high when the cells were grown in the presence of d-xylose, whereas the arabinofuranosidase activity was high when grown in media containing l-arabinose. The arabinofuranosidase was purified 59-fold with an 80% yield by DEAE Sephacel and Sephadex G-100 chromatography. The purified enzyme had an apparent molecular mass of 110 000 kDa and consisted of two subunits of 52 500 kDa and 57 500 kDa. Using p-nitrophenyl-a-l-arabinofuranosidase as the substrate, the enzyme had a Michaelis constant (Km) of 2.2 × 10-4m, maximum reaction velocity (Vmax) of 11o µmol min-1 mg-1, temperature optimum of 70°C and pH optimum of 7.0 (50% activity at pH 8.0). The enzyme was specific for the furanoside configuration. The purified enzyme partially delignified softwood Kraft pulp. Treatment of the pulp with 38 units ml-1 of a-l-arabinofuranosidase at 65°C for 2 h at pH 8.0 and 9.0 led to lignin releases of 2.3% and 2.1%, respectively. The enzyme acted synergistically with a thermophilic xylanase in the delignification process, yielding a 19.2% release of lignin.

The cross‐species specificity of the cohesin–dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin‐containing segments from the cellulosomes of two different species, Clostridium thermocellumand Clostridium cellulolyticum,were allowed to interact with cellulosomal (dockerin‐containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium‐dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin–dockerin interaction seems to be species‐specific. Based on intra‐ and cross‐species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino‐acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium‐binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Proteins 29:517–527, 1997. © 1997 Wiley‐Liss, Inc.

The enzymatic subunits of the cellulosome of Clostridium thermocellumare integrated into the complex by a major non-catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose-binding domain (CBD) and nine subunit-binding domains, or cohesin domains. Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits. Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.

Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 106 cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 106 cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production.

Acinetobacter calcoaceticus

Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ+ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ+ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of β-galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ+ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.

The enzymatic subunits of the cellulosome of Clostridium thermocellumare integrated into the complex by a major non‐catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose‐binding domain (CBD) and nine subunit‐binding domains, or cohesin domains. Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits. Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.

ABSTRACTLignocellulosic biomass, the most abundant polymer on Earth, is typically composed of three major constituents: cellulose, hemicellulose, and lignin. The crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix are three major factors that contribute to the observed recalcitrance of lignocellulose. By means of designer cellulosome technology, we can overcome the recalcitrant properties of lignocellulosic substrates and thus increase the level of native enzymatic degradation. In this context, we have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffoldin engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome represents the most elaborate artificial enzyme composite yet constructed, and the fully functional complex achieved enhanced levels (up to 1.6-fold) of degradation of untreated wheat straw compared to those of the wild-type free enzymes. The action of these designer cellulosomes on wheat straw was 33 to 42% as efficient as the natural cellulosomes of Clostridium thermocellum. In contrast, the reduction of substrate complexity by chemical or biological pretreatment of the substrate removed the advantage of the designer cellulosomes, as the free enzymes displayed higher levels of activity, indicating that enzyme proximity between these selected enzymes was less significant on pretreated substrates. Pretreatment of the substrate caused an increase in activity for all the systems, and the native cellulosome completely converted the substrate into soluble saccharides.IMPORTANCECellulosic biomass is a potential alternative resource which could satisfy future demands of transportation fuel. However, overcoming the natural lignocellulose recalcitrance remains challenging. Current research and development efforts have concentrated on the efficient cellulose-degrading strategies of cellulosome-producing anaerobic bacteria. Cellulosomes are multienzyme complexes capable of converting the plant cell wall polysaccharides into soluble sugar products en route to biofuels as an alternative to fossil fuels. Using a designer cellulosome approach, we have constructed the largest form of homogeneous artificial cellulosomes reported to date, which bear a total of six different cellulases and xylanases from the highly cellulolytic bacterium Thermobifida fusca. These designer cellulosomes were comparable in size to natural cellulosomes and displayed enhanced synergistic activities compared to their free wild-type enzyme counterparts. Future efforts should be invested to improve these processes to approach or surpass the efficiency of natural cellulosomes for cost-effective production of biofuels.

ABSTRACTIn nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases of Thermobifida fusca(Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates—a distinction previously reserved for cellulose-degrading enzymes.IMPORTANCEXylanases are important enzymes for our society, due to their variety of industrial applications. Together with cellulases and other glycoside hydrolases, xylanases may also provide cost-effective conversion of plant-derived cellulosic biomass into soluble sugars en route to biofuels as an alternative to fossil fuels. Xylanases are commonly found in multienzyme cellulosome complexes, produced by anaerobic bacteria, which are considered to be among the most efficient systems for degradation of cellulosic biomass. Using a designer cellulosome approach, we have incorporated the entire xylanolytic system of the bacterium Thermobifida fuscainto defined artificial cellulosome complexes. The combined action of these designer cellulosomes versus that of the wild-type free xylanase system was then compared. Our data demonstrated that xylanolytic designer cellulosomes displayed enhanced synergistic activities on a natural recalcitrant wheat straw substrate and could thus serve in the development of advanced systems for improved degradation of lignocellulosic material.

ABSTRACTDesigner cellulosomes are precision-engineered multienzyme complexes in which the molecular architecture and enzyme content are exquisitely controlled. This system was used to examine enzyme cooperation for improved synergy among Thermobifida fuscaglycoside hydrolases. Two T. fuscacellulases, Cel48A exoglucanase and Cel5A endoglucanase, and two T. fuscaxylanases, endoxylanases Xyn10B and Xyn11A, were selected as enzymatic components of a mixed cellulase/xylanase-containing designer cellulosome. The resultant mixed multienzyme complex was fabricated on a single scaffoldin subunit bearing all four enzymes. Conversion of T. fuscaenzymes to the cellulosomal mode followed by their subsequent incorporation into a tetravalent cellulosome led to assemblies with enhanced activity (~2.4-fold) on wheat straw as a complex cellulosic substrate. The enhanced synergy was caused by the proximity of the enzymes on the complex compared to the free-enzyme systems. The hydrolytic properties of the tetravalent designer cellulosome were compared with the combined action of two separate divalent cellulase- and xylanase-containing cellulosomes. Significantly, the tetravalent designer cellulosome system exhibited an ~2-fold enhancement in enzymatic activity compared to the activity of the mixture of two distinct divalent scaffoldin-borne enzymes. These results provide additional evidence that close proximity between cellulases and xylanases is key to the observed concerted degradation of the complex cellulosic substrate in which the integrated enzymes complement each other by promoting access to the relevant polysaccharide components of the substrate. The data demonstrate that cooperation among xylanases and cellulases can be augmented by their integration into a single designer cellulosome.IMPORTANCEGlobal efforts towards alternative energy programs are highlighted by processes for converting plant-derived carbohydrates to biofuels. The major barrier in such processes is the inherent recalcitrance to enzymatic degradation of cellulose combined with related associated polysaccharides. The multienzyme cellulosome complexes, produced by anaerobic bacteria, are considered to be the most efficient systems for degradation of plant cell wall biomass. In the present work, we have employed a synthetic biology approach by producing artificial designer cellulosomes of predefined enzyme composition and architecture. The engineered tetravalent cellulosome complexes contain two different types of cellulases and two distinct xylanases. Using this approach, enhanced synergistic activity was observed on wheat straw, a natural recalcitrant substrate. The present work strives to gain insight into the combined action of cellulosomal enzyme components towards the development of advanced systems for improved degradation of cellulosic material.