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Journal of Bacteriology, January 2003, p. 504-512, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.504-512.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from Clostridium thermocellum CelJ: Importance of the CBM to Cellulose Hydrolysis

Takamitsu Arai, Rie Araki, Akiyoshi Tanaka, Shuichi Karita, Tetsuya Kimura, Kazuo Sakka,^* and Kunio Ohmiya

Faculty of Bioresources, Mie University, Tsu 514-8507, Japan

Received 2 July 2002/ Accepted 21 October 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety. To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized. Isothermal titration calorimetric studies showed that the association constants (K_a) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10⁴ and 6.4 x 10⁴ M^-1, respectively, and that the binding of CBM30 to these ligands is enthalpically driven. Qualitative analyses showed that CBM30 had strong affinity for cellulose and ß-1,3-1,4-mixed glucan such as barley ß-glucan and lichenan. Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley ß-glucan and lichenan. By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Most cellulases are modular enzymes which consist of two or more discrete modules, such as catalytic and carbohydrate-binding modules (CBMs), connected to each other via a linker sequence (26). Catalytic modules, which are engaged in the hydrolysis of cellulose, are classified in 14 groups in glycoside hydrolase families on the basis of amino acid sequence similarities (12a) (http://afmb.cnrs-mrs.fr/cazy/CAZY/index.html). On the other hand, CBMs, many of which bind preferentially to cellulose, are also classified into 30 families on the basis of amino acid sequence similarities (http://afmb.cnrs-mrs.fr/cazy/CAZY/index.html). It is thought that a CBM in a cellulase molecule enhances the hydrolytic activity of a catalytic domain adjacent to the CBM by increasing the enzyme concentration on the surface of an insoluble substrate (17) or by supplying the catalytic module with a more easily degradable substrate, i.e., amorphous cellulose (36).

A number of cellulases classified in family 9 from various organisms have been reported. Microbial enzymes in this family can be broadly divided into three groups. The first group includes enzymes containing a family 3c CBM immediately downstream of a catalytic module, the second group includes enzymes containing an immunoglobulin-like module (Ig) upstream of a catalytic module, and the third group includes enzymes comprising a single catalytic module only (9). For example, cellulase E4 from Thermomonospora fusca belongs to the first group and cellulase CelD from Clostridium thermocellum is included in the second group. The crystal structures of the catalytic domains of E4 (31) and CelD (16) were determined to be similar (/)₆-barrel folds. In general, a catalytic module and a CBM in a cellulase molecule can function independently, and therefore many catalytic modules and CBMs were expressed independently of the other functional modules and were studied biochemically and structurally investigated. However, the family 3c CBMs, which are always found with family 9 catalytic modules, are unusual in that the removal of CBMs from the catalytic modules affects their enzyme activity to some extent; e.g., the removal of the CBM from T. fusca E4 decreased the catalytic activity toward CMC and the bacterial microcrystalline cellulose to 23 and 1%, respectively, of the activity of the original enzyme (31); the absence of the CBM led to the complete inactivation of C. cellulolyticum CelG (10); and removal of the CBM from C. thermocellum CelQ reduced the enzyme activity to about 1/1,000 of the original activity (4).

C. thermocellum secretes a high-molecular-mass cellulase complex, termed cellulosome, composed of various catalytic components such as cellulases and hemicellulases, and at least one noncatalytic scaffolding protein known as scaffoldin or CipA (5, 32). Catalytic components and CipA are assembled into the cellulosome by the interaction between a dockerin module of the former and one of the cohesin modules of the latter. CelJ, a 178-kDa major component of the C. thermocellum cellulosome, is a typical modular enzyme (1, 28). It consists of an N-terminal signal peptide and five modules in the following order: a module of unknown function (UM-1), a family 9 cellulase module, a family 44 cellulase module, a dockerin module, and another module of unknown function. Recently, the N-terminal module of Fibrobacter succinogenes endoglucanase CelF that is homologous to UM-1 of CelJ has been found to be a novel CBM, and this CBM, along with a few homologous proteins including UM-1 of CelJ, have been compiled in family 30 of CBMs, suggesting that CelJ contains a CBM in addition to two catalytic modules belonging to different families.

While we were characterizing the enzymatic properties of the N-terminal moiety of CelJ including the family 30 CBM and the family 9 catalytic module, we found that the hydrolytic activity of the catalytic module depended completely on the presence of the CBM. In this paper, we describe the characterization of the family 9 cellulase as an integrate form of the CBM and the catalytic module. We also deal with the function of the CBM elucidated by qualitative binding assay and thermodynamic studies, and we discuss the importance of the CBM to cellulose hydrolysis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and growth conditions. C. thermocellum strain F1 was used for isolation of the genomic DNA (30). The Escherichia coli strains used in this study were XL1-Blue MRF' (Stratagene) and JM109 (Stratagene). Recombinant E. coli strains were cultured at 37°C in Luria broth supplemented with ampicillin (50 µg/ml).

Plasmids and plasmid constructions. The plasmid vectors used in this study were pT7 Blue (Novagen), pBluescript II KS(+) and KS(-) (Stratagene), and pQE-30T (3). The plasmids used to produce the truncated derivatives of CelJ were constructed as follows: DNA fragments encoding respective derivatives were amplified by PCR from C. thermocellum F1 genomic DNA with LA Taq DNA polymerase (Takara) and an appropriate combination of primers (see Fig. 1) containing artificial BglII or SalI restriction sites for cloning the PCR fragments into plasmid vectors. The resulting PCR fragments were cloned into pT7 Blue as specified by the supplier. After sequencing the inserted DNA fragments for confirmation of the absence of mutation, the inserted fragments were transferred to pQE-30T. The combinations of the primers were as follows: celJF-Bgl706 and celJR-Sal2868 to construct pCBM-CM, yielding a polypeptide composed of the family 30 CBM, the Ig-like module, and the family 9 catalytic module (CBM30-Ig-CM9); celJF-Bgl706 and celJR-Sal1248 to construct pCBM, yielding a polypeptide composed of the CBM (CBM30); celJF-Bgl706 and celJR-Sal1561 to construct pCBM-Ig, yielding a polypeptide composed of the CBM and the Ig-like module (CBM30-Ig); celJF-Bgl1269 and celJR-Sal2868 to construct pIg-CM, yielding a polypeptide composed of the Ig-like module and the catalytic module (Ig-CM9); celJF-Bgl1558 and celJR-Sal2868 to construct pCM, yielding a polypeptide composed of the family 9 catalytic module (CM9). A schematic diagram of these proteins is shown in Fig. 1. Plasmid pTS307 was used for producing C. thermocellum endoglucanase CelC (29).

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FIG. 1. (A) PCR primers used for the amplification of the derivatives of celJ. (B and C) Molecular architecture of CelJ and its derivatives used in this study (B) and family 9 cellulases from C. thermocellum (C). Amino acid sequences were from DDBJ: CbhA, BAA20861; CelD, CZCLDM; CelF, CAA43035; CelI, A47704; CelK, AAC06139; CelN, CAB76935; CelQ, BAB33148; CelT, BAB79196.

Purification of truncated derivatives of CelJ and CelC. To produce the recombinant proteins, 1.5-liter cultures of E. coli recombinants were grown to mid-log phase (absorbance at 600 nm, 0.6) and then isopropyl-ß-D-thiogalactopyranoside was added to the cultures to give a final concentration of 1 mM. After an additional incubation of 3 h, the cells were harvested, washed, and disrupted by sonication. Cell debris was removed by centrifugation, and the cell extracts thus obtained were used for the purification of the derivatives of CelJ. These proteins were purified by HiTrap Chelating HP (Amersham Pharmacia Biotech), RESOURCE PHE (Amersham Pharmacia Biotech), and RESOURCE Q (Amersham Pharmacia Biotech) column chromatographies. The His₆ tag was removed from the recombinant proteins by thrombin digestion after the HiTrap Chelating HP column chromatography. The purity of each fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. thermocellum CelC was purified as described previously (29). The protein concentration was determined with bovine serum albumin as the standard using the Micro BCA protein assay reagent kit (Pierce).

Enzyme assays. The cellulase activity was measured by a 10-min incubation at 60°C in 10 mM sodium phosphate buffer (pH 6.3) or Britton and Robinson's universal buffer (50 mM phosphoric acid, 50 mM boric acid, and 50 mM acetic acid [the pH was adjusted to 2 to 12 with 1 M NaOH]) in the presence of 1.5% carboxy methyl cellulose [CMC] [low viscosity] [Sigma]). The reducing sugars released from the substrate were determined with the 3',5'-dinitrosalicylic acid reagent as described by Miller (22a). One unit of activity was defined as the amount of enzyme releasing 1 µmol of glucose equivalent per min from CMC. Enzyme activities toward Avicel (Merck), acid-swollen cellulose (ASC) prepared in this laboratory (20), ball-milled cellulose (BMC), lichenan (Sigma), laminarin (Nacalai Tesque), and oat spelt xylan (Fluka) were assayed as described above, except that CMC was replaced by each substrate. The viscometric assay was done using a viscometer (Tokimec) at 25°C in a 10 mM sodium phosphate buffer (pH 6.3). Enzyme (100 µl) was added to 1.2 ml of 0.3% CMC (medium viscosity [Sigma]).

Analysis of hydrolysis products. Cellooligosaccharides (cellobiose to cellohexaose, 5 µg each) were incubated with 0.1 U of the purified enzyme in 1 ml of 50 mM sodium succinate buffer (pH 5.5) at 60°C. Thin-layer chromatography (TLC) of the hydrolysis products was performed on a DC-Fertigplatten SIL G-25 plate (Macherey-Nagel) developed with a solvent of 1-propanol-water (85:15, vol/vol). Cellooligosaccharides were visualized by spraying the plate with an aniline-diphenylamine reagent (11).

Qualitative carbohydrate-binding assays. The binding of CBM30-Ig and CBM30 to the insoluble polysaccharides was determined as follows. CBM30-Ig or CBM30 (35 µg) was mixed with insoluble polysaccharides (1.5 mg) in a 10 mM sodium phosphate buffer (pH 6.3) in a final volume of 0.2 ml and incubated on ice for 1 h with occasional stirring. After centrifugation, the supernatant and the precipitate were analyzed SDS-PAGE. The polysaccharides tested were Avicel, ASC, lichenan, chitin (Nacalai Tesque), Sephadex G-100 (Amersham Pharmacia Biotech), and oat spelt xylan.

The affinities of these proteins for soluble polysaccharides, i.e., CMC (low viscosity [Sigma]), hydroxyethylcellulose (Fluka), methylcellulose (Nacalai Tesque), barley ß-glucan (Sigma), and birchwood xylan (Sigma), were examined by native affinity gel electrophoresis as described by Meissner et al. (22), with some simplifications.

We used the Laemmli system for electrophoresis, excluding SDS from all solutions. The separating gel contained 10% acrylamide. The polysaccharides were incorporated into the gel at a concentration of 0.1% prior to polymerization. A control gel without polysaccharides was prepared and run simultaneously. Protein samples were loaded onto gels in a standard loading buffer without SDS. Electrophoresis was run at 4°C and 100 V for 2.5 h. Proteins were visualized by Coomassie blue staining.

Isothermal titration calorimetry. The isothermal titration calorimetric experiments were done using VP-ITC (MicroCal LLC). The reaction cell was filled with CBM30-Ig or CBM30 solution, and the reference cell was filled with water. The injection syringe was filled with one of the cellooligosaccharide solutions. Then 1 to 10 µl of a ligand solution (3.0 to 3.5 mM) was injected 29 times into a CBM30-Ig or CBM30 solution with intervals of 3 min, and the binding heat was recorded. The injection syringe stirred the solution at 400 rpm. The control dilution of these ligands into the buffer was also observed to correct the observed binding heat. The observed data were analyzed using the ORIGIN software for the VP-ITC developed by MicroCal LLC. Titration experiments were done at pH 7.5 (50 mM Tris buffer) and 10°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction and production of the CelJ derivatives. Truncated forms of CelJ were constructed as described in Materials and Methods. CBM30-Ig-CM9 consists of the family 30 CBM, the Ig-like module, and the family 9 catalytic module; Ig-CM9 consists of the Ig-like module and the family 9 catalytic module; CM9 consists of the family 9 catalytic module only; CBM30-Ig consists of the family 30 CBM and the Ig-like module; and CBM30 consists of the family 30 CBM only (Fig. 1). These proteins were purified from whole-cell lysates of E. coli recombinants with column chromatography. Each of the purified preparations gave a single band on SDS-PAGE, and their molecular sizes were in good agreement with those deduced from the nucleotide sequences (Fig. 2).

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FIG. 2. SDS-PAGE of the purified derivatives of CelJ. The gel was stained with Coomassie brilliant blue. Lanes: M, protein molecular mass standard (molecular masses shown at the left); 1, CBM30-Ig-CM9; 2, Ig-CM9; 3, CM9; 4, CBM30-Ig; 5, CBM30.

Enzymatic properties of CBM30-Ig-CM9, Ig-CM9, and CM9. The specific activities of the truncated derivatives of CelJ toward various substrates are shown in Table 1. The purified CBM30-Ig-CM9 had relatively high specific activity toward CMC (13,100.00 IU/µmol), lichenan (7,920.00 IU/µmol), and barley ß-glucan (8,710.00 IU/µmol) and low activity toward ASC (163.00 IU/µmol), Avicel (9.80 IU/µmol) and p-nitrophenol cellulose (PNPC) (4.08 IU/µmol); there was no activity toward oat spelt xylan. The initial rates of the reactions were measured at 60°C in various concentrations of CMC. From a double-reciprocal plot, the V_max value was estimated to be 164 µmol/min/mg and K_m was estimated to be 4.06 mg/ml for CMC. The enzyme was maximally active around a pH of 6.0 when the enzyme activity was assayed after a 10-min incubation at 60°C in Britton and Robinson's universal buffer solutions at various pHs. The enzyme was stable in the pH range of 5 to 10 when incubated at 60°C for 30 min in the same buffer solutions without any substrates. The effects of temperature on the activity and stability of the enzyme were examined. The optimum temperature for activity was found to be 60°C. The enzyme was stable at 70°C for 10 min at pH 6.0 in the absence of substrates.

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TABLE 1. Activities of CBM30-Ig-CM9, Ig-CM9, and CM9 toward various substrates

The action of the enzyme on cellooligosaccharides, ASC, and Avicel was qualitatively analyzed by thin-layer chromatography (TLC) (Fig. 3). When cellotetraose, cellopentaose, and cellohexaose were treated with CBM30-Ig-CM9, cellobiose was the major product. The enzyme was not active toward cellotriose and cellobiose. CBM30-Ig-CM9 hydrolyzed ASC and Avicel to yield cellobiose as the major product with cellotriose as a minor product. These results suggest that CBM30-Ig-CM9 has a tendency to hydrolyze the substrates to liberate cellobiose; i.e., this enzyme is not a typical endoglucanase. Therefore, we compared the relationship between the reducing sugars released from CMC and the decrease in the viscosity of the CMC solution caused by CBM30-Ig-CM9 and that by C. thermocellum endoglucanase CelC. As shown in Fig. 4, although both enzymes obviously reduced the viscosity of the CMC solutions and resulted in production of reducing sugars, the ratio of the reduction of viscosity to the production of reducing sugars by CBM30-Ig-CM9 is smaller that that by CelC, supporting the suggestion described above.

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FIG. 3. TLC analysis of hydrolysis products from cellooligosaccharides, ASC, and Avicel. Each cellooligosaccharide (5 µg, G2 to G6) was incubated with the purified CBM30-Ig-CM9 (0.1 U) for 14 h, and the hydrolysates were analyzed by TLC. ASC and Avicel was incubated with the enzyme for 14 h. S, authentic oligosaccharides; G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6, cellohexaose.

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FIG. 4. Relationship between the release of reducing sugars from CMC solutions and the reduction of their viscosity by CBM30-Ig-CM9 and C. thermocellum CelC.

Surprisingly, the purified Ig-CM9 and CM9 lacking the N-terminal CBM showed only negligible activity toward CMC and other substrates; e.g., Ig-CM9 showed a specific activity of 0.18 IU/µmol for CMC (Table 1). This observation indicates that the hydrolytic activity of the family 9 catalytic module of CelJ depends completely on the presence of the CBM coterminous to the catalytic module.

Synergistic interactions between CBM30-Ig-CM9 and CelC. Synergistic interactions between CBM30-Ig-CM9 and C. thermocellum CelC toward ASC were investigated using different ratios of the CBM30-Ig-CM9 and the CelC concentrations. The degree of synergistic effect (DSE) was defined as the ratio of the observed activity of the combined enzymes to the sum of the observed individual activities (7). As shown in Fig. 5, synergism between CBM30-Ig-CM9 and CelC was observed for all ratios; i.e., DSE values varied between 1.4 and 1.9, and the highest DSE value (1.9) was obtained in the mixture containing 3 parts of CBM30-Ig-CM9 and 7 parts of CelC.

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FIG. 5. Synergistic interaction of CBM30-Ig-CM9 and CelC in the degradation of ASC. Reaction mixtures containing CBM30-Ig-CM9 and CelC in different ratios were incubated for 120 min at 60°C; e.g., 10+0 denotes the ratio of 10:0 of CBM30-Ig-CM9 and CelC. DSE values are shown above the respective bars. , activity of CBM30-Ig-CM9; , activity of CelC; , sum of the theoretical activities of respective enzymes; , observed activity of the combined enzymes.

Binding of CBM30-Ig and CBM30 to insoluble and soluble polysaccharides. To investigate the function of the family 30 CBM of CelJ, we qualitatively examined the ability of CBM30-Ig and CBM30 to bind insoluble polysaccharides by incubating the proteins with the polysaccharides and comparing the protein concentrations in the supernatant fraction (unbound protein) and in the precipitate fraction (bound protein) by SDS-PAGE. As shown in Fig. 6I, both the proteins bound to insoluble cellulose allomorphs such as Avicel, BMC, ASC, and lichenan. On the other hand, they showed no affinity for the insoluble fraction of agarose, chitin, oat spelt xylan, starch, and Sephadex G-150.

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FIG. 6. Adsorption of CBM30 to insoluble (I) and soluble (II) polysaccharides. In the experiment in panel I, CBM30 was incubated with insoluble polysaccharides including Avicel (A), ASC (B), BMC (C), insoluble fraction of oat spelt xylan (D), lichenan (E), agar (F), starch (G), Sephadex G-150 (H), and chitin (I). After centrifugation, proteins in the supernatant (lane 1) and the precipitate (lane 2) were analyzed by SDS-PAGE. In the experiment in panel II, affinities of CBM30-Ig (lane 1) and CBM30 (CBM30) for various soluble polysaccharides including hydroxyethylcellulose (b), methylcellulose (c), soluble fraction of oat spelt xylan (d), birchwood xylan (e), barley ß-glucan (f), lichenan (g), and soluble starch (h) were analyzed by native affinity gel electrophoresis. Lane M contains bovine serum albumin as a control protein. A gel without a polysaccharide served as a reference (a).

The affinities of CBM30-Ig and CBM30 for a series of different soluble polysaccharides were also qualitatively evaluated by native affinity gel electrophoresis (Fig. 6II). The migration of the proteins was significantly retarded by the inclusion of hydroxyethylcellulose, methyllcellusose, lichenan, and barley ß-glucan in gels but was not affected by birchwood xylan, oat spelt xylan, or soluble starch.

Isothermal titration calorimetry of CBM30-Ig and CBM30 with cellooligosaccharides. Calorimetric titration studies were carried out using CBM30-Ig and CBM30 with cellooligosaccharides as ligands at 10°C and at pH 7.5, where 1 to 10 µl of ligand solution (3.0 to 3.5 mM) was injected 29 times into a CBM-Ig or CBM30 solution. Figure 7 shows examples of the calorimetric titration of CBM30-Ig and CBM30 by cellohexaose. The reaction heat of each injection was evaluated from each peak area. From these results, the number of ligand molecules (n) bound to the CBM, the association constant K_a and the binding enthalpy (H) were evaluated. The standard Gibbs free energy change (G) and the standard entropy change (S) as (TS) were calculated using G = -RT ln K_a and G = H - TS, where R is the gas constant and T is the absolute temperature, with the assumption that H is equal to the standard enthalpy change, H. Table 2 summarizes the results for the binding of CBM30-Ig and CBM30 to cellooligosaccharides. We also examined the binding of CBM30 to cellotriose and cellotetraose, in addition to the longer cellooligosaccharides listed in Table 2. For cellotriose, no binding heat was observed under the experimental conditions used. For cellotetraose, the binding heat was fairly low (G° = -5.0 kcal/mol) and the K_a and n values calculated were 7.1 x 1 0³ M^-1 and n = 7.77, respectively, which are not listed in Table 2 because of their low reliability. The stoichiometry for cellopentaose and cellohexaose bound to CBM30-Ig and CBM30 at saturation was approximately 1:1, indicating that there was only one ligand-binding site per protein molecule. The lager K_a value of cellohexaose than of cellopentaose suggests that CBM30-Ig and CBM30 prefer larger cellooligosaccharides to smaller cellooligosaccharides; i.e., the K_a value of CBM30-Ig for cellohexaose is about 3.5 times higher than that for cellopentaose. The K_a value of CBM30-Ig for each ligand was similar to that of CBM30, indicating that the presence of the Ig-like module does not influence the cellulose-binding ability of the CBM.

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FIG. 7. Isothermal titration calorimetry of the binding of CBM30 to cellohexaose (A and C) and cellopentaose (B and D). (A and B) Thermogram; (C and D) integrated injection heats calculated from panels A and B, respectively.

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TABLE 2. Thermodynamic parameters of the binding of CBM-Ig and CBM30 to cellooligosaccharides as determined by ITC

Top Abstract Introduction Materials and Methods Results Discussion References

 
Traditionally, cellulases have been broadly divided into endoglucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91) based on their different modes of action on polymeric substrates. True cellobiohydrolases attack polymeric cellulose chains at a reducing end or a nonreducing end in a processive manner to release cellobiose. On the other hand, endoglucanases attack the polymers at random inside the cellulose chain. However, the classification of ß-1,4-glucanases into two mutually exclusive groups, endoglucanases and cellobiohydrolases, may be inappropriate. For example, Cellulomonas fimi CenC, a cellulase of family 9, first attacks an internal ß-1,4-glucosidic bond and removes only a few successive cellobiosyl residues from the new reducing end before it jumps to a new site (35). Clostridium cellulolyticum cellulase Cel9E (formerly CelE) and Cel48F (formerly CelF) also have a similar hydrolytic activity (12, 27). These enzymes, having both endo- and exoglucanase activities, were designated semiprocessive enzymes or processive endoglucanases. CBM30-Ig-CM9 was highly active on CMC but caused a relatively small decrease in the viscosity of the CMC solution per unit of reducing sugar released compared to a typical CelC endoglucanase from C. thermocellum (Fig. 4). These data closely resemble those obtained for CenC (35), suggesting that CBM30-Ig-CM9 hydrolyzes CMC by a semiproessive mechanism; i.e., the enzyme is a semiprocessive enzyme with both endo- and exoglucanase activities. Synergistic action between CBM30-Ig-CM9 and the endoglucanse CelC suggests that these two cellulases hydrolyze the insoluble substrate in different ways. Previously, it was reported that C. cellulolyticum Cel9E showed a synergistic action with Cel5A toward Avicel and bacterial microcrystalline cellulose (12). CBM30-Ig-CM9 showed strong activity toward CMC, barley ß-glucan, and lichenan. Such broad substrate specificity is common in the enzymes classified in family 9 (6). Semiprocessive enzymes can be divided into two types on the basis of their hydrolytic activity toward CMC. One type has a low activity toward CMC. The enzymes of this type, represented by C. cellulolyticum Cel9E (12) and Cel48F (27), may resemble cellobiohydrolases. Another type shows a high activity toward CMC. C. fimi CenC and CBM30-Ig-CM9 are classified in the latter type; i.e., these enzymes may be semiprocessive enzymes with less processivity.

Family 9 cellulases can be classified into three groups, i.e., enzymes consisting a catalytic module and a family 3c CBM, enzymes consisting of an Ig-like module and a catalytic module, and enzymes consisting of a catalytic domain only (Fig. 1). Belaich et al. (6) divided the enzymes containing an Ig-like module into two categories depending on the presence or absence of an N-terminal family 4 CBM. For example, C. thermocellum CbhA and CelK contain an N-terminal family 4 CBM but CelD does not. CelJ (CBM30-Ig-CM9), which contains a family 30 CBM as an additional module, is a new type of family 9 cellulases containing an Ig-like module. The N-terminal region of CelJ was first recognized as a domain of unknown function. Since the N-terminal domain of F. succinogenes CelF (23), which was homologous to that of CelJ, was identified as a CBM, it has recently been compiled and classified in family 30 of CBMs along with the homologous sequences from Clostridium cellulovorans endoglucanase Eng Y (33) and Fusobacterium mortiferum phospho-ß-glucosidase PbgB (34). Family 30 CBMs appear to be closely related to a family 9 catalytic module, although the first example, F. succinogenes CelF, coexists with the family 51 catalytic module. The catalytic module of CelJ shows relatively high sequence identities to EngY (59%) and PbgB (43%), although PbgB is not a cellulase. On the other hand, CelJ shows lower sequence identities to the other family 9 cellulases, e.g., 29, 28, and 30% sequence identities to C. thermocellum CbhA, CelD, and CelK, respectively. High sequence homology among the family 9 cellulases containing a family 30 CBM suggests a strong relationship between the catalytic modules and the CBMs in catalytic action. In actuality, the significance of the family 30 CBM in the hydrolytic action of the catalytic module was found by comparing the enzyme activities of CBM30-Ig-CM9 and CM9: the family 9 catalytic domain without the family 30 CBM showed a negligible activity toward all substrates tested. A similar phenomenon was observed for C. cellulolyticum Cel9E, which is composed of a family 4 CBM, an Ig-like domain, a family 9 catalytic domain, and a dockerin domain. Deletion of the CBM4 induced a total loss of activity (12). These facts are in marked contrast to the observations that C. fimi CenC (35) and C. thermocellum CelK (18), composed of a family 4 CBM, an Ig-like module, and a family 9 catalytic module, did not lose their activity by removal of the N-terminal CBM. Many family 9 cellulases such as C. thermocellum CbhA, CelD, and CelK do not contain a family 30 CBM in nature. Therefore, the reliance of family 4 catalytic domains related to an Ig-like domain upon a CBM is apparently different. Some family 9 cellulases contain a family 3c CBM, e.g., the removal of the CBM from T. fusca E4 decreased the catalytic activity toward bacterial microcrystalline cellulose to 1% of the activity of the original enzyme (31); the absence of the CBM led to the complete inactivation of C. cellulolyticum CelG (10); and the removal of the CBM from CelQ reduced the enzyme activity to about 1/1,000 of the original activity (4). In general, a catalytic module and a CBM in the same polypeptide can function independently; e.g., artificial removal of the CBM from the catalytic domain does not affect the enzyme activity toward soluble substrates, and the CBM isolated from the catalytic domain retains the substrate-binding ability. Therefore, the inactivation of CBM30-Ig-CM9 and some other family 9 cellulases by separation of a CBM from respective catalytic modules is an exceptional phenomenon. Since two catalytic residues, a nucleophile and a proton donor, are conserved as Asp-377 and Glu-735, respectively, in the family 9 catalytic module and since both Ig-CM9 and CM9 showed extremely weak but detectable enzyme activity, it is unlikely that additional residues existing in the CBM are necessary for enzymatic activity. Although the cause of this phenomenon has not yet been clarified, it is possible that the removal of the CBM from the enzyme affects the tertiary structure of catalytic modules, resulting in complete or partial inactivation of the enzymes. Recently, Kataeva et al. (19) showed that the interaction between the family 4 CBM and the family 9 catalytic module of C. thermocellum CelK affected the thermostability of both the CBM and the catalytic module although it did not reduce the enzyme activity and cellulose-binding ability at lower temperatures. If there is a stronger interaction between the family 9 catalytic module and the family 30 CBM in CBM-Ig-CM9, artificial separation may make each module unstable or inactive. Although we examined the secondary structure of CBM30-Ig-CM9, Ig-CM9, and CM9 by determining their circular dichroism spectra, we could not observe the apparent disorder of their secondary structure (data not shown). The slight change in the tertiary structure may cause the inactivation of the enzyme.

On the other hand, CBM30 retained its cellulose-binding ability after artificial separation from the catalytic module (Fig. 6; Table 2). This fact contrasts strikingly with the observations that the family 3c CBMs coterminous with the family 9 catalytic module showed very low, if any, cellulose-binding ability (13). To investigate the thermodynamics of binding, the K_a values of CBM30-Ig and CBM30 for some ligands and the change in enthalpy (H°) were used to determine the change in the Gibbs free energy (G°) and TS° for each ligand-binding event. The data (Table 2) showed that a one-binding-site model fits the binding of cellopentaose and cellohexaose to the CBMs. Although the minimal binding requirement of CBM30 appears to be cellotetraose, accurate values for it could not be obtained. The H and TS values for ligand binding were negative, and thus the interaction of CBM30 with carbohydrates is exothermic. The change in enthalpy makes a positive contribution to ligand binding, while the increase in entropy has a detrimental influence on the interaction of CBM30 with the soluble oligosaccharides.

CBMs are now classified into 30 families on the basis of amino acid sequence homology (http://afmb.cnrs-mrs.fr/cazy/CAZY/index.html). Among them, 11 families include CBMs that have a preferential affinity for cellulose. These CBMs appear to be divided into two categories, one consisting of CBMs with affinity for crystalline cellulose and the other consisting of CBMs having affinity for amorphous cellulose but not for crystalline cellulose. CBMs of families 1, 2, and 3 with affinities for crystalline cellulose have common structural characteristics, although their tertiary structures are different from one another (35); i.e., they have a planar cellulose-binding surface including a set of aromatic residues and a group of polar residues. CBMs of this type do not seem to have an affinity for small oligosaccharides such as cellobiose. Thermodynamic studies have not been reported for these CBMs with cellooligosaccharides, although the interaction between cellohexaose and family 1 CBMs from Trichoderma reesei cellulases was studied by nuclear magnetic resonance spectroscopy (21). It was reported that the binding of the family 2 CBM of C. fimi Cex to insoluble cellulose was entropically driven (8). On the other hand, CBMs with preference to amorphous cellulose and/or soluble saccharides have a carbohydrate-binding cleft where aromatic and polar amino acid residues are conserved (14, 24, 25). These modules often show affinities for oligosaccharides and even monosaccharides; e.g., the family 4 CBM of C. fimi CenC has an affinity for cellotetriose with a K_a of 180 M^-1 (15), the family 9 CBM of Themotoga maritime xylanase Xyn10A has an affinity for glucose with a K_a of 3.2 x 10³ M^-1 (25), and the family 17 CBM of Clostridium cellulovorans has an affinity for cellotetraose with a K_a of 1.2 x 10³ M^-1 (24). It was found that the interactions of these CBMs with oligosaccharides are driven by enthalpic forces. The thermodynamics of CBM30 binding to cellooligosaccharides is consistent with those of CBMs with affinities for soluble polymers and oligosaccharides.

CBM30 of C. thermocellum CelJ showed a wide ligand specificity, including insoluble and soluble cellulosic materials, ß-1,3-1,4 mixed glucan such as lichenan and barley ß-glucan. It seems reasonable that the family 30 CBM has an affinity for ß-1,3-1,4 mixed glucan because both the family 9 and 44 catalytic modules of CelJ have hydrolytic activity toward this substrate, and therefore the presence of the CBM is expected to enhance their activity. It is possible that the family 9 catalytic module mutated to be dependent on the family 30 CBM during their evolution process in C. thermocellum CelJ.

In conclusion, this is the first report about a family 9 cellulase (which absolutely requires a family 30 CBM for maintaining the enzyme activity) and about thermodynamic studies of a family 30 CBM. It remains to be determined why the catalytic module depends on the CBM for its activity.

 
This work was supported in part by a grant-in-aid for university and society collaboration, The Ministry of Education, Culture, Sports, and Technology of Japan.

 
* Corresponding author. Mailing address: Faculty of Bioresources, Mie University, 1515 Kamihamacho, Tsu 514-8507, Japan. Phone: 81-59-231-9621. Fax: 81-59-231-9684. E-mail: sakka{at}bio.mie-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, January 2003, p. 504-512, Vol. 185, No. 2
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.2.504-512.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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