Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose

Abstract

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.

Figure 1. Schematic representation of CbCel9A/Cel48A wild-type (WT) and its truncational mutants.

The signal peptide is represented by the filled rectangle. GH9: glycoside hydrolase family 9 module; GH48: glycoside hydrolase family 8 module; CBM3b: carbohydrate-binding module family 3 type B; CBM3c: carbohydrate-binding module family 3 type C.

Figure 2. Time course hydrolysis of Avicel, Filter paper, and PASC by CbCel9A/Cel48A and its truncational mutants analyzed by a reducing sugar assay (A) and HPLC analysis (B).

The reactions were performed in citrate buffer (pH 6.0) at 75°C. Substrates were 10 mg ml^-1 of Avicel or 16 discs of Whatman No. 1 filter paper (0.6 cm in diameter) or 2.5 mg ml^-1 of PASC. Enzyme concentrations on Avicel and filter paper were 0.5 μM and 0.2 μM on PASC, or 0 μM for the control.

Figure 3. Hydrolysis of Avicel by TM1, TM3, TM4 and site-directed mutants of TM4.

All of TM4 and its site mutants (TM4/W775A, TM4/W830A, and TM4/W775A/W830A) were expressed in E. coli. The experiment was performed by incubating 0.2 µM of the purified TM1, TM3, TM4 or the site-directed mutants of TM4 respectively with 10 mg ml^-1 Avicel in citrate buffer (pH 6.0) at 75°C for 13 h.

Figure 4. Time course hydrolysis of cellotetraose (G4) and cellopentaose (G5) by CbCel9A/Cel48A.

The reactions were performed by incubating 0µM (for the control) or 0.2 µM of each enzyme with 2.5 mg ml^-1 of cellotetraose (G4) or cellopentaose (G5) in a citrate buffer (pH 6.0) at 75°C for 24 h. Samples were taken at different time points and analyzed using an HPLC method.

Figure 5. Hydrolysis of Avicel, Filter paper and PASC by CbCel9A/Cel48A, its truncational mutants, and their binary combinations as analyzed by a reducing sugar assay.

The experiments were carried out in citrate buffer (pH 6.0) at 75°C for 16 h by incubating 0.4 µM of each enzyme with 10 mg ml^-1 of Avicel or with 16 discs of Whatman No. 1 filter paper (0.6 cm in diameter) or by incubating 0.2 µM of each enzyme with 2.5 mg ml^-1 of PASC as the substrate. The numbers on top of the columns represent the degrees of synergy calculated as described in Materials and Methods.

Figure 6. Synergistic action of TM1 and TM2 in hydrolysis of Avicel.

The experiments were carried out in a citrate buffer (pH 6.0) at 75°C for 18 h. In (A), the substrate concentration was constant (10 mg ml^-1 of Avicel), and the enzymes are combinations of TM1 and TM2 at different concentrations as shown in the figure. In (B), the concentrations of Avicel are different (0.4, 2, 10 mg ml^-1) and the enzyme concentration (0.5 µM) is constant for TM1 and TM2. The numbers on top of the columns in Panels A and B represent the degrees of synergy calculated as described in Materials and Methods.

Figure 7. Effect of cellulose hydrolysis by four endoglucanases in the presence of either CbCel9A/Cel48A WT or the TM2 mutant.

4EGs: a mixture of four recombinant endoglucanses from C. bescii (CbMan5B/Cel44A-TM2, CbCel9B/Man5A-TM1, CbMan5C/Cel5A-TM2, and CbCel5B-TM1). The reactions were carried out in a citrate buffer (pH 5.5) at 70°C for 16 h, by incubating different concentrations (0, 0.5, 1.0, and 2.0 µM) of WT or TM2 with 5 mg ml^-1 of Avicel, 5 mg ml^-1 of PASC, or 10 mg ml^-1 of Miscanthus, in the presence of the four endoglucanases (4EGs, each at 0.5 µM).