Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis

Abstract

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 10(4) and 6.4 x 10(4) 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 beta-1,3-1,4-mixed glucan such as barley beta-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 beta-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.

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.

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.

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.

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.

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.

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 β-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).

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.