Cleavage of cellulose by a CBM33 protein

Abstract

Bacterial proteins categorized as family 33 carbohydrate-binding modules (CBM33) were recently shown to cleave crystalline chitin, using a mechanism that involves hydrolysis and oxidation. We show here that some members of the CBM33 family cleave crystalline cellulose as demonstrated by chromatographic and mass spectrometric analyses of soluble products released from Avicel or filter paper on incubation with CelS2, a CBM33-containing protein from Streptomyces coelicolor A3(2). These enzymes act synergistically with cellulases and may thus become important tools for efficient conversion of lignocellulosic biomass. Fungal proteins classified as glycoside hydrolase family 61 that are known to act synergistically with cellulases are likely to use a similar mechanism.

Figure 1

The action of CelS2. Panels A and B show soluble native (Glc_3–6) and oxidized (Glc_2–6GlcA) cello-oligosaccharides generated by CelS2 activity on Avicel, as detected by HPAEC (A) and MALDI-TOF MS (B). In panel B, only the major peaks in each oligosaccharide cluster are labeled. Panel (C) shows details of the mass spectrum for Glc₅GlcA. The oxidized oligosaccharide is observed as sodium and potassium adducts, and as sodium and potassium adducts of the oligosaccharide sodium/potassium salts, as is commonly seen for carbohydrates containing carboxylic groups. Observed masses (m/z) in the Glc₅GlcA cluster are 1011.22 (Glc₅GlcLA+Na), 1013.25 (Glc₆+Na), 1029.24 (Glc₅GlcA+Na), 1045.21 (Glc₅GlcA+K), 1051.24 (Glc₅GlcA-H+2Na), and 1067.23 (Glc₅GlcA-H+K+Na). GlcLA indicates the lactone form of the oxidized cello-oligosaccharide.

Figure 2

Degradation of high-molecular weight filter paper cellulose by Celluclast (CC), in the presence or absence of CelS2 and reduced glutathione (RG) as shown by the increase of soluble cello-oligosaccharides (Glc and Glc₂; converted to total Glc) over time. Under these conditions, reactions with only CelS2 did not yield detectable amounts of Glc or Glc₂ (not shown). The chitin-active CBM33, CBP21 did not affect CC efficiency (not shown). RG had no effect in reactions with only CC; only one of the two overlapping curves is shown. Data are mean ± SD (N = 3); error bars indicate SD. See the Materials and Methods section for experimental details.