Regulation of Adsorption Behavior of Carbohydrate-Binding Module Family 1 and Endo-β-1,4-Glucanase onto Crystalline Cellulose

Most fungal cellulases consist of a catalytic domain and a small cellulose-binding domain, which belongs to the CBM1. 1,2) The 2 domains are connected by a fl exible linker peptide. Both domains bind to cellulose, but the affi nity of CBM1 is known to be much higher than that of the catalytic domain. 3) CBM1 may be found at either the N-or C-terminus of the protein. All CBM1 members show high sequence similarity regardless of the type of cellulase. CBM1 is made up of around 30 amino acids with 3 aromatic residues located on a fl at surface, which are essential for hydrophobic interactions with glucose pyranose rings in a single cellulose chain. 4-6) Both the intact enzyme and the catalytic domain show the same activity against soluble substrate; however, the latter shows decreased adsorption to, and activity against, crystalline cellulose. 1,3,7,8) During degradation of crystalline cellulose, CBM1 gives the catalytic domain a chance to approach the substrate, thereby enhancing degradation at liquid-solid interfaces. The adsorption of CBM1 is thus necessary for effi cient attack on insoluble substrates. In the production of biomass ethanol, strong adsorption makes it diffi cult to recover enzymes bound in the solid phase. Therefore, in general, residual components are recovered along with the enzymes, and reused for the following round of saccharifi cation. 9-13) In this case, however, hydrolysates and salts recovered with the enzymes cause inhibition of saccharifi cation. 14,15) Revovery of the enzymes alone would therefore allow degradation to proceed effectively, resulting in a signifi cant reduction in enzyme costs. 16) The interaction between CBM1 and cellulose is diminished by the addition of ethylene glycol, 17) cellodextrins 17) and an increase in temperature. 18) However, the additives have to be removed before the following round of saccharifi cation, while taking care not to denature the enzymes. On the other hand, temperature is easy to regulate, but does not result in aggressive desorption. 18) A simple and effective process, with mild conditions for enzymes and the environment, is required for the desorption of the enzyme. Some chaotropic agents, such as ammonium sulfate, acetate, urea and iodine, are known to affect hydrophobic interactions. 19,20) These have been used in hydrophobic chromatography 21) and purification of DNA fragments, 22) among other uses. DNA is separated easily from cell lysates by increasing the hydrophobic interaction between nucleic acids and silica particles. In the same manner, the interaction between CBM1 and cellulose would also be controllable by changing the concentration of chaotropic agent. In this study, the adsorption behavior of CBM1 was studied using the CBM1-GFP fusion protein, and the possibility of recycling the enzyme was investigated.