Cellulase-lignin interactions-the role of carbohydrate-binding module and pH in non-productive binding.

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme-lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase-lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM-cellulose interaction were also shown to contribute to lignin-binding.

Determination of the carboxylic acids in acidic and basic process samples by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) with indirect UV detection was used in developing a method for the simultaneous determination of inorganic anions, aliphatic and heterocyclic organic acids in various processed samples. The analytes were determined simultaneously in 10 min using an electrolyte containing 20mM 2,3-pyrazine dicarboxylic acid, 65mM tricine, 2mM BaCl(2), 0.5mM cetyltrimethylammonium bromide, and 2M urea at pH 8.06. Linear plots for the analytes were obtained in the concentration range of 2-150 mg L(-1). Relative standard deviations (RSDs) of peak areas during a 3-day analysis period varied from 5.5% for glycolate to 9.5% for oxalate. RSDs of migration times varied between 0.4% and 1.1%. The detection limit (at S/N 3) was 1 mg L(-1) for all the analytes studied. The proposed method was successfully demonstrated for the determination of carboxylic acids in eight oxygen treated samples of commercial softwood and hardwood kraft lignin and two red wine samples of Pinot Noir grapes. In the kraft lignin samples the concentrations of carboxylic acids correspond to the oxidation time. The acid concentrations of wine varied considerable.