Partial and complete alteration of surface charges of carboxymethylcellulase by chemical modification: Thermostabilization in water-miscible organic solvent

Carboxymethylcellulase was purified from Aspergillus niger to homogeneity level. The native and subunit molecular weights were found to be 42 and 45 kDa, respectively. The purified CMCase was modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of dimethylamine hydrochloride (DIMAM) and ethylenediamine dihydrochloride (EDAM) as nucleophiles. The amino groups of DIMAM were further modified by acetic anhydride for the complete elimination of surface charges (double modification, DM). The specificity constants (V(max)/K(m)) of EDAM (1.1), DIMAM (1.2) and DM-A (1.0) were increased as compared with native enzyme (0.16). Partial neutralization of surface negative charges (DIMAM), reversal of surface negative charges (EDAM) and complete neutralization of negative plus positive charges (DM-A), of CMCase had a drastic effect on the thermostability determined in the aqueous buffer, pH 5.2. Gibbs activation free energies of denaturation (ΔG*) of native, EDAM, DIMAM and DM-A at 80°C were 110, 107, 102 and 103 kJ mol^-1, respectively, whereas enthalpy of denaturation (ΔH*) of native, EDAM, DIMAM and DM-A at 80°C were 143, 144, 213 and 197 kJ mol^-1, respectively. The entropies of denaturation (ΔS*) of native, EDAM, DIMAM and DM-A at 80°C were 91, 105, 316 and 265 Jmol^-1 K^-1, respectively, indicating highly disordered conformations of all the transition states of modified CMCases. On the other hand, the thermostability of doubly modified CMCase in 50% (v/v) aqueous dioxan (DM-D) was dramatically increased with concomitant activation with increasing temperatures up to 95°C as compared with the native CMCase under similar conditions. The analysis of thermodynamic parameters revealed that ΔS* of denaturation became negative implying a highly ordered transition state of DM-D in the presence of a solvent of higher hydrophobicity. We wish to propose that at least monomeric enzymes could be made significantly more thermostable in water-miscible organic solvents by totally converting the charged surface groups to non-polar ones by double modification. Copyright (C) 1999 Elsevier Science Inc. All rights reserved.