Purified glucoamylase (GA) from Fusarium solani was chemically modified by cross-linking with aniline hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 [aniline-coupled glucoamylase-1 (ACG-1)], 7 (ACG-7), and 13 min (ACG-13). The aniline coupling of GA had a profound enhancing effect on temperature, pH optima, and pK(a)'s of active site residues. The specificity constants (K-cat/K-m) of native, ACG-1, ACG-7, and ACG-13 were 136, 244, 262, and 208 at 55 C for starch, respectively. The enthalpy of activation (Delta H*) and free energy of activation (Delta G*) for soluble starch hydrolysis were lower for the chemically modified forms compared to native GA. Proteolysis of ACGs by alpha-chymotrypsin and subtilisin resulted in activation.