A strategy for the hyperstabilization of a thermophilic esterase from Bacillus stearothermophilus by multipoint covalent attachment of the enzyme through amino groups to glyoxyl agarose gels has been developed. All immobilized esterase derivatives, whether multipoint or one-point linkage, showed improved operational stability under conditions in which the free enzyme was inactivated by interprotein interactions (e.g., aggregation at the isoelectric point) or by interaction with surfaces (adsorption at hydrophobic surfaces, denaturation by liquid-liquid interfaces, etc.). However, only via optimization of the major variables that control the extent of the enzyme-support interaction (contact time between the immobilized enzyme and the activated support, temperature, etc.) was it possible to obtain enzyme derivatives with a significantly increased stability with respect to denaturing reagents. The optimized multipoint covalent immobilization process increased the stability of the esterase preparations by factors of up to 600-fold (depending on the pH) compared to single-point covalent derivatives, with a retention of 65% of the initial activity after immobilization. Apparent stabilization factors of up to 30,000-fold were obtained when comparing the stability of the multipoint immobilized esterase with the soluble enzyme. A clear correlation between thermostabilization and enhanced stability in the presence of organic cosolvents was observed. Multipoint covalently linked esterase derivatives retained more than 70% of the initial activity after 1 week in 50% DMF or DMS at 30 degrees C.