title: Immobilization of thermolysin to polyamide nonwoven materials.
abstract: In the last few years, an increasing number of biotechnological techniques have been applied to the restoration and conservation of works of art, paintings, old maps, and papers or books. Enzymes can solve problems that give restorers difficulties, although for many applications it is not possible to use soluble enzymes; therefore, it is necessary to look for suitable carriers for immobilization. Different methods for covalent immobilization of enzymes to polyamide nonwovens were tested, using thermolysin as an example. Two distinct strategies were pursued: (1). controlled, partial hydrolysis of the polymer and subsequent binding of the enzyme to the released amino and carboxy groups; and (2). attachment of reactive groups directly to the polyamide without disintegrating the polymeric structure (O-alkylation). Different spacers were used for covalent fixation of the enzyme in both cases. The enzyme was fixed to the released amino groups by glutaraldehyde, either with or without a spacer. Either way, active enzyme could be immobilized to the matrix. However, intense treatment caused severe damage to the stability of the nonwoven fabric, and reduced the mechanical strength. Conditions were investigated to conserve the nonwoven fabric structure while obtaining near-maximum immobilized enzyme activity. Immobilization of the enzyme to the released carboxy group after acid hydrolysis was performed using dicyclohexylcarbodiimide. In comparison to the enzyme bound via the amino group, the yield of immobilized enzyme activity was slightly lower when benzidine was taken as spacer and still lower with a 1,6-hexanediamine spacer. O-alkylation performed with dimethylsulfate caused severe damage to the nonwoven fabric structure. Considerably better results were obtained with triethyloxonium tetrafluoroborate. As the spacers 1,6-hexanediamine and adipic acid dihydrazide were used, activation for immobilizing thermolysin was performed with glutaraldehyde, adipimidate, and azide. With the exception of azide, all combinations of spacers and activation reagents gave high yields of immobilized enzyme activity. Thermolysin immobilized by this technique showed a remarkably improved stability with respect to elevated temperature, extreme pH values, and reduced polarity. The nonwoven fabric can be stored for weeks without loss of enzyme activity by washing with distilled water and drying.
authors.affiliation: Physiologisch-chemisches Institut der Universität Tuebingen, Hoppe-Seyler-Strasse 4, D-72076 Tuebingen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Moeschel
authors.forename: Meryem
authors.initials: M
authors.lastname: Nouaimi
authors.forename: Christa
authors.initials: C
authors.lastname: Steinbrenner
authors.forename: Hans
authors.initials: H
authors.lastname: Bisswanger
mesh_terms: D004356:Drug Storage / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009757:Nylons / Q000737:chemistry*; D013784:Textiles; D013820:Thermolysin / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D009757:Nylons; D000431:Ethanol; D013820:Thermolysin