Immobilization of fuculose-1-phosphate aldolase from Escherichia coli to glyoxal-agarose gels by multipoint covalent attachment

Recombinant fuculose 1-phosphate aldolase (FucA) from Escherichia coli has been immobilized by multipoint covalent attachment to glyoxal-agarose gels. Experiments, varying the main parameters that control the immobilization process (surface density of aldehyde groups, temperature, pH), were carried out. An immobilization yield of 80-90% and FucA retained activity on immobilized derivative of 10-20% can be achieved when pH 10, 20 degrees C and 200 mu moles cm(-3) of aldehyde groups was used. The observed activity loss in the immobilization process might be related to the fact that the complex quaternary structure of the enzyme could not be maintained. A short contact-time enzyme support is required to obtain high ratio of active to total immobilized enzyme. A highly loaded derivative of immobilized FucA (65 AU cm(-3) of support) has been prepared to use in aldol condensation reactions. Reactions catalyzed by these aldolases involve the use of non-conventional media because of substrate solubility. For instance, the condensation of dihydroxyacetone phosphate (DHAP) and Z-amino-propanal, Z-(R)-alaninal and Z-(S)-alaninal in highly concentrated water-in-oil emulsions gave synthetic yields of 40, 25 and 29% respectively.