Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Abstract

Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzyme's thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three β-galactosidases from Escherichia coli, Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.