Simple enzyme immobilization inside glass tubes for enzymatic cascade reactions

Abstract

The two enzymes Aspergillus nigerglucose oxidase (GOD) and horseradish peroxidase (HRP) were immobilized with a simple and quick procedure inside micropipette glass tubes with the help of the avidin–biotin system and of a biotinylated second generation polycationic dendronized polymer (de-PG2). De-PG2 strongly adheres to SiO2 surfaces and served as soft organic layer for non-covalently gluing the biotinylated enzyme–avidin complexes to the solid glass surface. The immobilized enzymes remained highly active for a period of several weeks. Furthermore, after connecting a glass tube containing immobilized GOD (first tube) with a glass tube containing immobilized HRP (second tube), this system was applied as a simple flow reactor for the spectrophotometric quantification of D-glucose in aqueous solution via a cascade reaction catalysed by the two immobilized enzymes. Moreover, preliminary measurements showed that the general procedure developed for the immobilization of HRP and GOD inside the glass tubes can also be applied to E. coli β-galactosidase, allowing the spectrophotometric determination of lactosevia a three-enzyme cascade reaction in a system composed of three sequentially connected tubes. The success of the work is based on two factors: (i) on the use of the dendronized polymerde-PG2 which combines properties of polycationic dendrimers as well as polycationic polymers in one and the same molecule, leading to efficient and reproducible binding to SiO2 surfaces; and (ii) on the monitoring of the immobilization on SiO2 with the transmission interferometric adsorption sensor (TInAS) for elaborating the optimal experimental conditions for the immobilization of the enzymes inside the tubes.