Covalent immobilisation of β-Galactosidase from Escherichia coli to commercially available magnetic nanoparticles for the removal of lactose from milk

[摘要] ENGLISH ABSTRACT:ß-Galactosidase of Escherichia coli is the equivalent of lactase in humans andhas the ability to bind and hydrolyse lactose. Lactase de ciency is a commonphenomenon present in almost 70% of the world's population. This hasresulted in greater than before demands on the food processing industry todevelop a method that will allow for the hydrolysis of the disaccharide lactosein milk but will also allow for the removal of the remaining active enzyme.In this thesis, a new method, that is bio-speci c and well characterizedfor the removal of lactose from a lactose containing solution, is described.The E537D mutated version ofß-Galactosidase, which has a much loweractivity compared to the wildtype and is able to bio-speci cally bind lactosefor longer periods, was covalently immobilised to commercially availablemagnetic nanoparticles (fl uidMAG-Amine) via two coupling strategies. Glutaraldehydeis a cross-linking agent that reacts with amine groups, while N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) is a couplingagent that activates carboxylic groups. These agents are widely used forthe coupling of biomolecules to solid supports.The covalently coupledfluidMAG-E537Dß-Galactosidase particles werecharacterized regarding retained enzymatic activity and ability to bind andphysically remove lactose from a lactose containing solution by applying anexternal magneticeld, after lactose binding, to the enzyme-particle complexin solution.Each component aimed at yielding this functionally immobilised enzymecomplex was studied and optimized to contribute to the development of thisnovel technique, which is a ordable and simple, for the removal of lactose fromsolution for the ultimate production of lactose free milk.Results indicated the glutaraldehyde method ofß-Gal cross-linking tofluidMAG-Amineto be the preferred strategy since it allowed an increased carrier capacityof protein to the particles. The glutaraldehyde cross-linked protein also exhibiteda two-fold higher activity than the EDC coupled protein. Furthermore,the glutaraldehyde cross-linkedfluidMAG-E537Dß-Gal was able to physicallyremove 34 % of the lactose from a 0.2 nmol/L lactose in solution. This, therefore,con rmed the potential use of this novel technique in the food processingindustry.

[发布机构] Stellenbosch University