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

Pretorius, Chantelle (2012-12)

Thesis (MSc)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: ß-Galactosidase of Escherichia coli is the equivalent of lactase in humans and has the ability to bind and hydrolyse lactose. Lactase de ciency is a common phenomenon present in almost 70% of the world's population. This has resulted in greater than before demands on the food processing industry to develop a method that will allow for the hydrolysis of the disaccharide lactose in 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 characterized for the removal of lactose from a lactose containing solution, is described. The E537D mutated version of ß-Galactosidase, which has a much lower activity compared to the wildtype and is able to bio-speci cally bind lactose for longer periods, was covalently immobilised to commercially available magnetic nanoparticles (fl uidMAG-Amine) via two coupling strategies. Glutaraldehyde is a cross-linking agent that reacts with amine groups, while N- (3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) is a coupling agent that activates carboxylic groups. These agents are widely used for the coupling of biomolecules to solid supports. The covalently coupled fluidMAG-E537D ß-Galactosidase particles were characterized regarding retained enzymatic activity and ability to bind and physically remove lactose from a lactose containing solution by applying an external magnetic eld, after lactose binding, to the enzyme-particle complex in solution. Each component aimed at yielding this functionally immobilised enzyme complex was studied and optimized to contribute to the development of this novel technique, which is a ordable and simple, for the removal of lactose from solution for the ultimate production of lactose free milk. Results indicated the glutaraldehyde method of ß-Gal cross-linking to fluidMAG-Amine to be the preferred strategy since it allowed an increased carrier capacity of protein to the particles. The glutaraldehyde cross-linked protein also exhibited a two-fold higher activity than the EDC coupled protein. Furthermore, the glutaraldehyde cross-linked fluidMAG-E537D ß-Gal was able to physically remove 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 processing industry.

AFRIKAANSE OPSOMMING: ß-Galaktosidase vanaf Escherichia coli is dieselfde as laktase in mense en beskik oor die vermoë om laktose te bind en te hidroliseer. 'n Gebrek aan laktase kom algemeen voor en ongeveer 70 % van die wêreldbevolking ly hieraan. Laasgenoemde het daartoe gelei dat daar meer druk as vantevore op die voedselproduksie industrie is om 'n metode te ontwikkel waarmee die hidrolise van die disakkaried laktose in melk moontlik sal wees asook die verwydering van die oorblywende aktiewe ensiem. In hierdie tesis word 'n nuwe metode beskryf wat biospesi ek en goed gekarakteriseer is vir die verwydering van laktose vanuit 'n laktose bevattende oplossing. Die E537D gemuteerde weergawe van ß-Galaktosidase, wat beskik oor 'n baie laer aktiwiteit as die wildetipe asook die vermoë om laktose biospesi ek vir langer periodes te bind, is kovalent geïmmobiliseer op kommersieel beskikbare magnetiese nanopartikels (fluidMAG-Amine) via twee koppelingsstrategieë. Glutaraldehied is 'n kruisbindingsagent wat met amino groepe reageer, terwyl EDC 'n koppelingsagent is wat karboksie groepe aktiveer. Hierdie agente word algemeen gebruik vir die binding van biomolekules aan soliede matrikse. Die kovalent gekoppelde fluidMAG-E537D ß-Galaktosidase partikels is gekarakteriseer met betrekking tot behoue ensimatiese aktiwiteit en vermoë om laktose te bind en sies te verwyder vanuit 'n oplossing wat laktose bevat deur 'n eksterne magneetveld op die ensiem-partikel kompleks in oplossing toe te pas, nadat die binding van laktose plaasgevind het. Elke komponent van hierdie funksioneel geïmmobiliseerde ensiemkomplekse is ondersoek en geoptimaliseer met die doel om by te dra tot die ontwikkeling van 'n nuwe tegniek wat bekostigbaar en eenvoudig is vir die verwydering van laktose vanuit 'n oplossing vir die uiteindelike gebruik in die produksie van laktose-vrye melk. Resultate het getoon dat die glutaraldehied metode van ß-Gal kruisbinding op fluidMAG-Amine verkies word aangesien dit 'n verhoogde draerkapasiteit van proteïene op die partikels moontlik maak. Die glutaraldehied gekoppelde proteïene beskik ook oor twee keer meer aktiwiteit as die EDC gekoppelde proteïene. Die glutaraldehied gekoppelde fluidMAG-E537D ß -Gal kon 34 % van die laktose teenwoordig in 'n 0.2 nmol/L laktose oplossing sies verwyder. Hierdie het dus die potensiële gebruik van hierdie nuwe metode in die voedselproduksie industrie bevestig.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/71958
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