Thesis (MSc)--Stellenbosch University, 2012. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/71958 |
| Date | 12 1900 |
| Creators | Pretorius, Chantelle |
| Contributors | Swart, Pieter, Storbeck, Karl-Heinz, Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 148 pages : illustrations |
| Rights | Stellenbosch University |
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