Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2001. / Kluyveromyces sp. have adapted to existence in milk due to the evolution of
permeabilisation and hydrolytic systems that allow the utilisation of lactose, the sugar
most abundant in milk. Lactose hydrolysis, to equimolar units of glucose and galactose,
is facilitated by a glycoside hydrolase, i.e., β-galactosidase (EC 3.2.1.23). The versatility
of this enzyme allows its application in numerous industrial processes, amongst the most
significant of which, is its role in the alleviation of lactose intolerance, one of the most
prevalent digestive ailments, globally. In this study, β-galactosidase production by
Kluyveromyces lactis UOFS y-0939 was initially optimised in shake flask culture with
lactose as the sole carbon source, and thereafter, production was scaled up to batch, fedbatch
and continuous culture. Shake flask studies revealed optimum conditions of 30°C,
pH 7 and a 10% inoculum ratio, to be most favourable for β-galactosidase synthesis,
producing a maximum of 0.35 ± 0.05 U.ml-1 when cell lysates were prepared by
ultrasonication with glass beads. Batch cultivation in 28.2 and 40 g.L-1 lactose revealed
that elevated levels of the carbon source was not inhibitory to β-galactosidase production,
as maximum enzyme activities of 1.58 and 4.08 U.ml-1, respectively, were achieved. Cell
lysates prepared by ultrasonication and homogenisation were compared and homogenised
cell lysates were more than 3.5 fold higher that those prepared by ultrasonication, proving
homogenisation to be the superior method for cell disruption. The lactose feed rate of
4 g.L-1.h-1 in fed-batch culture operated at ± 20.4% DO, appeared to be inhibitory to
biomass production, as indicated by the lower biomass productivity in fed-batch
(0.82 g.L-1.h-1) than batch culture (1.27 g.L-1.h-1). Enzyme titres, however, were favoured
by the low DO levels as a maximum of 8.7 U.ml-1, 5.5 fold more than that obtained in
batch culture, was achieved, and would be expected to increase proportionally with the
biomass. Continuous culture operated at a dilution rate of 0.2 h-1, under strictly aerobic
conditions, revealed these conditions to be inhibitory to the lactose consumption rate,
however, the non-limiting lactose and high DO environment was favourable for
β-galactosidase synthesis, achieving an average of 8 ± 0.9 U.ml-1 in steady state.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:dut/oai:localhost:10321/726 |
| Date | January 2011 |
| Creators | Ram, Elaine C. |
| Contributors | Singh, Suren, Jackson, J., Obiero, G. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 121 p |
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