ß-galactosidase production by Kluyveromyces lactis in batch and continuous culture
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 18.104.22.168). 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.