Lactose hydrolysis by immobilized whole cells of K. lactis CBS 2357 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Bioprocess Engineering at Massey University
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The application of immobilized yeast for lactose hydrolysis was investigated. The enzyme stability was tested as a function of pretreatment. The stability of K. lactis CBS 2357 cells after treatment with glutaraldehyde (GA) and the β-galactosidase activity of whole cells after immobilization in alginate bead and corn particles were studied. Permeabilization using ethanol and chloroform (10% and 2%, respectively) at 37 °C and 120 rpm for 5 min, followed by stabilization with 10 mM glutaraldehyde at 30 °C for 1 hour with gently shaking deactivated 2.5% of the initial whole cells β-galactosidase activity, tested with the ONPG method. The glutaraldehyde treatment could significantly maintain β-galactosidase activity in phosphate buffer pH 6.5 containing 0.1 mM MnCl2. Manganese and potassium ions in the Mn-Buffer were found to be essential to enhance the activity. The biomass activity of GA stabilized cells in Mn-Buffer can be maintained above 70% during 72 hours of incubation at 30 °C. An increase of incubation temperature from 30 to 37 °C deactivated 10% of biomass activity after 72 hours. Direct stabilization of alginate biocatalyst with glutaraldehyde caused a significant reduction of β-galactosidase activity with the resulting deactivation depending on glutaraldehyde and alginate concentrations. When 40 g of biocatalyst containing 2x109 cells/g alginate was stabilized in 100 ml of 0 to 4 mM glutaraldehyde, the optimum range of glutaraldehyde concentration was between 0.5 to 1.0 mM. When this concentration range was applied to stabilize 2%- to 3%-alginate biocatalyst, the average biocatalyst activity remained within 56-74% of the initial activity. It was shown that the adsorption of K. lactis on corn particles through a "double liquid cultivation stage" followed by permeabilization of biocatalyst gave a higher activity. The activity obtained was 0.84 μmol lactose hydrolyzed /min/g biocatalyst under the conditions tested. This activity was about 5 times higher than the case without permeabilization and about 2 times higher than that of the permeabilized biocatalyst prepared with a "single liquid cultivation stage". When tested in the packed-bed reactor, during the initial stages the degree of hydrolysis (d.h.) was 45% within the operational conditions tested. Free enzyme was detected during the first 5 hours of operation, especially when non-stabilized corn biocatalyst was used. After 5 hours, free enzyme was no longer detected in the reactor outlet, suggesting that direct adsorption might have rendered good cell confinement inside the corn particles.