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

Abstract

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.