The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate.

AIMS: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on beta-galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. METHODS AND RESULTS: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum beta-galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0.06, 0.09, 0.12, 0.18 to 0.24 h(-1). The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum beta-gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum beta-galactosidase activity at 0.24 h(-1) was observed. Following that stage beta-gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. CONCLUSION: The maximum beta-galactosidase activity per unit of cell mass was found to be 341.18 mmol ONP min(-1) g(-1) at a dilution rate of 0.24 h(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in beta-gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.

Configuration of a bioreactor for milk lactose hydrolysis.

Permeabilized microbial cells can be used as a crude enzyme preparation for industrial applications. Immobilization and process recycling can compensate for the low specific activity of this preparation. For biomass immobilization, the common support is alginate beads; however, its low surface area and the low biomass concentration limit the activity. We here describe a biocatalyst consisting of a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen. A ratio of wet permeabilized biomass to alginate of 50:4 (wt/wt) resulted in a paste with maximum immobilized beta-galactosidase activity and maximum gel biomass retention. The biocatalysts retained activity better when stored in milk at 4 degrees C than in 50% glycerol. The unused biocatalysts stored in milk did not lose activity after 50 d. However, repeated use of the same biocatalyst 40 times resulted in almost 50% loss of activity. A bioreactor design with two different conditions of operation were tested for milk lactose hydrolysis using this biocatalyst. The bioreactor was operated at 40 degrees C as packed bed or with recirculation, similar to a continuous stirred tank reactor. The continuous system with recirculation resulted in 82.9% lactose hydrolysis at a residence time of 285.5 min (flow of 2.0 ml/min), indicating the potential of this system for processing low lactose milk, or even in processing other substrates, using an appropriate biocatalyst.