Translocation of cytoplasmic β-galactosidase across the inner membranes of Kluyveromyces lactis (original) (raw)
Related papers
2009
The translocation behaviour of cytoplasmic β-galactosidase to periplasmic space and through the outside cell wall across the inner membranes of Kluveromyces lactis has been investigated to optimize the cell disruption process by ultrasonication for the production and separation of intracellular target biomolecule i.e. β-galactosidase. The translocation of β-galactosidase in the cells was judged by a concept of location factor (LF), which allows the location of the enzymes to be judged within the cell and has been determined using the relative rates of the enzyme and protein during the cell disruption process. The temperature was found to be useful external stimuli for the translocation of target enzyme (LF could be increased to one or more). The LF values were maximum when cells were subjected to heat stress between 45-50°C for a specified time. The enzyme activity was also found to decrease with an increase in the temperature. Maximum enzyme activity was found to be at 45°C of the ...
Research in microbiology
The beta-galactosidase activity of Kluyveromyces fragilis cells immobilized in a kappa carrageenan gel was studied in a bioreactor functioning under isothermal and non-isothermal conditions. We observed an increase in enzyme activity which was found to be proportional to the intensity of the temperature gradient applied across the biocatalytic membrane, as well as to the average temperature of the bioreactor. The efficiency of such a non-isothermal bioreactor was calculated with respect to the yield of a bioreactor working under comparable isothermal conditions and was evaluated in terms of reduction of processing times in industrial applications. The possibility that enzyme activity in living cells is affected by non-isothermal conditions naturally existing owing to metabolic heat production is also discussed.
New secretory strategies for Kluyveromyces lactis β-galactosidase
Protein Engineering, 2001
We examined several strategies for the secretion of Kluyveromyces lactis β-galactosidase into the culture medium, in order to facilitate the downstream processing and purification of this intracellular enzyme of great industrial interest. We constructed ...
Process Biochemistry, 2004
Whole cell immobilization is one of the major immobilization methods due to cost advantages. Extracellular enzyme producing cells can be immobilized directly, but intracellular enzyme producing cells should be treated first to increase cell permeability. In this work -galactosidase-producing Kluyveromyces lactis (ATCC 8583) cells were used. Since -galactosidase is an intracellular enzyme permeabilized dead cells were immobilized into gelatin using glutaraldehyde as cross-linker. Two chemical and one physical disruption processes were tested and the physical method was determined to be better because of the probable risk of chemical toxicity accompanied with the chemical methods. Thirty percent activity was obtained by immobilized cells relative to free disrupted cells.
Whole cell immobilization is one of the major immobilization methods due to cost advantages. Extracellular enzyme producing cells can be immobilized directly, but intracellular enzyme producing cells should be treated first to increase cell permeability. In this work -galactosidase-producing Kluyveromyces lactis (ATCC 8583) cells were used. Since -galactosidase is an intracellular enzyme permeabilized dead cells were immobilized into gelatin using glutaraldehyde as cross-linker. Two chemical and one physical disruption processes were tested and the physical method was determined to be better because of the probable risk of chemical toxicity accompanied with the chemical methods. Thirty percent activity was obtained by immobilized cells relative to free disrupted cells.
Current Microbiology, 2002
In this paper we report on the effect of different concentrations of lactose and galactose in the production of β-galactosidase by Kluyveromyces marxianus CBS6556. The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the β-galactosidase activity. Therefore, maximum induction of β-galactosidase in K. marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM. Those informations can help to design low cost medium with higher β-galactosidase productivity by K. marxianus cells.
Indian Journal of …, 2008
Whey containing 4.4% (w/v) lactose was inoculated with Kluyveromyces marxianus MTCC 1389 for carrying out studies related to β-galactosidase production. β-galactosidase activity was found to be maximum after 30 h and further incubation resulted in decline in activity. The maximum cell biomass of 2.54 mg mL-1 was observed after 36 h of incubation. Lactose concentration dropped drastically to 0.04 % from 4.40% after 36 h of incubation. Out of the four methods tested for extraction of enzyme, SDS-Chlorofom method was found to be best followed by Toluene-Acetone, sonication and homogenization with glass beads in that order. It could be concluded through this study that SDS-Chloroform is cheap and simple method for enzyme extraction from Kluyveromyces cells, which resulted in higher enzyme activity as compared to the activity observed using the remaining extraction methods. The study could also establish that whey could effectively be utilized for β-galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.
Indian Journal of Microbiology, 2008
Whey containing 4.4% (w/v) lactose was inoculated with Kluyveromyces marxianus MTCC 1389 for carrying out studies related to β-galactosidase production. β-galactosidase activity was found to be maximum after 30 h and further incubation resulted in decline in activity. The maximum cell biomass of 2.54 mg mL-1 was observed after 36 h of incubation. Lactose concentration dropped drastically to 0.04 % from 4.40% after 36 h of incubation. Out of the four methods tested for extraction of enzyme, SDS-Chlorofom method was found to be best followed by Toluene-Acetone, sonication and homogenization with glass beads in that order. It could be concluded through this study that SDS-Chloroform is cheap and simple method for enzyme extraction from Kluyveromyces cells, which resulted in higher enzyme activity as compared to the activity observed using the remaining extraction methods. The study could also establish that whey could effectively be utilized for β-galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.
Electroinduced extraction of β-galactosidase from Kluyveromyces lactis
Applied Microbiology and Biotechnology, 2001
A new methodology for the extraction of β-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation. The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4-4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75-80% without any further treatment. Most of the enzyme was extracted within 8 h after electropulsation. This release was dependent on the growth phase. The specific activity of β-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5-1.7 in comparison with crude extract.