Construction of a flocculent Saccharomyces cerevisiae fermenting lactose (original) (raw)
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Aspergillus niger β-galactosidase production by yeast in a continuous high cell density reactor
Process Biochemistry, 2005
The continuous production of extracellular heterologous -galactosidase by a recombinant flocculating Saccharomyces cerevisiae, expressing the lacA gene (coding for -galactosidase) of Aspergillus niger was investigated. A continuous operation was run in a 6.5 l airlift bioreactor with a concentric draft tube using lactose as substrate. Data on the operation with semi-synthetic medium with 50 and 100 g/l initial lactose concentrations are presented. The best result for -galactosidase productivity-6.2 × 10 5 U/1 h-was obtained for a system operating at 0.24 h −1 dilution rate and for a lactose concentration in the feed of 50 g/l. This value represents a 11-fold increase in -galactosidase productivity when compared to batch culture. Together with extracellular -galactosidase production an ethanol productivity of 9 g/1 h was obtained for the bioreactor fed with 50 g/l initial lactose concentration at 0.45 h −1 dilution rate. In addition to -galactosidase and ethanol production, this system allowed for complete lactose metabolism. The feasibility and advantages of using continuous high-cell-density systems operating with flocculent yeast cells for extracellular protein production is clearly shown.
Construction of a flocculent brewer's yeast strain secreting Aspergillus niger β-galactosidase
Applied Microbiology and Biotechnology, 2000
One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the¯occulating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of¯occulent and non-¯occulent brewer's yeast strains secreting b-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular b-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by¯occulent cells.
Construction of a flocculent brewer's yeast strain secreting Aspergillus niger β-galactosidase
Applied Microbiology and Biotechnology, 2000
One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the¯occulating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of¯occulent and non-¯occulent brewer's yeast strains secreting b-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular b-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by¯occulent cells.
Food Science and Technology, 2019
The cheese whey shows an organic nutrient charge that can be used to obtain metabolites of interest by biotechnology of microorganisms. Thus, fermentative processes for enzyme production, in particular beta-galactosidase becomes feasible. The enzyme plays an important role in the biotech food industry to obtain milk and dairy products with low lactose content for consumption by intolerant individuals. The objective of this work was to determine the enzyme activity of the concentrated beta-galactosidase (CBG) and the permeabilized cells (PC) both obtained from Saccharomyces fragilis OZ 275. The enzyme beta-galactosidase obtained from the fermentation of Saccharomyces fragilis OZ 275 in cheese whey was used to determine the optimal conditions for the hydrolysis of lactose solution at 1% (w/v). Response Surface Methodology (RSM) by Box-Behnken Design (BBD) was employed to determine beta-galactosidase activity for such factors pH, temperature and enzyme concentration suitable for the lactose hydrolysis. Based on the statistical analysis, the optimum operational conditions for maximizing lactose hydrolysis thus optimizing the enzyme activity for CBG were, temperature 30 °C, pH 6.0 and enzyme concentration 3% (v/v) and for PC was temperature 44 °C, pH 7.0 and enzyme concentration 4% (v/v).
Biocatalysis and Agricultural Biotechnology, 2019
Microfiltrated cheese whey permeate (CWP) was used as substrate by Saccharomyces fragilis IZ 275 yeast for the production of beta-galactosidase. Yeast growth kinetics was evaluated by lactose consumption, biomass production and, enzyme production. Lactose was almost depleted within 12 h of cultivation, only 11% of the substrate was left in the culture medium, indicating an average intake rate of 2.63 g.L 1 .h 1 (lactose consumed/ fermentation time). Biomass production increased with increasing cultivation time, reaching 11.06 mg.mL 1 and a specific growth rate (μ) of 0.317.h 1 in 20 h of cultivation. The maximum yield of volumetric and specific betagalactosidase activity was 14.28 U.mL 1 and 0.039 U.mg 1 , respectively, and maximum lactose hydrolysis was obtained at 24 h of cultivation. This work evaluated the importance of the microfiltration process of cheese whey in Saccharomyces fragilis IZ 275 fermentation for beta-galactosidase production.
Metabolic engineering for direct lactose utilization by Saccharomyces cerevisiae
2002
A recombinant strain of Saccharomyces cerevisiae, secreting β-galactosidase from Kluyveromyces lactis, grew efficiently with more than 60 g lactose l −1. The growth rate (0.23 h −1) in a cheese-whey medium was close to the highest reported hitherto for other recombinant S. cerevisiae strains that express intracellular β-galactosidase and lactose-permease genes. The conditions for growth and β-galactosidase secretion in this medium were optimized in a series of factorial experiments. Best results were obtained at 23 • C for 72 h. Since the recombinant strain produced less than 3% ethanol from the lactose, it was also assayed for the production of fructose 1,6-bisphosphate from cheese whey, and 0.06 g l −1 h −1 were obtained.
Journal of Biotechnology, 2004
Secretion of the heterologous Kluyveromyces lactis -galactosidase into culture medium by several Saccharomyces cerevisiae osmotic-remedial thermosensitive-autolytic mutants was assayed and proved that new metabolic abilities were conferred since the constructed strains were able to grow in lactose-containing media. Cell growth became independent of a lactose-uptake mechanism. Higher levels of extra-cellular and intra-cellular -galactosidase production, lactose consumption and growth were obtained with the LHDP1 strain, showing a thermosensitive-autolytic phenotype as well as being peptidase-defective. The recombinant strain LHDP1 presented the highest -galactosidase yields from biomass and the lowest ethanol levels from lactose. This strain is effective for the heterologous production and release of K. lactis -galactosidase into the extra-cellular medium after osmotic shock.