World Journal of Microbiology and Biotechnology 8, 42-44. 0 -Glucosidase synthesis in batch and continuous culture of Saccharomyces cerevisiae. G. Ramesh ...
World Journal of Microbiology and Biotechnology 8, 42-44
0 -Glucosidase synthesis in batch and continuous culture of Saccharomyces
cerevisiae G. Ramesh, M.S. Deshpande, S.B. Maggirwar, M.V. Hegde and V.B. Rale* Glucose prevented maltose utilization in batch culture of Saccharomyces cerevisiae whereas in a mixed carbohydrate-limited system, maltose and glucose were consumed simultaneously. The specific activity of a-glucosidase depended on the dilution rate as well as the proportion of maltose in the mixture. The chemostat provides a way of reaching the low residual concentrations of glucose in the broth that are necessary to release catabolite repression and permit maltose induction of c~-glucosidase. Key words: Saccharomyces cerevisiae, maltose induction, catabolite repression, chemosfaL ~-glucosidase, permease.
Continuous cultivation of microorganisms enables the growth rate, nutritional and physical environment to be separated and defined. Maltase is a good model system for investigating the production of an enzyme under dual control by induction and catabolite repression. The intracellular enzyme, 0~-l-4glucosidase, in Saccharomyces is induced by maltose or some metabolic derivatives of maltose. Its induction is prevented by glucose-mediated catabolite repression (Zimmermann & Eaton 1974). cr from S. cerevisiae is not inactivated when cells are exposed to glucose (20 g/l) or ethanol (Gorts 1969). Ethanol (20g/l) does not affect activity of the permease (Gorts 1969; Van Rijn & Van Wijk 1972). In addition maltose permease that is involved in the transport of maltose is inactivated and repressed by glucose (Siro & Lovgren 1979). Mixtures of glucose and maltose are often encountered during the hydrolysis of starch and other fermentation substrates. Environmental manipulations affecting the growth rate, such as fed-batch and continuous culture were employed to study ~-glucosidase production in chemostat cultures of S. cerevisiae on both single and mixed carbohydrate substrates. The functional M A L loci have been mapped on different chromosomes (Mortimer & G. Ramesh, M.S. Deshpande and V.B. Rale are at the Department of Microbiology, University of Poona, Pune 411 007, India. Fax: 91 0212 333899. M.V. Hegde and S.B. Maggirwar are at the Division of Biochemistry, Department of Chemistry, University of Poona, Pune 411 007, India. *Corresponding author.
~) 1992 Rapid Communications of Oxford Ltd
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WorldJournalof Microbiologyand Biotechnolog!r Vol 8, 1992
Schild 1985; Sollitti & Marmur 1988). From the relative rates of induction in the five locally isolated strains it seems quite likely that they possibly carry different MAL loci.
Materials and Methods Yeast and Growth Saccharomyces cerevisiae CBI1 (adel mallp MALIg mal2p MAL2g MAL6p MAL6g) (Federoff et al. 1982) (received as S. car[sbergensis from J. Marmur, Albert Einstein College of Medicine, New York) was used throughout. It was grown on YEP (2% peptone plus I% yeast extract), YEPG (YEP medium with 1% glucose), YEPM (YEP medium with 1% maltose), YNB (6.5 g of yeast nitrogen base of Difco per litre), YNBB (YNB medium with 2.7 g Na2HPOa/litre and 4.3 g NaHzPO4.HzO/litre) all at pH 5.5 adjusted with 2 M NaOH. YEPG and YEPM were used for inoculum preparation and for maintaining the organism. YNBB was used in frhe batch and continuous fermentation. The batch cultures were with 100 ml medium in 500 ml shake flasks using YNBB medium supplemented with either glucose or maltose. Shake-flask cultures were inoculated with a single loop from the slant. After I8 h of growth a 5% (v/v) inoculum was used for the batch and continuous culture studies. The cultures were incubated at 28~ at 220 rev/min. For continuous culture, 750 mI of fresh medium was held in a 1.5 litre fermenter (Biostat M, Braun, Germany). The agitation was set at 400 rev/min and the temperature at 28~ Medium was pumped into the fermenter at a constant rate to give dilution rates (D) from 0.02 to 0.16 h ~. Glucose was added (17, 67 and 83%, w/v) to the reservoir containing 100% maltose for mixtures of glucose and maltose after fixed intervals of time (12 h; one generation cycle at
G. Ramesh et al. D = 0.08 h-l). Samples were routinely taken from the outlet of the fermenter to measure culture turbidity, residual glucose and maltose concentrations and cr activity. Steady state was assumed when these values converged to constant values. Analytical Procedures Growth was followed turbidometrically at 660 nm. Glucose concentration was measured with a glucose oxidase-peroxidase kit. Maltose was determined by subtracting the glucose from the total reducing sugars measured using the dinitrosalicylic acid method. ~-Glucosidase activity was determined using cells that were de-cryptified with dimethyl sulphoxide (DMSO) (Geeta et al. 1990): 2 mg of cells were added to 4 ml of cold water, centrifuged for 15 rain at 10,000 x g, and the pellet then suspended in 0.5 ml of 40% DMSO, incubated at 30~ for 25 rain and then recentrifuged as before. The ce[ls were washed in 3 ml of 0.I M sodium phosphate buffer, pH 6.8, centrifuged for 10 rain at 10,000 x 8 and the pellet was resuspended in 1 ml of the same buffer, a-Glucosidase activity was determined by measuring the hydrolysis of p-nitrophenyl glucose (2 mM) at 400 nm (Halvorson & Ellias 1958). A unit of enzyme activity is defined as an increase in absorbance of 0.001 rain 1 at 400 nm.
Results Batch Culture 5. cerevisiae C B l l grown on glucose, had a specific activity of a-glucosidase of only i 0 U / g cell, but when grown on maltose (Figure 1) this increased to 950 U / g cell. When the organism was grown on a mixture of maltose and glucose, glucose consumption began first (Figure 2). cr was synthesized while the glucose concentration was still fairly high, and maltose was utilized slowly until glucose was depleted. The specific enzyme activity reached a maximum value of I000 U / g cell. Thus, increasing the ratio of maltose to glucose results in increase in cr activity. In these experiments the maximum specific growth rate on glucose as well as maltose was 0.5 h -~. When maltose was depleted, c~-glucosidase synthesis stopped and activity then declined.
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TIME ( h ) Figure 1. Growth and cr activity in batch culture of S. cerevisiae CB11 on maltose (5g/I). O, c~-Glucosidase; O, cell density; A , maltose).
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Figure 3. Specific activity of 0r and residual maltose concentration in a maltose-limited chemostat of S. cerevisiae at steady state. Maltose concentration in the feed is 3 g/l. 0 , c~-Glucosidase; A , maltose.
World Journal of Microbiology and Biotechnology, Vol 8, 1992
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Figure 4. c~-Glucosidase activity, residual maltose and glucose concentration in a maltose plus glucose limited chemostat of S. cerevisiae CB11 at steady state. Concentrations of glucose and maltose in the feed are 3g/I and 2.5g/I, respectively. O, c
