Abstract
The effect of pH (from 4.0 to 5.0), temperature (T) (from 30 oC to 40 oC) and dissolved oxygen concentration (DO) (from 0.2 to 6.0 mg O2/L) on glucose 6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) and Invertase (EC 3.2.1.26) formation by S. cerevisiae were studied. The best culture conditions for G6PDH and Invertase formation were: 2.55 L culture medium (yeast extract, 3.0 g/L; 5peptone, 5.0 g/L; glucose, 2.0 g/L; sucrose, 15.0 g/L; Na2HPO4.12 H2O, 2.4 g/L; (NH4)2SO4, 5.1 g/L and MgSO4. 7H2O, 0.075 g/L); 0.45 L inoculum (0.70 g dry cell/L); pH = 4.5; T = 35 oC and DO = 4.0 mg/L. G6PDH was highly sensitive to pH, T and DO variation. The increase in G6PDH production was about three times when the DO ranged from 0.2 to 4.0 mg O2/L. Moreover, by shifting pH from 5.0 to 4.5 and temperature from 30 oC to 35 oC, G6PDH formation increased by 57% and 70%, respectively. Invertase activity (IA) of whole cells decreased at least 50% at extremes values of DO (2.0 and 6.0 mg O2/L) and pH (4.0 and 5.0). Furthermore, IA oscillated during the fermentation due to the glucose repression/derepression mechanism
Glucose 6-phosphate dehydrogenase; invertase; Saccharomyces cerevisiae; baker,s yeast
INFLUENCE OF pH, TEMPERATURE AND DISSOLVED OXYGEN CONCENTRATION ON THE PRODUCTION OF GLUCOSE
6-PHOSPHATE DEHYDROGENASE AND INVERTASE BY Saccharomyces cerevisiae
J. Abrahão-Neto, P. Infanti and M. Vitolo * * To whom correspondence should be addressed.
Dept. of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences
University of São Paulo - P.O. Box: 66083, 05389-970 - São Paulo, SP - Brazil
(Received: July 10, 1996; Accepted: October 14, 1996)
Abstract: The effect of pH (from 4.0 to 5.0), temperature (T) (from 30 oC to 40 oC) and dissolved oxygen concentration (DO) (from 0.2 to 6.0 mg O2/L) on glucose 6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) and Invertase (EC 3.2.1.26) formation by S. cerevisiae were studied. The best culture conditions for G6PDH and Invertase formation were: 2.55 L culture medium (yeast extract, 3.0 g/L; 5peptone, 5.0 g/L; glucose, 2.0 g/L; sucrose, 15.0 g/L; Na2HPO4.12 H2O, 2.4 g/L; (NH4)2SO4, 5.1 g/L and MgSO4. 7H2O, 0.075 g/L); 0.45 L inoculum (0.70 g dry cell/L); pH = 4.5; T = 35 oC and DO = 4.0 mg/L. G6PDH was highly sensitive to pH, T and DO variation. The increase in G6PDH production was about three times when the DO ranged from 0.2 to 4.0 mg O2/L. Moreover, by shifting pH from 5.0 to 4.5 and temperature from 30 oC to 35 oC, G6PDH formation increased by 57% and 70%, respectively. Invertase activity (IA) of whole cells decreased at least 50% at extremes values of DO (2.0 and 6.0 mg O2/L) and pH (4.0 and 5.0). Furthermore, IA oscillated during the fermentation due to the glucose repression/derepression mechanism.
Keywords: Glucose 6-phosphate dehydrogenase, invertase, Saccharomyces cerevisiae, baker,s yeast.
INTRODUCTION
Glucose 6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49), which is an enzyme largely found in Saccharomyces cerevisiae, presents great interest as an analytical reagent for the measurement of hexokinase and creatin-kinase activities, ATP and hexose concentration (Bergmeyer, 1984). The use of G6PDH for measuring glucose in the presence of fructose constitutes an important tool, for the detection of illegal sugar addition in the final products of the wine and fruit juice industries, both highly developed in Brazil (Whitaker, 1991). The G6PDH is a specialty product (with a market price of around US$ 70/mg ), being imported the entire amount consumed in Brazil . Thus studies related to the culture conditions of S. cerevisiae for G6PDH production should become an important matter. In this communication the effect of pH, temperature and dissolved oxygen on G6PDH production by S. cerevisiae were studied. In addition, the effect of adopted culture conditions on the invertase activity (IA) (EC 3.2.1.26) of intact cells is also reported.
MATERIALS AND METHODS
Inoculum Preparation
S. cerevisiae (isolated from pressed yeast cake) was maintained in slant tubes containing nutrient-agar (Difco), 23.0 g/L and glucose, 1.0 g/L. The cells were transferred to test tubes containing 2.5 mL of growth medium (GM) (glucose,10.0 g/L; peptone, 5.0 g/L and yeast extract, 3.0 g/L; pH = 4.5) and incubated for 48 h at 33 oC. Following this procedure, one tube was used to inoculate 50 mL of culture medium (CM) in a 250 mL Erlenmeyer flask, followed by incubation at 30 oC for 22h in a rotary shaker (100 min-1) (Super-Hohm). The composition of CM was: yeast extract, 3.0 g/L; peptone, 5.0 g/L; glucose, 2.0 g/L; sucrose, 15.0 g/L; Na2HPO4.12H2O, 2.4 g/L; MgSO4.7H2O, 0.075 g/L and (NH4)2SO4, 5.1 g/L. All media were sterilized at 120 oC for 20 min.
Batch Fermentation
A volume of 0.45 L of inoculum (0.70 g dry cell/L) was poured into a 5L NBS-MF 200 bench fermenter (coupled to a NBS dissolved oxygen controller, DO-81) containing 2.55 L of CM. The tests were carried out under the following conditions: Temperature of 30 oC, 35 oC and 40 oC; pH of 4.0, 4.5 and 5.0 and dissolved oxygen (DO) of 0.2, 2.0, 4.0 and 6.0 mg O2/L. Antifoam (dimethylpolysiloxane, 0.1 mg/L) was added when needed. An aliquot of 20.0 mL of the fermenting medium was taken for analysis every hour. During each test the pH was maintained at the desired value by the automatic addition of 1M NaOH or 0.5M H2SO4.
Measurement of Glucose and Cell Concentrations
Five milliliters of fermenting medium were filtered through a Millipore membrane (pore diameter = 0.45m m). The cell concentration, expressed as g dry cell/L, was measured by drying the cell cake until constant weight (105 oC for 2h). The glucose concentration of the filtrate was measured by the enzymatic method described by Bergmeyer (1984). The generation time (gt) was calculated (Borzani, 1975) for yeast cells harvested during exponential growth, which occurred at the interval of fermentation time between 2h and 5h.
Measurement of Enzymes Activities
Invertase
The invertase activity determinations (always in duplicate) were carried out at 37 oC in a mixture of 1.5 mL of 0.010 M acetate-acetic acid buffer (pH 4.6), 2.5 mL 0.3 M sucrose solution and 0.5 mL of cell suspension. After 3 minutes, the hydrolysis was stopped by adding 1.0 mL of the Somogyi reagent (Somogyi, 1952), quickly followed by immersion in a boiling water bath for 10 minutes. The reducing sugar concentration (RS) was then measured as previously described (Vitolo and Borzani, 1983).
One invertase unit (U) was defined as the amount of enzyme catalyzing the formation of 1 gram of RS per minute at the assay conditions. Specific invertase activity (IA) was expressed as U/g dry cell.
The cell suspension was prepared as follows: 5 mL of the fermenting medium was centrifuged at 3000 x g for 15 minutes and the sediment was washed with distilled water, centrifuged again and suspended in distilled water in order to obtain a known suspension volume.
Glucose 6-Phosphate Dehydrogenase
A volume of 10 mL of fermenting medium was centrifuged at 5000 x g for 10 min, and the sediment was washed with 3.0 mL of TRIS-HCl buffer (50 mM, pH 7.5), centrifuged again and resuspended in the same buffer (3.0 mL) containing 2 mM MgCl2, 0.2 mM EDTA, 2 mM aminocaproic acid, 2 mM DTT (dithiothreitol) and 1mM PMSF (phenylmethylsulfonyl fluoride). The cells were disrupted by submission to a vortex (PHOENIX AT56) in the presence of 3.0 mL glass beads (0.5 mm diameter). Cell debris and glass beads were removed by centrifugation at 5000 x g for 10 min and the supernatant was collected.
The G6PDH activity was measured in the supernatant by the spectrophotometric determination of reduced NADP at 30 oC, according to the method described by Bergmeyer (1984).
One G6PDH unit (U) was defined as the amount of enzyme catalyzing the reduction of 1 micromol of NADP per minute at the assay conditions. Specific G6PDH activity was expressed as U/mg protein.
The coefficient of variation (CV) that affects the measurement of RS concentration is 2.6% (Vitolo and Borzani, 1983), meanwhile the CV for the reduction of NADP was less than 5% (a mean of five determinations was taken into account).
Measurement of Protein
The concentration of protein was determined by Bradford,s method (1976) using bovine serum albumin as a standard.
RESULTS AND DISCUSSION
Dissolved oxygen (DO) affects the specific G6PDH activity of S. cerevisiae in the DO range from 0.2 to 6.0 mg O2/L (Table 1). The highest G6PDH activity (0.510 U/mg protein) was attained at a DO value of 4.0 mg O2/L. Nevertheless, the activity at the DO values of 0.2 and 6.0 mg O2/L diminished 50% at least, due to the reduced growth rate under these oxygen concentrations. The gt (generation time) at 0.2 and 6.0 mg O2/L were, respectively, 40% and 12%
higher than at 4.0 mg O2/L, undoubtedly the best DO condition regarding G6PDH and invertase specific activities (Table 1). The connection between growth rate and G6PDH activity occurs via pentoses (Voet and Voet, 1994), a pathway from which the cell synthesizes ribose-6P, a key constituent of nucleotides and nucleic acids. Since the demand for nucleic acids during fast growth is high, the specific G6PDH activity will be also.
Since sucrose (a non-fermentable sugar) was the main carbon source present in the culture medium, the availability of glucose for the yeast depends on sucrose hydrolysis by invertase, an enzyme located at the yeast cell wall (Trumbly, 1992). The invertase activity was also taken into account, due to its use in the inverted syrup production and in biosensors (Layman, 1986; Barlikova et al., 1991).
The highest specific invertase activity (IA) (2.70 U/g dry cell) and the lowest gt (1.39 h) occurred at DO 4.0 mg O2/L (Table 1). Moreover, IA decreased around 55% at a DO value of 2.0 or 6.0 mg O2/L and the average variation of gt was about 9% as DO was changed from 2.0 to 6.0 mg O2/L. Nevertheless, such gt variation is rather small to justify such a sharp decrease in IA, although some correlation between IA and cell growth would be expected, since invertase is attached to the cell wall.
* The generation time (gt) was calculated according to Borzani (1975).
**X = Final cell concentration, expressed as g dry cell/L.
*X = Final cell concentration, expressed as g dry cell/L.
Table 2 shows that at 35 oC the highest IA (2.70 U/g dry cell) was attained at pH 4.5. However, the specific invertase activity was about 50% and 82% lower at pH 4.0 and 5.0, respectively, at the same temperature. At first, the effect of pH on IA could have occurred either intracellularly or extracellularly. As the pH was varied from 4.0 to 5.0, a change in the intracellular pH would be unlikely because S. cerevisiae has an effective internal buffering capability (Weitzel et al., 1987). Probably, the pH affected IA by altering the tertiary and/or quaternary structures of the protein, leading to an inadequate insertion of the macromolecule in the cell wall during budding (Vitolo et al., 1995; Reddy et al., 1990).
The reducing sugar concentration (RS) in the medium during yeast growth depends on the DO (Figure 1). After 2.0 h of cultivation at DO 4.0 mg O2/L, the maximum RS concentration in the culture medium was 7.3 g/L, at least 20% lower than those observed at DO 0.2 mg O2/L (Figure 1). As can be seen from Table 1 the highest final activities of G6PDH (0.51 U/mg prot) and invertase (2.70 U/g dry cell) occurred at DO 4.0 mg O2/L. Furthermore under a constant DO the invertase activity oscillated markedly, reaching a minimum value always around 2.5 h after the start of fermentation (Table 1). This may be due to a glucose repression / derepression mechanism, which can be circumvented by adding sucrose to the culture medium step by step (Vitolo et al., 1995; Reddy et al., 1990). Moreover, The G6PDH activity was also affected by RS concentration in the culture medium (Table 1 and Figure 1).
The maximum in the RS concentration at 2 h of cultivation could be the result of sucrose hydrolysis by the invertase present in the inoculum cells. Since cell concentration is low at this phase of growth, glucose and fructose may be consumed at a lower rate than they are produced, resulting in the increase of RS up to 2 h. The low RS accumulation observed in the test carried out at DO 4.0 mg O2/L leads to the assumption that hexose uptake was more efficient than at other aeration rates employed. According to Abrahão-Neto et al. (1996), this result should be related to the increase of hexokinase activity.
Figure 1: RS concentration versus time for batch cultures of S.cerevisiae carried out at pH 4.5, 35 oC and with the following DO (mg O2/L) values: 0.2 (n); 2.0 (l); 4.0 (¡) and 6.0 (o).
Figure 2: RS concentration versus time for batch cultures of S. cerevisiae carried out at 35 oC, DO = 4.0 mg O2/L and at the following pH values: 4.5 (l) and 5.0 (n).
Undoubtedly, RS uptake and invertase and G6PDH activities are interconnected events, in which pH has a significant role (Table 2, Figure 2). Setting the DO and temperature at 4.0 mg O2/L and 35 oC, respectively, the low RS accumulation and the high G6PDH and invertase activities were observed at pH 4.5. An increase of 0.5 in the pH caused at least a 50% decrease in the activity of both enzymes. Moreover, Table 2 shows that the invertase was more strongly influenced by temperature than the G6PDH. Tests are being carried out to explain this result.
From Tables 1 and 2 it is clear that invertase and G6PDH activities depended markedly on the yeast growing (expressed as the generation time) instead of the final cell concentration. Moreover, the highest activity occurred under well determined growth conditions and similarly for both enzymes, in spite of the invertase and G6PDH are located at the cell wall and in the cytoplasm, respectively.
The data presented lead to setting the main parameters for invertase and G6PDH production at 35 oC, pH 4.5 and DO 4.0 mg O2/L. On one hand, having a well-defined value for the dissolved oxygen concentration, makes KLa calculation feasible, which in turn, allows to be the process adequately scaled up (Dunn et al., 1992). On the other hand, knowing how to satisfactorily explain an experimental observation like invertase oscillation, allows the overall process to be carried out efficiently.
REFERENCES
Abrahão-Neto, J.; Infanti, P. and Vitolo, M. Hexokinase production from S. cerevisiae: culture conditions. Appl. Biochem. Biotechn., Golden, 57/58: 407-412 (1996).
Barlikova, A.; Svorc, J. and Miertus, S. Hybrid biosensor for the determination of sucrose. Anal. Chim. Acta, Baltimore, 247: 83-87 (1991).
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Borzani, W., Cinética de processos fermentativos. In: Engenharia Bioquímica, Edgard Blucher Ltda, São Paulo (1975).
Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., Baltimore, 72: 248-254 (1976).
Dunn, I.J.; Heinzle, E.; Ingham, J. and Prenosil, J.B., Biological reaction engineering. Weinhein, VHC, p.113 (1992).
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Patkar, A. and Seo, J.H., Fermentation kinetics of recombinant yeast in batch and fed-batch cultures. Biotech. Bioeng., New York, 40: 103-109 (1992).
Reddy, A.V.; Marcoll, R. and Maley, F., Effect of oligossaccharides and chloride on the oligomeric structures of external, internal and deglycosylated invertase. Biochemistry, Washington, 29 (10): 2482-2487 (1990).
Somogyi, M., Notes on sugar determination. J. Biol. Chem., Baltimore, 195: 19-23 (1952).
Trumbly, R.J., Glucose repression in the yeast S. cerevisiae. Mol. Microbiol., London, 6:15-21 (1992).
Vitolo, M. and Borzani, W., Measurement of invertase activity of cells of S. cerevisiae. Analyt. Biochem., Baltimore, 130: 469-470 (1983).
Vitolo, M.; Duranti, M.A. and Pellegrim, M.B., Effect of pH, aeration and sucrose feeding on invertase activity of intact S. cerevisiae cells grown in sugarcane blackstrap molasses. J. Ind. Microbiol, Basingstoke, 15: 75-79 (1995).
Voet, D. and Voet, J.G., Biochemistry, 2nd ed., John Willey & Sons, Inc., New York (1994).
Weitzel, G.; Pilatus, U. and Rensing, L., The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatment in yeast. Exper. Cell Res. 170: 64-79 (1987).
Whitaker, J.R., Enzymes in analytical chemistry. In: Fox, P.F., Food Enzymology, Elsevier Applied Science, New York, pp. 287-308 (1991).
- Abrahăo-Neto, J.; Infanti, P. and Vitolo, M. Hexokinase production from S. cerevisiae: culture conditions. Appl. Biochem. Biotechn., Golden, 57/58: 407-412 (1996).
- Bergmeyer, H.U., Methods of Enzymatic Analysis, 3rd ed., Verlag Chemie, Weinheim (1984).
- Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., Baltimore, 72: 248-254 (1976).
- Layman, P.L., Industrial enzymes: battling to remain specialities. Chem. Eng. News, New York, 15: 11-14 (1986).
- Patkar, A. and Seo, J.H., Fermentation kinetics of recombinant yeast in batch and fed-batch cultures. Biotech. Bioeng., New York, 40: 103-109 (1992).
- Somogyi, M., Notes on sugar determination. J. Biol. Chem., Baltimore, 195: 19-23 (1952).
- Trumbly, R.J., Glucose repression in the yeast S. cerevisiae Mol. Microbiol., London, 6:15-21 (1992).
- Weitzel, G.; Pilatus, U. and Rensing, L., The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatment in yeast. Exper. Cell Res. 170: 64-79 (1987).
- Whitaker, J.R., Enzymes in analytical chemistry. In: Fox, P.F., Food Enzymology, Elsevier Applied Science, New York, pp. 287-308 (1991).
Publication Dates
-
Publication in this collection
06 Oct 1998 -
Date of issue
Mar 1997
History
-
Accepted
14 Oct 1996 -
Received
10 July 1996
