Abstract: The effect of different mineral nutrients was studied on the production of extracellular invertases by Saccharomyces cerevisiae GCB-K5. Nutritional studies were carried out in 250ml Erlenmeyer flasks by submerged fermentation. The culture medium (pH 6.0) was incubated for 48 hours at 30oC. The optimum levels of peptone and dipotassium hydrogen phosphate were found to be 4.0 and 0.2 g/l, respectively. Maximum invertase activity was found to be 12.68 U/ml. The amount of sugar consumed and dry cell mass were 21.08 and 5.88g/l, respectively.
Introduction
Invertases are special kind of enzymes that catalyze the hydrolysis of sucrose into glucose and fructose. Invertase finds uses in the production of confectionery with liquid or soft centers, fermentation of cane molasses into ethanol, in calf feed preparation and also in manufacture of inverted sugars as food for honeybees. (Sanchez et al., 2001). The organism showing greatest ability to secrete invertase is yeast (Moreno et al., 1979; Silveira et al., 2000). Different substrates can be used in submerged fermentation for the production of invertase (Gomez et al., 2000). Different organic nitrogen sources and their concentrations have a major effect on the ability of yeast to synthesize the invertase. There exists a specific physiological response of sucrose metabolism to the presence of nitrogen source (Silveira et al., 1996). The present study describes the relation between different nutrient sources and invertase secretion in submerged culture.
Materials and Methods
Organism and inoculum preparation: The project was performed in Biotechnology Laboratories, Govt. College, Lahore. in six months duration. Yeast strain Saccharomyces cerevisiae GCB-K5 was used for the production of invertase. The culture was maintained on sucrose (2%), yeast extract (0.3%), peptone (0.5%) and agar (2.0%) medium and stored at 4°C in the refrigerator. Inoculum was
Shafiq et al.: Invertase producation by Saccharomyces cerevisiae prepared from 2-3 days old slant culture of Saccharomyces cerevisiae GCB-K5. Ten ml of sterilized distilled water was added to the slant having optimal growth. Cells were then scratched with sterilized inoculating needle and the tube was shaken gently. The number of cells (1.0 x 107 cells/ml) was counted with the help of Hemocytometer slide.
Fermentation conditions: Production of yeast invertase was studied by shake flask technique using 250 ml Erlenmeyer flasks. Twenty-five ml of fermentation medium (sucrose 30.0 g/l, peptone 5.0 g/l and yeast extract 3.0 g/l) was transferred to each cotton wool plugged Erlenmeyer flask. The flasks were sterilized in an autoclave at 15 lbs/inch2 pressure (121°C) for 15 minutes and cooled at room temperature. The fermentation media was varied in composition according to parameters under study. Different nitrogen and phosphate sources were tested for their effect on the production of invertase. Nitrogen sources and phosphate sources were added at the rate of 0.5 and 0.015g/100 ml fermentation medium, respectively. Enhanced production of invertase in response to applied supplements was recorded. One ml of inoculum was aseptically transferred to each flask. Flasks were then rotated in a rotary incubator shaker (Model: GLSC 051.HR.196-11) at 30°C for 48 hours. The agitation rate was kept at 200 rev/min. After 48 hours, fermented broth was subjected for the estimation of invertase produced.
Analytical methods: “One invertase unit is defined as the amount of enzyme, which releases one milligram of inverted sugar in 5 minutes at 20°C, at pH 4.5”. Invertase activity was determined according to the method of Sumner and Howell (1935). Dry cell mass of yeast was determined by centrifugation of fermented broth at 5000 rev/min, using weighed centrifuge tubes. The tubes were oven dried at 105°C for one hour. Sugar was estimated colorimetrically by DNS method (Tasun et al., 1970). Transmittance was measured at 546nm on photoelectric colorimeter (Model: AE-11M Erma, Japan).
Results and Discussion
Effect of different nitrogen sources: Nitrogen constituent has a profound effect on the production of invertase in culture medium because there exists a strong correlation between nitrogen equilibrium and productivity of yeast cells (Rouwenhorst et al., 1991; Neto et al., 1996). The data in Table 1 shows the effect of different nitrogen sources (Nutrient broth, peptone, yeast extract) on invertase production by Saccharomyces cerevisiae GCB-K5. Among all the nitrogen sources tested, peptone gave maximum production of invertase (8.75 U/ml). It enhanced the growth of yeast and in turn increased the invertase production. The dry cell mass and sugar consumption were 5.32 and 20.54g/l, respectively.
Effect of different concentrations of peptone: The effect of different concentrations of peptone (0.30-0.60 g/100 ml) on the production of invertase was studied (Table 2). Maximum production of invertase (9.13 U/ml) was achieved at 0.40 g/100 ml level of peptone. Dry cell mass and sugar consumption were 5.61 and 20.60 g/l, respectively. Maximum production of invertase at optimized concentration of peptone might be due to favourable nutrient supply for yeast growth (Silveira et al., 2000). At low concentration, less invertase production might be due to lower supply of nitrogen, which was insufficient for yeast growth. Dworschack and Wickerham (1960) used 0.5 g/100 ml peptone in culture medium for maximum invertase production.
Effect of different phosphate sources: The data in Table 3 shows the effect of different phosphate sources like NaH2PO4, (NH )4 H2PO4 , KH2PO4 , KH2PO4 and Na2HPO4 on invertase production by Saccharomyces cerevisiae GCB-K5. Among all the phosphate sources tested, K2HPO4 gave maximum production of invertase i.e., 12.04 U/ml. Dry cell mass and sugar consumption were 5.84 and 20.42 g/l, respectively. Final pH of medium was 5.8. Maximum production of invertase by using K2HPO4 as phosphate source might be due to fact that phosphate was readily available to yeast. Similar type of work was reported by Gomez et al. (2000).
Effect of different concentrations of K2HPO4: The data in Table 4 shows the effect of different concentrations (0.010-0.030 g/100 ml) of dipotassium hydrogen phosphate (K2HPO4) on the production of invertase by Saccharomyces cerevisiae GCB-K5. Maximum production of invertase (12.68 U/ml) was achieved when 0.020 g/100 ml K2HPO4 was used. The dry cell mass and sugar consumption were 5.88 and 21.08 g/l, respectively. Any increase or decrease in the phosphate concentration beyond optimum, greatly reduced the invertase units due to overgrowth or improper growth of yeast. At high concentrations, phosphate reduces cell mass and increases the alkalinity of medium, which is highly unfavourable for yeast growth and enzyme stability (Underkofler and Hickey, 1954).
It was estimated that the strain of Saccharomyces cerevisiae GCB-K5 gave 12.68 units of invertaseper ml of fermentation medium, when supplied with 4.0g/l peptone as nitrogen supplement and 0.2 g/l dipotassium hydrogen phosphate as phosphate source n the fermentation m edium.
| Table 1: | Effect of organic nitrogen sources on the production of invertase by Saccharomyces cerevisiae GCB-K5. |
| Sucrose concentration, 25 g/l; incubation period, 48 hours; temperature, 30°C; initial pH, 6.0; agitation rate, 200 rev/min. | |
| Table 2: | Effect of different concentrations of peptone on the production of invertase by Saccharomyces cerevisiae GCB-K5. |
| Sucrose concentration, 25 g/l; incubation period, 48 hours; temperature, 30°C; initial pH, 6.0; agitation rate, 200 rev/min. | |
| Table 3: | Effect of different phosphate sources on the production of invertase by Saccharomyces cerevisiae GCB-K5. |
| Sucrose concentration, 25 g/l; incubation period, 48 hours; temperature, 30°C; initial pH, 6.0; agitation rate, 200 rev/min. | |
| Table 4: | Effect of different concentrations of K2HPO4 Saccharomyces cerevisiae GCB-K5. |
| Sucrose concentration, 25 g/l; incubation period, 48 hours; temperature, 30°C; initial pH, 6.0; agitation rate, 200 rev/min. | |
The incubation temperature and initial pH were 30°C and 6.0, respectively while incubated for 48 hours. All these conditions were concluded as optimal for invertase biosynthesis by the yeast strain.