PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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FSB1 – 2004 Food Science and Biotechnology in Developing Countries RESULTS AND DISCUSSION Induction experiments: Figure 1 shows the results obtained for enzymatic activities at several inducer concentrations through 72 hours of culture. Activity (U/L*min) 2500 2000 1500 1000 500 0 Conc 6.25 Conc 12.5 Conc 25 Conc 50 Conc 100 0 12 24 36 48 60 72 Tme (Hours) Figure 1. Invertase production at several sucrose concentrations. Maximal invertase production was reached in cultures with 50 g/L at 60 hours. Results obtained were higher than those reported by Ashokkumar et al (2001). Repression experiments: Figure 2 shows the results obtained for enzymatic activities using the same inducer concentration (50 g/L) and several glucose concentrations through 72 hours of culture. Activity (U/L*min) 45000 40000 35000 30000 25000 20000 15000 10000 5000 conc 0 conc 6,25 conc 12,5 conc 25 conc 50 conc 100 0 0 12 24 36 48 60 72 84 96 Time (Hours) Figure 2. Invertase production at several glucose concentrations and 25 g/L of sucrose.
FSB1 – 2004 Food Science and Biotechnology in Developing Countries It is important to make a mention that experiments were called as “repression experiments” however, it is very clear that in this study, glucose has a double role on invertase expression, because at lowest initial concentrations it acts like activator of biomass production and it has a direct effect on invertase production. In contrast, at higher than 12.5 g/L of glucose, this molecule acts as repressor of the invertase production. To evaluate the degree of modification on invertase production by two molecules used, it was decided to stablish a induction or repression ratio. Both ratios were calculated dividing the maximal invertase level at each experiment by the basal activity obtained in experiments with glucose as sole carbon source at 30 g/L. Figure 3 shows the results of induction and repression ratios for the invertase in different initial concentrations of inductor and repressor enzymatic. Repretion Ratio 250,0 200,0 150,0 100,0 50,0 0,0 R.R. I.R. 0 6.25 12.5 25 50 100 Initial Concentration (g/L) Figure 3. Induction ratio at several sucrose concentrations (white rhombus) and repression ratio at several glucose concentrarions and 25 g/L of sucrose (black squares). Use of glucose in culture medium stimulated significantly the invertase production; however, this increasing capacity of glucose in enzyme activity is reverted at higher initial concentration of 50g/L. Cultures with inducer and glucose at initial concentration lower than 50 g/L favored the invertase expression in comparison with those results obtained with sole inducer. This behavior has been described previously by Aguilar et al (2001) using as study model the tannase enzyme produced by the same fungus. The best initial concentration of enzymatic inducer was 50g/L, obtain maximum activity at 60 hours with 2170.8U/L*min and the best initial concentration of repressor was at 6.25g/L with an 15 10 5 0 Induction Ratio
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PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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