PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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FSB1 – 2004 Food Science and Biotechnology in Developing Countries Inoculum and culture medium Inoculum was prepared by transferring the spores to flasks with Potato Dextrose Agar and incubating at 30°C for 3-5 days. The spores were then scraped into a 0.01% Tween 80 solution and counted in a Neubauer chamber. Culture medium for tannase production included (g/L): KH2PO4 (2.19), (NH4)2SO4 (4.38), MgSO4.7H2O (0.44), CaCl2.7H2O (0.044), MnCl2.6H2O (0.009), NaMoO4.2H2O (0.004), FeSO4.7H2O (0.06) and tannic acid (12.5) as sole carbon source (Aguilar et al. 2001). Culture conditions Two culture systems were used to produce tannase enzyme. Submerged culture (SmC) and solid state culture (SSC). SmC conditions included agitation at 250 rpm, temperature 30ºC, initial pH 5.5 and several incubation times. Inoculum level was 1 x10 7 spores per ml of culture medium. The solid support used in SSC was polyurethane foam (PUF) pulverized. An additional culture condition in SSC was: 70 % initial humidity. SmC and SSC were kinetically monitored. Crude enzymatic extract from SSC was obtained by compression of the fermented material. Tannase assay The tannase activity was evaluated by the method reported by Sharma (2000). One tannase unit was defined as enzyme amount that release one µmol of gallic acid per min under assay conditions. Substrate degradation Total sugar content was determined by the method reported by Dubois (1956). Whilst reducing sugar content was evaluated by the DNS method. Concentration and partial purification of tannase Tannase was concentrated using two methods with acetone and polyethylenglycol (PEG) and partially purified through ionic exchange chromatography (IEC) using three different columns and isoelectrofocusing using a Rotofor ® . Tannase activity and protein content were evaluated in each step of protocol. Electrophoresis gels (SDS-PAGE) were analyzed at the end of each step. RESULTS AND DISCUSSION Evaluating the two culture systems as observed in the table 1. It is possible to observe that the strain of A. niger GH1 in the solid culture, presented a higher production of the tannase enzyme. For the fermentation time, to a high concentration of substrate the strain consumes the majority in the first hours of the fermentation (Figure 1) and the tannase enzyme production of is observed at 24 h (Figure 2). These results favored the purification of the enzyme therefore as can be observed in the Figure 3 the production of the proteases shoot up after the 24 h of the fermentation and this would be able to affect the tannase activity since Aguilar et al. (2000) showed that the protease activity influences in the production of the tannase activity causing the decrease of enzyme activity. The incubation time required for the production of the tannase enzyme by A. niger GH1 in solid state culture is lowest than the time reported by Sharma (1999), that describes for A. niger Van Tieghem a time of incubation for the production of the tannase enzyme of 120 h for which the reduction of the time is favorable for the case from A.niger GH1.
FSB1 – 2004 Food Science and Biotechnology in Developing Countries TANIC (g/L) 14 12 10 8 6 4 2 0 Table 1. Comparison in the production of tannase in liquid and solid cultures at 30º C (30 h). Strain Cultures Kind GH1 SmC GH1 SSC *SmC: Submerged Culture. *SSC: Solid State Culture. 0 8 16 24 32 40 48 56 64 72 80 88 TIME (h) Figure 1. Substrate uptake by A. niger at 30° C in SSC with 12.5g/L of tannic acid. Proteasse Activity U/ L*min 10 9 8 7 6 5 4 3 2 1 0 Tannase U/L Total Tannase U/L 537 537 2291 2291 Tannase Activity (U/L) 1800 1600 1400 1200 1000 800 600 400 200 0 6 12 18 24 30 36 42 48 Time (hours) Figure 3. Protease activity of GH1 strain at 30° C in SSC with concentrations of 25(♦) y 50 (■) g/L of tannic acid. 0 0 6 12 18 24 30 36 42 48 hours Figure 2. Tannase enzyme expression by the GH1 strain at 30° C in SSC with concentrations of 25(♦) and 50 (■) g/L of tannic acid.
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PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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