PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

More documents

Recommendations

Info

Food Science and Biotechnology for Deveoping Countries. 2004 exchange column, P-III, was assayed for specific absorptivity by the dry weight method (3). It was determined the P-III molecular mass for both samples by vertical electrophoresis in SDS-polyacrilamide gel (4), the one from cationic exchange and that from hidrophobic interaction chromatography, The pH effect on proteolytic activity with 1% casein as substrate was carried out by Kunitz method, modified by Ortega and del Castillo (5), at a pH range from 6.0 to 11, at 35 ºC, the pH effect on enzyme activity with (10.0 mM in 0.1 M phosphate buffer) BAPNA was determined as Dubois et al. (1988), at 4.0 a 9.0 pH values with Cys and 25 mM EDTA at 40 °C. Temperature effect on proteolytic activity was measured by Kunitz method modified by Ortega and del Castillo (5), with 1% casein in 0.1 M phosphate buffer pH 7.0, at temperatures from 30 °C to 80 °C. Temperature effect on enzyme activity with BAPNA was made by Dubois et al. (6) method, with 10.0 mM BAPNA in 0.1 M pH 7.0 phosphate buffer, Cys and 25 mM EDTA; at different temperature values from 30 °C to 80 °C. The pH stability asay was made by incubating the enzyme at pH values ranging from 3.0 to 10.0, for 2, 12 and 24 hours, the residual activity percentage was determined by (5) at 20 °C. with 1% casein in 0.05 M pH 7.6 phosphate buffer at 35 °C. RESULTS AND DISCUSION. Purification started with strong cationic exchange chromatography of the J. mexicana 2X extract, which rendered 5 peaks, with proteolytic activity (Figure 1); the peak III was marked as Protease III (P-III), and it was passed separately through two kinds of chromatography: a) cationic exchange, with a profile that shows peak III accompanied by P-II and P-IV fraction remains, the peak with the greater proteolytic activity and hidrophobic interaction was the peak III, with three components, from which the main peak presented the proteolytic activity. Compared to b) cation exchange column fractions, these peaks are better defined, as ocurred for the performed purification of P-IV and P-V with this chromatography (7). Specific absorptivity for P-III, was 3.2 g -1 L cm -1 ; a value within the limit of the specific absorptivity highest values found for another cysteine proteases from plant sources. The results from SDS-polyacrilamide electrophoresis of P-III are showed in figure 2, it can be seen clearly a single band of protein for P-III, so we can tell that the purification was sufficient to make the biochemical characterization studies.
Food Science and Biotechnology for Developing Countries.2004 2 8 0 n m A b s I II III IV V No. Fracción No. Fraction FIGURE 1. Chromatography profile of the elution of 2X extract of J. mexicana in Econo-pac® High S, 0.05 M phosphate buffer , pH 6.3 and NaCl 0.0-1.0 M linear gradient. 1 .0 N a C l M 0 .0 - m n 0 8 2 s A III Fracción No. Fracción No. FIGURE 2. 12.5% Poliacrilamide gel electrophoresis of P-III: lane 1, P-III in Econo-pac Methyl, lane 2: P-III rec in Econo-pac High S, lane 3: P-IV M .0 -0 .5 O 2S 4) 4 1 O 2S 4) 4 1 H (N
Page 1 and 2: FSB1 - 2004 Food Science and Biotec
Page 3 and 4: RESULTS AND DISCUSSION FSB1 - 2004
Page 5 and 6: UT/L 25000 20000 15000 10000 5000 F
Page 32 and 33: Food Science and Biotechnology for
Page 34 and 35: Food Science and Biotechnology for
Page 40: FSB1 - 2004 Food Science and Biotec

PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

Create successful ePaper yourself

Delete template?

Save as template?