PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

More documents

Recommendations

Info

FSB1 – 2004 Food Science and Biotechnology in Developing Countries Therefore according to the results, the optimum values of production of the tannase enzyme by A. niger GH1 and for the partial purification are: 30º C of incubation for 24 h in solid state culture and with a substrate concentration of 50 g/L of tannic acid. For the partial phase of purification was carried out the evaluation of two methods of purification of the tannase enzyme, besides two proteins concentration methods. From the two methods evaluated to concentrate proteins, the one that it was obtained greater performances was of polietilenglicol (PEG) and dialysis membrane reported by Sharma (1999) since was obtained almost four times more of tannase activity that with the acetone method (Table 2). Table 2. Comparison of proteins precipitation methods evaluating the tannase activity and the extract of the protein obtained in the production at preparative scale by A. niger GH1. Technique Activity Tannase (U/L) Protein (mg/L) Specific Activity (U/mg) Total Activity (U) PEG 2327.88 1198.07 2.0 11.63 Acetone 1:1 620.76 780.76 0.8 3.90 Acetone 1:2 0 767.30 0 0 Acetone 1:3 0 740.38 0 0 Results obtained of IEC showed that it is possible to separate the tannase from the rest of proteins, however two isoforms of tannase were revetaled (figure 4). These results are not according with the data reported by Barthomeuf et al., (1993), due they detected and purified one isoform of tannase by this kind of chromatography. Results obtained for isoelectrofocusing with the Rotofor ® revelated two isoforms of tannase (figure 4), one of these showed a isoelectric point already to 7 and other one at 4. It is important to note that results obtained in this study are according with the results published by Garcia-Peña et al (1999) using the same technique. This study permits to establish the first bases for the purification of tannase produced by Aspergillus niger GH1 by solid state culture using polyurethane foam as inert support.
FSB1 – 2004 Food Science and Biotechnology in Developing Countries kD 250 150 100 75 50 37 25 10 Ec PEG 15 ANX DEAE Q K Figure 4. Comparison of purification steps on electrophoresis gels (SDS-PAGE). (Ec) crude extract, (PEG) concentrated with polyethylenglycol, (15) fraction 15 from Rotofor ® , (ANX) fraction from ANX column (DEAE) fraction from DEAE column (Q) fraction from Q column, (K) tannase of reference (from Kikkoman). Finally, it is important to note that tannase enzyme was produced at high levels at 30ºC for 24 hours of culture with 50 g/L of inducer (tannic acid) Two isforms were reveled with pre-protocol developed. Both isoforms presented molecular weights of 75 and 100 kDa. Acknowledgements This work is part of the research project on tannase and tannins economically financed by SEP- CONACYT program and FOMIX COAH-CONACYT. REFERENCES Aguilar,C. Augur, C. Viniegra-González, G. and Favela, E. (1999). A Comparasion of Methods to Determine Tanin Acyl Hidrolase Activity. Brazilian Archives of Biology and tecnology, 42, 355-361. Aguilar C. N., Favela–Torres, E., Viniegra-Gonzáles G. and C. Augur. (2000). Culture conditions dictete the production of proteases by Aspergillus niger Aa-20 reporte interno UAM1. Aguilar y Gutiérrez-Sánchez, (2001). Review: Sources, Properties, Applications and Potencial uses of Tannin Acyl Hidrolase. Food Science Technology Int. 7;(5) 373-382. Aguilar, C. Augur C. Favela, E. Viniegra- González, G. (2001a) Induction and repression patterns of fungal tannase in solid-state and submerged cultures. Process Biochemistry, 36, 565-570. Aguilar y col., (2001b). Production of tannase by Aspergillus niger Aa-20 in sumerged and solid state fermentation: influence of glucose and tannic acid. Journal of Industrial Microbiological and Biotechnology. 26. 296-302. Barthomeuf, C. Regerat, F., Pourrat, H. (1994). Improvement in tannase recovery using enzymatic disruption on micelium in combination with reserve mycellar enzyme extraction. Biotechnol Tech 8: 137- 142. Chae, S. Yu, T. (1983). Experimental manufacture of a com wine by fungal tannase. Hanguk Sipkum Kwahakoechi 15: 326- 332.
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 30 and 31: 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?