PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
MATERIALS AND METHODS
Microorganism and inoculum preparation
Spores of Aspergillus niger GH1 (DIA-UAdeC collection) stored at -20ºC were used in all experiments.
Inoculum of microorganism was prepared on potato dextrose agar and spores were harvested with
tween 80 (0.01%).
Solid state fermentation (SSF)
For the SSF process, Erlenmeyer flasks (250 mL) were used like reactor. These flasks contain dried
pulverized polyurethane foam (PUF) like solid support, moisturized with inoculated liquid medium
(inoculation level 2x10 7 spores/L). The reactors are incubated at 30ºC and the samples were taken at
24 hours of culture).
Enzymatic assay for tannase
Tannase activity was assayed following the Rodanine-method reported by Sharma et al (2000). It is
important to state that time of enzyme reaction was determined under conditions used.
pH and Temperature stability
Dialyzed extracts were conditioned at several values of pH (3 -7) and temperature (20 – 80ºC) and its
tannase activity determined.
RESULTS AND DISCUSSION
In this study, tannase from the strain Aspergillus niger GH1 was produced by solid state fermentation
using polyurethane foam as support. After 24 hours of culture, fermented material was compressed to
obtain the crude enzyme extract, which wwas dialyzed against buffer at 4ºC.
Obtained results demonstrated that this fungal enzyme is thermo-labile with a stability of 30 at 40ºC,
and it had a maximum temperature value of 30ºC (Figure 1). To values higher than 50ºC the tannase
activity is seriously affected. It is important to note that to values lower than 30ºC enzyme is also, highly
sensible to temperature. This result is according with the literature.
Results obtained of the effect of pH to enzyme activity showed that the optimal pH is 5, and the
dialyzed tannase extract is stable to pH values from 4 to 7. Values higher than pH / presented several
inconvenient to analyze due the hydrolysis of substrate by alkali condition.