PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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.