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