PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
begin operation technique, for this reason the major of companies now uses the liquid
fermentation in their production processes. However, some physiological aspects of enzyme
production in this kind of processes have not been studied to detail, still.
In this study the effect of glucose and sucrose concentration on invertase production of
Aspergillus niger Aa-20 in submerged culture was evaluated.
MATERIAL AND METHODS
Microorganism and inoculum preparation
Spores of Aspergillus niger Aa-20 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%).
Induction experiments
All experiments were carried out at the same conditions. Volume, 1 liter of culture media
Czapek-Dox: NaNO3 0.765%, KH2PO4 0.304%, MgSO4 0.152%, KCl 0.152, and several initial
sucrose concentrations of sucrose (6.25, 12.5, 25, 50 and 100g/L). It is important consider that
the rest of components were adjusted for maintain the ratio C/N at 10. Incubating temperature
of 30ºC for 72 hours, with a monitoring each 12 hours, inoculation level of 1*10 8 spore/L and
shaking at 200 r.p.m. were conditions used for experiments.
Repression experiments
Repression experiments were carried out at same conditions of volume, temperature, time,
monitoring, shaking and inoculation level that those experiments of induction, In this set of
experiments, a fixed concentration of inducer (sucrose at 50 g/L) and several concentration of
enzymatic repressor (glucose: 6.25, 12.5, 25, 50, 100g/L) were added.
Enzymatic assay for invertase
Invertase activity was assayed following the DNS-method reported by Ashokkumar et al (2001).
Total sugars determination:
Total sugars content was evaluated with phenol-sulfuric acid method as reported by Aguilar
(2000).
Reducing sugar determination
Reducing sugar content was determined with the DNS method (Miller, 1959).
Biomass produced determination
Biomass concentration was evaluated gravimetrically.