PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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FSB1 – 2004 Food Science and Biotechnology in Developing Countries EFFECT OF INVERTASE PRODUCTION OF Aspergillus niger Aa 20 IN SUBMERGED CULTURE Cuitláhuac Aranda-Vásquez 1 , Octavio Loera-Corral 2 , Raul Rodríguez-Herrera 1 , Juan Carlos Contreras-Esquivel 1 and Cristóbal Noé Aguilar 1 *. 1 Food Research Dept. School of Chemistry. Universidad Autónoma de Coahuila. Saltillo, Coahuila, México. *Corresponding author: cag13761@mail.uadec.mx 2 Department of Biotechnology. Universidad Autónoma Metropolitana. Iztapalapa, México, D.F. ABSTRACT Invertase production kinetics were studied in submerged culture with or without an enzymatic inducer (sucrose) or repressor (glucose). The maximum production of the enzyme (2170.8 U/Lmin) was reached in experiments with 50g/L of inducer in 60 hours. Maximal invertase production (37639.58 U/L*min).was obtained with 6.25 g/L of glucose and 50 g/L of sucrose in 72 hours of culture. Key words: invertase, glucose, sucrose, submerged culture. INTRODUCTION The invertase (called sucroase or β-fructofuranosidase), is an enzyme widely used in food industry over all in confectionery. Action of this enzyme is breaking the sucrose in glucose and fructose for obtain glucoside and fructoside syrup, which are more expensive that the sucrose itself, for this reason is important the production and utilization of the enzyme (Romero-Gomez et al., 2000). The microbial metabolism in particular the fungus, allows to synthesize great variety of proteins or enzymes, but do not make all in the same time. Only if an inducer is in contact with the microorganism the transcript gene level is up, this make that the fungus begin to synthesize the indicated enzyme for metabolize the inducer molecule. In the same form that a microorganism synthesize a enzyme which can metabolize a near molecule, too avoid synthesize enzymes in which case end product is available free in the culture media, this kind control is called enzymatic repression for feed-back or for end product disposition. There are other form of enzymatic repression which consist in the prevention of the contact between the microorganism with the molecule that actives the corresponding operón in this form the structural gene levels stay in her minimum value and enzyme neither is synthesized, this type of repression is called for feed-forward or for absence of initial activator. The technologies for enzyme production in submerged culture had their best age in the First World War; in that moment the penicillin production in great quantities was priority for the companies, the major part of investigation that was available in moment was with respect to liquid culture for this reason used this technique for satisfied the enormous demand of penicillin and drugs. After of First World War the companies follow produced enzymes with the same
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.
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PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

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