PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

FSB1 – 2004
Food Science and Biotechnology in Developing Countries
PRODUCTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF A FUNGAL TANNASE
PRODUCED BY SOLID STATE FERMENTATION.
Mario Cruz-Hernández, Juan Carlos Contreras-Esquivel,
Raúl Rodríguez-Herrera and Cristóbal Noé Aguilar*
Food Research Dept. School of Chemistry. Universidad Autónoma de Coahuila. Saltillo, Coahuila,
México. *Corresponding author: cag13761@mail.uadec.mx
ABSTRACT
Production and partial characterization of a fungal tannase was studied. Aspergillus niger GH1 was
selected by its capacity for tannase production in the solid state culture (SSC). Several culture
conditions were evaluated. A protocol for tannase pre-purification was developed. It included
concentration by polyethilenglycol (PEG), ionic exchange chromatography (IEC) and isoelectric
focusing Rotofor ® . Two tannase isoforms were found in the culture broth.
Key words: tannase, production, partial purification, partial characterization, solid state fermentation.
INTRODUCTION
Tannase or tannin acyl hydrolase (EC, 3.1.1.20) catalyzes the hydrolysis reaction of the ester bonds
present in the hydrolysable tannins and gallic acid esters. Its production at industrial level is by
microbial way using SmC, where the activity is expressed mainly of intracellular form, implying
additional costs in its production (Lekha and Lonsane, 1994). At the moment, the main commercial
applications of the tannase are given in the elaboration of instantaneous tea or of acorn liquor and in
the production of the gallic acid (Coggon et al., 1975; Chae and Yu, 1983; Pourrat et al., 1985; García-
Najera et al., 2002), which is an important intermediary compound in the synthesis of the antibacterial
drug, trimetroprim, used in the pharmaceutical industry (Sittig, 1988) and also in the food industry;
gallic acid is a substrate for the chemical or enzymatic synthesis of the propylgallate, a potent
antioxidant. Also, the tannase is used as clarifying agent in some wines, juices of fruits and in
refreshing drinks with flavour to coffee (Lekha et al., 1993).
Several studies have reported interesting advantages between the tannase produced by SSC in
relation with that produced by SmC. On this topic there are few reports, but they are clearly interesting
(Chaterjee et al., 1996; Lekha and Lonsane 1997; García-Peña et al., 1999; Ramírez-Coronel et al.,
1999; Aguilar et al., 1999; Aguilar et al., 2001a, 2001b and 2002a; Van de Lagemaat and Pyle, 2001).
In these, attractive advantages indicated, are the high production titles (up to 5.5 times more than in
SmC), the nature extracellular of the enzymes and the stability to wide pH and temperature ranges
(Lekha and Lonsane, 1994).
This study was aimed to evaluate several culture conditions for production partial purification and
characterization of fungal tannase
MATERIAL AND METHODS
Microorganism
The strain of Aspergillus niger GH1 (DIA/UAdeC-Collection) was selected after several comparative
studies between tannin-degrading strains (Cruz-Hernández et al., 2002). Spores were maintained at -
20ºC under a crio-protector system.