Effects of temperature, pH and additives on the activity of tannase produced by Paecilomyces variotii
support: This work was supported by CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível
Keywords: characterization, enzyme, fermentation, fungi, Paecilomyces variotii, tannase.
biochemical characterization of the tannase from a Paecilomyces
variotii strain isolated in
Tannin acyl hydrolase, commonly referred to as tannase (E.C: 126.96.36.199), is an enzyme that cleaves ester linkages in hydrolysable tannins (Banerjee et al. 2001; Belmares et al. 2004), producing glucose and gallic acid (Banerjee et al. 2005). Tannase is an extracellular, inducible enzyme produced in the presence of tannic acid by fungi, bacteria and yeast (Aguilar and Gutiérrez-Sanchez, 2001). The first step in the development of microbial enzyme production is the lineage selection. Extracellular enzymes were preferred because they are easily extracted and do not require expensive extraction methods. Studies on the production of tannase using solid, liquid and submersed fermentation have been reported (Lekha and Lonsane, 1997) duction processes (Van de Lagemaat and Pyle, 2001). The fermentation broth can use by-products such as wheat bran, rice bran, sugar beet pulp, fruit pulps, banana waste, cassava waste and coffee residues, adding tannic acid. The use of by products or residues rich in sources of carbon for fermentation purposes is an alternative way of solving pollution problems that can be caused by incorrect disposal in the environment (Battestin et al. 2005). In the present work, a Paecilomyces variotii lineage obtained by fungal isolation procedures was used for the production of tannase using coffee husk and wheat bran residues.
Tannase is extensively used in wine, beer and coffee-flavoured soft drinks or as an additive in food detanification. Gallic acid is also used in the enzymatic synthesis of propyl gallate, which is mainly used as an antioxidant in fats and oils (Belmares et al. 2004; Vaquero et al. 2004; Yu et al. 2004). Usually the end products of a fermentation process contains some unwanted components, which have to be eliminated as far as possible by downstream processing (Mukherjee and Banerjee, 2006). Purification and characterization of tannase has been attempted earlier owing to its wide applications in various food, feed, leather and pharmaceutical industries. Various media preparations can be used with tannic acid as the sole carbon source for production of microbial tannase but biotransformation of tannin rich agro residue is cost-effective (Mukherjee and Banerjee, 2006).
This paper reports on the determinations of pH, temperature optima and stabilities of crude and partially purified tannase from the isolated strain Paecilomyces variotii. The effects of inhibitors, chelators and surfactants on the crude tannase activity were also determined.
variotii is a lineage obtained by means of fungal isolation procedures
and was used for the tannase production in coffee husk and wheat bran
residues. Five hundred fungal cultures were obtained from the departmental
stock culture collection (from Food Science Department-Unicamp), collected
from different places in
All the chemicals were of analytical grade. Tannic acid was from Ajinomoto OmniChem Division.
strain was maintained in potato dextrose agar (PDA) slants, stored
the fermentation process, a 250 mL conical flask was used containing
the following constituents:
sulphate Fractionation and dialysis. Ammonium sulphate was added
to the supernatant to give a final concentration of 80% saturation.
The ammonium sulphate was added with constant stirring at
chromatography (FPLC) on a DEAE sepharose column. The partially
purified enzyme was dissolved in acetate buffer (
Molecular mass determination by SDS-PAGE. The properties of the purified tannase were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were detected by Coomassie blue staining and then de-stained using a mixture of methanol, glacial acetic acid and distilled water. The molecular weights of the proteins were determined using the standard protein mixture of 94, 67, 43, 30, 20 and 14 kDa.
Determination of Km and Vmax. Km and Vmax were determined by plotting velocity against substrate concentration (0.17 - 1.76 µmol-tannic acid [S]). To calculate kinetic constants, data were plotted and fitted directly to the Michaelis-Menten equation. Calculations were also performed by using the linear transform method of Lineweaver and Burk (1934).
colorimetric assay was used to determine tannase activity, based on
measuring the residual tannic acid content after the enzymatic reaction
(Mondal et al. 2001). The reaction mixture consisted
of 0.3 mL of the substrate tannic acid (0.7% (w/v) in
Abs530 = Abscontrol − Abstest
optimum pH for tannase activity was determined at
stability of the enzyme was examined at different pH values by incubating
the enzyme in buffers at different pH values ranging from 3.5 to 9.0
for 12 hrs at
effects of inhibitors, chelators and surfactants on the tannase activity
of Paecilomyces variotii were also determined. The inhibitors
evaluated for their effects on tannase activity were sodium bisulphite,
iodoacetamide, 2-mercaptoethanol, 4-aminobenzoic acid, sodium azide,
n-bromosuccinimide and cysteine at a concentration of
the 500 tested strains, 6.75% of the fungi produced the enzyme. The
strains that showed the best activities were: LAB345G, LAB53G, and
LAB153G. These strains were tested in agro-industrial residues and
the best result was obtained using
The crude tannase produced by Paecilomyces variotii showed optimum activity at pH 6.5, whereas purified tannase showed pH optima at 5.5 (Figure 2). These results are in agreement with earlier reports by Batra and Saxena (2005) and Mahendran et al (2006). The enzyme was active at acidic pH and activity decreased as the pH approached the alkaline range. Any change in pH affects the protein structure and a decline in enzyme activity beyond the optimum pH could be due to enzyme inactivation or its instability. It could be concluded from the results that tannase from the new isolate needed an acidic protein environment to be active, fungal tannase is an acidic protein in general (Mahapatra et al. 2005). The effect of pH on the enzyme activity is determined by the nature of the aminoacids at the active site, which undergoes protonation and deprotonation, and by the conformational changes induced by the ionization of the amino acids. Enzymes are very sensitive to changes in pH and they function best over a very limited range, with a definite pH optimum (Sabu et al. 2005).
Crude tannase from Paecilomyces variotii showed 100% stability at pH 6.5 and 88% and 86% stability, respectively, at pH 4.0 and 7.5 after 24 hrs of incubation (Figure 2). This enzyme showed a wide range of pH stability. Similar results were reported for Candida sp (Aoki et al. 1976) and Penicilliun restricticum (Batra and Saxena, 2005).
functional temperature range of the tannase produced was 30-
crude tannase from Paecilomyces variotii was stable in a temperature
range from 20-
temperature for optimum activity of Paecilomyces variotii was
Tannase was produced extracellularly by isolated strain of Pecilomyces variotii using solid-state fermentation on wheat bran and coffee husk residues. A typical purification is shown in Table 2. The substrate tannic acid was used to monitor tannase activity throughout the purification procedure. A fractional precipitation with 80% ammonium sulphate removed some of the non-enzymatic proteins and about 34% of the total tannase was recovered (Table 2). The elution profile of the tannase extract obtained from the DEAE sepharose column showed five protein peaks, tannase activity being found in 2 of the peaks (Figure 5). These results agree with those of Beverini and Metche (1990), where a commercial tannase from Aspergillus oryzae was purified by affinity chromatography on Con A-Ultrogel and resulted in the separation of two fractions (tannase I and tannase II). The active fractions referring to the fourth peak were pooled and used for studying the biochemical properties of the tannase. DEAE sepharose column chromatography led to an overall purification of 10 fold with a yield of 3% (Table 2), results in agreement with those of Sharma et al. (1999), who purified a tannase from Aspergillus niger van Tieghem. The yield of 3% was lower than the value of 7-19% recovery reported by other authors (Rajakumar and Nandy, 1983; Farias et al. 1994). However, the purification factor was similar to that of the purified tannase obtained from various different fungi, as reported by other workers (Rajakumar and Nandy, 1983; Sharma et al. 1999). The molecular mass of the purified enzyme was determined by SDS-PAGE (Figure 4). The purified enzyme migrated as a single protein band corresponding to molecular masses of 87.3 kDa (major peak) and 71.5 kDa (minor peak).
see the effect of substrate concentration on tannase activity, assay
was performed at various concentrations of tannic acid. The graphical
analysis of the effect of substrate concentration on tannase activity
yielded Km of 0.61 µmol and Vmax of 0.55 U.mL-1
protein (Figure 6). The Km values
for tannase from Cryphonectria parasitica using tannic acid
as substrate have been found to be
the enzyme industry, the main importance of inhibitors is that they
reduce the efficiency of the enzyme reaction (Kar et
al. 2003). The inhibitors evaluated for their effects on tannase
activity were sodium bisulphite, iodoacetamide, 2-mercaptoethanol,
4-aminobenzoic acid, sodium azide, n-bromosuccinimide and cysteine.
Tannase activity was inhibited by sodium bisulphite, 2-mercaptoethanol,
4-aminobenzoic acid, sodium azide, n-bromosuccinimide and cysteine
at a concentration of
chelator EDTA disodium salt at a concentration
The effects of chemical substances on the activity of an enzyme are often precise and specific. In the present study, surfactants and chelators were chosen for an evaluation of their effects on tannase activity. The effects of Tween 80, Tween 20 and Triton X-100 (0.025-1% (v/v)) were studied, using enzyme solutions containing 0.12% (v/v) and the above chemical substances at the concentrations mentioned.
Tween 80 and Tween 20 caused a decrease in tannase activity at concentrations of 0.025, 0.5% and 1% (v/v) (Table 1). Tween 80 is predominantly composed of oleic acid (70%). Tween 20 consists of lauric acid. Due to the predominance of oleic acid and lauric acid in Tween 80 and Tween 20, they cause a decrease in tannase activity. Similarly, Tween 80 (1% (v/v)) caused an inhibition of the lipase activity from Pseudomonas sp. KWI-56 , and Tween 60 at 0.05 - 1.0% (v/v) and another anionic surfactant, SLS, at 0.05 - 0.7%, caused inhibition of tannase activity (Kar et al. 2003). This inhibition may be the result of a combined effect of factors such as the reduction in the hydrophobic interactions that play a crucial role in holding together the tertiary protein structure, and a direct interaction with the protein molecule (Kar et al. 2003).
Triton X-100 caused a decrease in tannase activity at concentrations of 0.5 and 1% (v/v). These results are in agreement with those of Kar et al. (2003), who used Triton X-100 at concentrations of 0.03 - 0.5% (v/v) and showed a reduction in tannase activity. In contrast, Triton X-100 did not significantly affect the α-amylase activity of the Bacillus strain GM 8901. The extent of stimulation by surfactants varies for the different enzymes (Kim et al. 1995).
most countries where the economy is largely depend on agriculture
and farming practice is intensive, accumulation of agricultural residues
is a serious problem. The presence of tannins and their derivatives
in agro residues is a major hurdle in their utilization as feed material.
Solid-state fermentation technology using non-pathogenic microorganisms
that can produce hydrolytic enzymes such as tannases would be advantageous
for the proper utilization of these residues. Our isolate, identified
as Paecilomyces variotii, was able to grow in media containing
a mixed substrate including coffee husk and wheat bran residues. Wheat
bran is a good substrate for tannase production and coffee husk is
a highly available, economically viable agro-industrial residue in
This work allowed for a better understanding of the effects of temperature, pH and inhibitors on the tannase activity of Paecilomyces variotii, presenting important data from a newly-isolated fungus that produces an interesting tannase. The tannase produced was functional at a wide range of temperature and pH values. Our inhibitory reagent studies suggest the presence of sulphur containing amino acids at the active site and also tryptophan residues. The partial purified tannase from Paecilomyces variotii is a unique one with low Km. These properties can be further exploited in developing tannase for a wider range of applications in the pharmaceutical, food, feed and leather industries and thus their production at higher levels should be sought for. Therefore, Paecilomyces variotii would provide a new source for the efficient production of tannase for industrial applications.
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