Tannase Activity Research Articles

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF.

Encapsulation of tannase for the hydrolysis of tea tannins.

Tannase was encapsulated in alginate, chitosan, carrageenan or pectin gel matrices, and in the case of alginate, coated with high or low molecular weight chitosan to reduce enzyme release. Cross-linking with glutaraldehyde also improved enzyme retention. Active enzyme preparations were obtained, although carrageenan gels were unstable in tea. Tannase activity was evaluated by reduction in centrifugable (flocculated) tea solids, and a reduction in tea cream measured turbidimetrically after removal of flocculated solids. Tannin interactions with the polysaccharide gels increased the level of centrifugable solids (flocculent) in the tea. An optimum bead formulation consisted of an alginate core, coated with chitosan and cross-linked with glutaraldehyde. Both core and coating materials contained active enzyme. Beads were prepared in a single step procedure involving extrusion of alginate/tannase solution into a hardening bath containing tannase-loaded, chitosan solution. Tannase retained hydrolytic activity through three successive batch cycles, for a total period of 39h processing, and tea cream was visibly removed by treatment with the immobilized tannase. Activity remained stable during 1-month bead storage under refrigeration.

Selection of tannase-producing Aspergillus niger strains

The aim of this work was to select strains of Aspergillus niger for tannase production. Growth of colonies in plates with tannic acid-containing medium indicated their ability to synthesize tannase. Tannase activity was also measured in solid-state fermentation. A. niger 11T25A5 was the best tannase producer (67.5 U.g-1/72 hours of fermentation).

Induction and repression patterns of fungal tannase in solid-state and submerged cultures

Induction and repression patterns of tannase production by Aspergillus niger Aa-20 in solid-state (SSC) and submerged culture (SmC) were established. Tannic acid and glucose were used as carbon sources. Induction and repression ratios were obtained with different concentrations of tannic acid and glucose, respectively. Tolerance to high concentrations of tannic acid by A. niger Aa-20 was lower in SmC than in SSC. In SSC an increase in tannic acid enhanced the expression of tannase activity. The addition of glucose (>20 g l −1) resulted in strong catabolite repression in SmC system. The tannase/biomass yield in SSC was at least 2 times higher than in SmC. The results presented demonstrate the capacity of SSC to minimize catabolite repression. The role of gallic acid in tannase regulation was also studied.

A new tannase substrate for spectrophotometric assay

A new tannase substrate, protocatechuic acid p-nitrophenyl ester, 5, was synthesized using modern synthetic methods. The synthesis was designed to be performed by non-specialized chemists. It only involves four steps, three of which are protection–deprotection, and uses standard methods of separation and purification, such as recrystallization and column chromatography over silica. Under tannase action, protocatechuic acid p-nitrophenyl ester, 5, releases p-nitrophenol, which is easily measured spectrophotometrically either at 350 nm for pH values<6 or at 400 nm for pH values of 6–7 (yellow). The pH-response and the catalytic parameters of a crude Penicillium sp. tannase preparation were determined using 5 as substrate, thus showing the usefulness of this substrate in determining tannase activity.

Isolation of tannin-degrading lactobacilli from humans and fermented foods.

Lactobacilli with tannase activity were isolated from human feces and fermented foods. A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.

Modeling gallic acid production rate by empirical and statistical analysis

For predicting the rate of enzymatic reaction empirical correlation based on the experimental results obtained under various operating conditions have been developed. Models represent both the activation as well as deactivation conditions of enzymatic hydrolysis and the results have been analyzed by analysis of variance (ANOVA). The tannase activity was found maximum at incubation time 5 min, reaction temperature 40ºC, pH 4.0, initial enzyme concentration 0.12 v/v, initial substrate concentration 0.42 mg/ml, ionic strength 0.2 M and under these optimal conditions, the maximum rate of gallic acid production was 33.49 mumoles/ml/min.

Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml−1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l−1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.

A comparison of methods to determine tannin acyl hydrolase activity

Six methods to determine the activity of tannase produced by Aspergillus niger Aa-20 on polyurethane foam by solid state fermentation, which included two titrimetric techniques, three spectrophotometric methods and one HPLC assay were tested and compared. All methods assayed enabled the measurement of extracellular tannase activity. However, only five were useful to evaluate intracellular tannase activity. Studies on the effect of pH on tannase extraction demonstrated that tannase activity was considerably under-estimated when its extraction was carried out at pH values below 5.5 and above 6.0. Results showed that the HPLC technique and the modified Bajpai and Patil methods presented several advantages in comparison to the other methods tested.

Parametric optimization and biochemical regulation of extracellular tannase from Aspergillus japonicus

Aspergillus japonicus produces maximum extracellular tannase activity after 24 h incubation. The enzyme is produced constitutively on simple and complex sugar substrates but activity is doubled in the presence of tannic acid as sole carbon source. Parametric optimization of the enzyme yielded 1·13-fold increase in enzyme production (33·06 U/ml) at 30°C and pH 6·6 with 0·2% glucose and 2% tannic acid in Czapek-Dox's minimal medium. The enzyme shows strong end-product repression with gallic acid.

Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae

We cloned the Aspergillus oryzae tannase gene using three oligodeoxyribonucleotide (oligo) probes synthesized according to the tannase N-terminal and an internal amino acid (aa) sequence. The nucleotide (nt) sequence of the tannase gene was determined and compared with that of a tannase DNA complementary to RNA (cDNA) by means of reverse transcriptase PCR. The results indicated that there was no intron in the tannase gene and that it coded for 588 aa with a molecular weight of about 64 000. The tannase low-producing strain A. oryzae AO1 was transformed with the plasmid pT1 which contained the tannase gene, and tannase activities of the transformants increased in proportion to the number of copies. Tannase consisted of two kinds of subunits, linked by a disulfide bond(s) with molecular weights of about 30 000 and 33 000, respectively. We purified these subunits and determined their N-terminal aa sequences. The large and small subunits of tannase were encoded by the first and second halves, respectively. Judging from the above results, the tannase gene product is translated as a single polypeptide that is cleaved by post-translational modification into two tannase subunits linked by a disulfide bond(s). We concluded that native tannase consisted of four pairs of the two subunits, forming a hetero-octamer with a molecular weight of about 300 000.

Production, purification and characterization of a Tannase from Aspergillus niger LCF 8

Tannase (Tannin acyl hydrolase EC 3.1.1.20) is produced in high yield by Aspergillus niger LCF 8. Optimal conditions of production were determined. Induction of the strain, pH and aeration are the main parameters influencing the production yield. A pure enzyme sample (1,980 nKat/mg protein) was obtained by physical disruption of the mycelium, ultrafiltration and high pressure size exclusion chromatography (HPSEC). The biochemical properties of the enzyme were investigated. It is a glycoprotein of about 186,000 Dal, containing 43% sugars. The pI is 4.3. The optimal temperature for activity was 35°C. Esterase activity peaked at pH 5.0 while tannase activity was optimal at pH 6.0. The tannase was stable between pH 3.5 and 8.0 at temperature below 50°C. An in situ immobilized enzyme was also obtained. This was highly active towards gallotannins and ellagitannins. Since A. niger is officially approved in numerous countries for enzyme production in the food industry, the in situ immobilised tannase can be used for removing tannins responsible for unwanted effects in food or cosmetology processing.

Visual Reading Method for Detection of Bacterial Tannase

Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free gallic acid and (ii) the green to brown coloration of gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes.

Tannase activity associated with growth of Cryphonectria parasitica on American and Chinese chestnut extracts and properties of the enzyme

Abstract Cryphonectria parasitica grew on American and Chinese chestnut total aqueous and tannin extracts from winter and summer bark. Differences in the amount of conidial germination and growth on extracts of the two species were small. C. parasitica tannase (tannin acyl hydrolase) was found to be more abundant intracellularly, had a pH optimum of 5.5, and a temperature optimum for activity of 30°C. Total tannase activities on American chestnut total aqueous and tannin extracts were higher than on the respective Chinese extracts.

Characterization of Tannase Activity on Tea Extracts

ABSTRACTAssay of tannase activity was performed directly, using extracts of black tea leaf, by a pH stat method at the pH optimum of the enzyme (6.0). Reaction rates were calculated from the initial rates of the volume of titrant vs. time plots, and activity was expressed as μmoles/min. The enzyme was unstable to pH levels above 6.0 and temperatures above 40°C. Optimum temperature was 35°C. Using saturating amounts of enzyme, the pH stat was also used to determine total hydrolyzable gallate for kinetic studies and to determine the effect of gallated polyphenols on loss of solids in instant tea processing.

Production of tannase (tannin acyl hydrolase E.C. 3.1.1.20) by a strain of Aspergillus niger

Culture of a strain of Aspergillus niger on medium containing 3 % tannic acid yielded tannase (tanin acylhydrolase), evidenced both in the culture medium and the mycelium. Fermentation in submerged culture at constant air flow gave mycelium with high tannase activity.

Studies on Tannin Acyl Hydrolase of Microorganisms

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity. The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added. After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol. The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation.u=114×Et1−Et2t2−t1 Where Et1 and Et2 mean the optical density of the ethanol solution at 310 mμ prepared after t1 and t2 minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute. The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid.

Studies on Tannin Acyl Hydrolase of Microorganisms

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. u=114×Et1−Et2t2−t1Where Et1 and Et2 mean the optical density of the ethanol solution at 310 mμ prepared after t1 and t2 minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.The substrate tannic acid used in this dete...