Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

2.1. Microorganism and Culture Conditions

Aspergillus niger GH1, utilized for tannase production, was obtained from the UAdeC-DIA culture collection. This strain was previously isolated from Mexican semidesert and characterized as tannase producer [29]. Fungal spores were stored at −40°C in a cryoprotectant medium composed of glycerol and skim milk.

Microorganism was propagated by transferring conserved spores to Erlenmeyer flasks with Czapek-tannic acid agar and incubating at 30°C for 4–6 days. After this, spores were harvested with Tween 80 (volume ratio of 0.01%) and counted in a modified Neubauer chamber before inoculation.

Tannase was produced by A. niger in submerged culture. The culture medium for tannase contained (g/L) KH₂PO₄: 2.19; (NH₄)₂SO₄: 4.38; MgSO₄·7H₂O: 0.44; CaCl₂·7H₂O: 0.044; MnCl₂·6H₂O: 0.009; NaMoO₄·2H₂O: 0.004; FeSO₄·7H₂O: 0.06. Salt solution was autoclaved at 12°C for 15 min and cooled at room temperature. Tannic acid was added to the salt solution to a final concentration of 12.5 g/L, then the pH was adjusted at 5.5 with 1 N NaOH, and then the culture media was sterilized again by filtration through 0.45 μm nylon membranes. Tannase production was carried out in 1 L Erlenmeyer flasks containing 250 mL of culture medium inoculated with 1 × 10⁷A. niger spores/mL. Flasks were incubated for 24 h at 30°C with constant agitation at 250 rpm.

2.2. Tannase Recovery

Crude enzymatic extracts from SmF were obtained by filtering the biomass through Whatman no. 41 filter paper. Mycelial cells retained were washed with physiological solution, frozen with liquid nitrogen and milled in a mortar. The macerate was recovered with acetate buffer (100 mM, pH = 5.5). Crude extract was put into a 10 kD MWCO cellulose membrane (Sigma, St. Louis, USA) and dialyzed against water. Dialyzed extract was concentrated (12.5-fold) with polyethylene glycol-6000 as described by Sharma and coworkers [30]. Concentrated extract was applied into a HiTrap G25 column, eluted with citrate buffer (100 mM, pH 5.0) and fractionated in an AKTA prime FPLC system (Amersham, Piscataway, USA).

2.3. Enzyme Immobilization

Tannase from A. niger was immobilized in Ca alginate beads. 4 mL of partially purified tannase (288 U/L) were mixed with 46 mL of 2.0% sodium alginate solution to get homogeneity. Then the mixture was added with constant agitation and a temperature of 4°C to 0.6 M CaCl₂ solution as droplets using a glass burette. The beads were kept in 0.1 M CaCl₂ at 4°C for about 2 h and then washed briefly with sterile water.

2.4. Kinetic Constants of Tannase

Values of K_M and V_max were calculated for free and immobilized tannase using methyl gallate and tannic acid as substrates in citrate buffer (50 mM, pH 5.5). Hydrolysis was carried out in a 250 mL glass jacketed bioreactor. 50 beads of immobilized tannase or the equivalent in free enzyme was added to 100 mL of substrate with constant agitation and controlled temperature at 30°C, and 1 mL samples were withdrawn at regular intervals. Reaction was stopped with 0.2 mL of 2 N HCl, and the reaction mixture was filtered through a 45 μm membrane and analyzed for gallic acid by an HPLC procedure [31]. Tannase activity rate was estimated as V (IU) following the Michaelis-Menten equation. Estimation of different parameters for the equation was obtained through the linearization method of Lineweaver-Burk, using the Solver utility of the program Excel (Microsoft, Redmond, USA).

2.5. Analytical Methods

Tannase activity was assayed using HPLC methodology, essentially as described by Beverini and Metche [31] with slight modifications. In brief, the enzyme (50 μL) was added to 1 mL of methyl gallate 3 mM. The reaction mixture was incubated at 30°C for 30 min. The reaction was stopped with 2M HCl. Each sample was filtered through a 45 μm membrane prior to HPLC analysis. One unit of enzyme (IU) was defined as the amount of enzyme able to release 1 μmol gallic acid per minute of culture filtered under the standard assay conditions. Protein estimation was done as described by Bradford [32].

3.1. Tannase Production and Recovery

Tannase production was carried out by A. niger GH1 in submerged fermentation. Under the described conditions, tannase was expressed mainly intracellularly (data not shown). At the end of incubation, 1.16 g of biomass were obtained from 2 L of culture broth. After cell disruption it were recovered 200 mL of a crude extract with 15.68 IU of tannase per liter and a specific activity of 7.29 IU per mg of protein.

Tannase production was significantly lower than previously reported by our group [28]. This may be related with the culture system utilized. In that case, Mata-Gómez and coworkers [28] utilized a tannase produced by A. niger GH1 in a solid-state fermentation system. The enzyme was produced mainly extracellularly, and the volumetric activity reached about 400 IU/L. Lekha and Lonsane [33] reported that in submerged fermentation, tannase production by A. niger is intracellular during the first 48 h and the enzyme is subsequently excreted. In contrast, tannase production in solid-state fermentation is completely extracellular.

On the other hand Barthomeuf and coworkers found that, during the first hours of submerged fermentation, tannase remains strongly bound to the mycelium of Aspergillus niger and no more than 5% of the enzyme may be removed by physical methods, such as cell disruption [34]. However, at industrial level tannase is produced mainly by submerged fermentation due to more standardized methodology and equipment.

Crude extract was subjected to a recovery process consisting of dialysis, concentration, and gel filtration chromatography. This protocol led to 5.4-fold purification and 5.6-fold concentration with a recovery yield higher than 90%.

We previously reported the purification of an extracellular tannase from A. niger. In that case, through a protocol consisting of ultrafiltration, ionic exchange, and gel filtration chromatography, a 42-fold purification was obtained with 0.3% yield [28]. In the present paper, we concentrated and partially purified the enzyme according with the needs of an enzyme for industrial use. Industrial enzymes require a certain degree of purity to avoid undesirable reactions and the presence of proteins and other foreign compounds in the final product. The degree of purity required is lower for immobilized enzymes due to entrapment of the biocatalyst [25].

Most of the reported protocols for tannase involve several precipitation and chromatographic steps. For example, Mahendran and coworkers purified a Paecilomyces variotii tannase by a treatment with activated charcoal followed by precipitation with ammonium sulfate, ionic exchange chromatography, and gel filtration. They obtained a 30.5-fold purification of the enzyme, but with a recovery yield of 17.6% [20]. On the other hand, Sharma and coworkers purified a Penicillium variable tannase through a two-step procedure. They utilized ultrafiltration and gel filtration and obtained a 135-fold purification with a recovery yield of 91% [21].

3.2. Enzyme Immobilization

Tannase was efficiently immobilized in Ca alginate beads. During immobilization, it was obtained 253 beads of 3.5 mm of diameter per mL of enzyme extract. Since no residual activity or protein was detected in CaCl₂ solution or in the wash water, it was assumed that the enzyme was completely trapped.

Recently, Schons and coworkers reported the immobilization of a Paecilomyces variotii tannase by entrapment into several polysaccharide matrixes [35]. Besides the fact that the best encapsulation efficiency (57%) was obtained with pectin, tannase immobilized in alginate beads (15% of encapsulation efficiency) was more efficient for hydrolysis of tannic acid.

3.3. Kinetic Constants of Tannase

Kinetic parameters of free and immobilized were evaluated at 30°C and a pH of 5.5 using tannic acid and methyl gallate as substrate. K_M and V_max were obtained with the linearization method of Lineweaver-Burk. The kinetic constants are summarized in Table 1, Lineweaver-Burk plots are showed in Figures 1 and 2.

K_M values for free enzyme using methyl gallate and tannic acid as substrate are very close among them (0.433 and 0.400 mM, resp.). When tannase was immobilized, these K_M values were notoriously increased (1.3- and 59-fold for methyl gallate and tannic acid, resp.).

These results indicate that A. niger GH1 tannase produced in submerged fermentation has almost the same affinity for methyl gallate and tannic acid. In contrast, the tannase produced by this microorganism in solid-state fermentation has more affinity for tannic acid than for methyl gallate [28]. These catalytical disparities among the tannases could be related to differences in glycosylation among the enzyme produced in solid-state and submerged fermentation, as suggested by Renovato and coworkers [36].

The K_M value of tannase increased significantly after immobilization in Ca alginate, especially when tannic acid was used as substrate. This is a common phenomenon during enzyme immobilization [14, 18, 22, 24]. The increase of K_M is partially due to mass transfer resistance of the substrate into the polymer matrix, and mass transfer resistance appears to be drastic in substrates such as tannins [14]. For example, Yu and coworkers [24] immobilized an A. niger tannase with a coacervate calcium alginate membrane. In that case, the K_M value increased 4 times with immobilization. Similar results were reported by other authors (Table 1).

Hydrolysable tannins have the ability to complex with macromolecules such as carbohydrates but, to maintain its binding capacity, tannins must have more than two acidic unit constituents esterified to the glucose core [4]. Thus, the interaction between alginate and tannic acid is much higher than that with methyl gallate, as indicated by these results.

On the other hand, the V_max value for intracellular tannase from A. niger is higher using tannic acid as substrate in both free and immobilized forms. In addition, immobilization resulted in an increase in V_max when tannic acid was used as substrate. Typically, immobilized enzymes have lower V_max values than their free counterparts [37], and this phenomenon is associated with conformational changes of the enzyme during immobilization [18]. Higher V_max values after immobilization have been reported for inulinase [38], invertase [39], and β-galactosidase [40]. However, this is an unusual behavior and the mechanistic basis for this positive feature requires further investigation [37].

The increase of the maximal velocity of reaction and the K_M value makes the immobilized tannase a better biocatalyst than free enzyme for applications on liquids with high tannin content, such as bioremediation of tannery or olive-mill wastewater. In addition, the immobilization technique allows the recovery and reutilization of the enzyme.