A single colony of Sporidiobolus ruineniae A45.2 was inoculated in yeast extract-malt extract broth (YMB) (3 g/L yeast extract, 3 g/L malt extract, 10 g/L glucose) and incubated at 30 °C on a 150-rpm rotary shaker for 24 h. Accordingly, 10% (v/v) of inoculum was transferred to YMB supplemented with 1% (w/v) filtered sterile tannic acid and incubated at the same conditions as have been described above. After 48 h of cultivation, the culture was harvested by centrifugation. Cell pellets were washed with 20 mM sodium phosphate buffer pH 6.5 supplemented with 0.1% (v/v) Triton X-100 to remove any gallic acid attached to the yeast cell wall and the residual tannic acid. The cell pellets were then suspended with 20% (w/v) sucrose in 30 mM Tris-HCl pH 8.0. To release the tannase associated with the cell envelope [19] (link), the suspension was supplemented with lysozyme solution prepared in 100 mM EDTA pH 7.3 to yield a final concentration of 0.1 mg/mL of lysozyme prior to being incubated on ice for 40 min. The lysozyme-EDTA treated suspension was centrifuged at 17,350 × g for 15 min at 4 °C. The supernatant was then dialyzed against 20 mM sodium phosphate buffer pH 7.0 at 4 °C until equilibrium was reached. The resulting dialyzed enzyme was then used in further experiments.
Tannase activity was determined according to the method described in a previous study [20] (link) with slight modifications. Briefly, 50 μL of the enzyme solution was mixed with 50 μL of the substrate (12.5 mM methyl gallate in 100 mM sodium phosphate buffer pH 6.5). The reaction was carried out at 37 °C for 20 min. Then, 60 μL of 0.667% (w/v) methanolic rhodanine solution was added into the reaction mixture to stop the reaction and to detect the release of gallic acid from tannic acid. After a 5 min period of incubation at room temperature (25 °C), a pinkish purple color was visualized by adding 40 μL of 0.5 M KOH and the mixture was left at room temperature for 5 min. Finally, 800 μL of distilled water was added, the mixture was vigorously mixed, and absorbance was measured at 520 nm. One unit of tannase was defined as the amount of enzyme that released 1 μmol of gallic acid in 1 min under the assay conditions.
Tannase activity was determined according to the method described in a previous study [20] (link) with slight modifications. Briefly, 50 μL of the enzyme solution was mixed with 50 μL of the substrate (12.5 mM methyl gallate in 100 mM sodium phosphate buffer pH 6.5). The reaction was carried out at 37 °C for 20 min. Then, 60 μL of 0.667% (w/v) methanolic rhodanine solution was added into the reaction mixture to stop the reaction and to detect the release of gallic acid from tannic acid. After a 5 min period of incubation at room temperature (25 °C), a pinkish purple color was visualized by adding 40 μL of 0.5 M KOH and the mixture was left at room temperature for 5 min. Finally, 800 μL of distilled water was added, the mixture was vigorously mixed, and absorbance was measured at 520 nm. One unit of tannase was defined as the amount of enzyme that released 1 μmol of gallic acid in 1 min under the assay conditions.
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