Tannase activity estimation

It was determined using a UV spectrophotometric method, as described by Ahmed and Abou-Taleb²⁰. One milliliter of the crude enzyme (filtrate) was added to a 4 mL solution (consisting of 0.35 g tannic acid dissolved in 100 mL citrate buffer (0.05M and pH 5.5) and mixed well. The mixture reaction was incubated in a water bath (WB series standard model, 12 l, WB-12; Germany) at 37 °C, and 0.2 mL of the reacting compound was withdrawn at zero time (t0) and after 30 min of incubation time (t1). The enzyme reaction was stopped by adding ethanol 95% (2 mL v/v). The absorbance of the t0 and t1 was measured using a UV spectrophotometer (Chrom Tech CT-2200 UV/Vis) at 310 nm. The absorbance of the t₁ and t₂ were scored at 310 nm using a UV spectrophotometer (Chrom Tech CT-2200 UV/Vis). One unit (U) of tannase activity was considered the enzyme needed to hydrolyze 1μmol of ester per 1 min per mL. The enzyme activity was calculated according to the following Eq. (1):

where A is the absorbance and t is the time in minutes.