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.