PURIFICATION, CHARACTERIZATION, AND SUBSTRATE RELATIONSHIPS OF THE TANNASE FROM CRYPHONECTRIA-PARASITICA

The tannase of Cryphonectria parasitica was isolated from the mycelium and purified 142-fold with a 10% yield by anion exchange chromatography and gel filtration. The estimated molecular weight was 240 kDa and the molecule may be a tetramer composed of four subunits with a molecular weight of 58 kDa. The enzyme was separated into six bands in the pH range of 4·6-5·1. Based on the Michaelis-Menten constant (Km) of the tannase for three substrates tested, aleppo tannic acid was the best substrate (Km= 0·95 mm). Hamamelitannin, present in blight-susceptible chestnut bark, was also a good substrate for the enzyme (Km= 5·07 mm). The ellagitannins, vescalagin and castalagin, were not good substrates for the enzyme and no inhibition of C. parasitica tannase by these compounds was found. Gallic acid was an effective competitive inhibitor of the tannase with all substrates and concentrations tested (Ki= 11·0-13·1 mm). The relative activities of the tannase on specific tannin substrates may be related to the number of ester and depside bonds in the substrates. © 1994 Academic Press, Inc.