A protein with an N-terminal sequence showing a much lesser extent of homology than French bean and kiwi fruit thaumatin-like proteins (TLPs) to other TLPs, and possessing a molecular mass of 30 kDa which is considerably higher than those of previously reported TLPs, has been purified from the seeds of the chestnut Castanopsis chinensis Hance. The protein was unadsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.3), and adsorbed on Affi-gel blue gel in the same buffer, on CM-cellulose in 10 mM ammonium acetate buffer (pH 4.5), and on Mono S in 20 mM ammonium acetate buffer (pH 5.5). A highly purified protein preparation was obtained after fractionation on the first three chromatographic media. Castanopsis TLP appeared as a single band (30 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a single peak (30 kDa) in gel filtration on Superdex 75 by fast protein liquid chromatography. The TLP exerted antifungal activity against Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola, and Physalospora piricola, with an IC50 of 0.5 muM against E oxysporum. Castanopsis TLP was more potent than French bean and kiwi fruit TLPs in its antifungal activity toward F. oxysporum and M. arachidicola. The antifungal activity of Castanopsis TLP remained essentially unaltered after incubation at 40degreesC for 10min, was reduced after incubation at 60degreesC, and disappeared after treatment at 80degreesC. The antifungal activity underwent a decline after treatment with trypsin (enzyme: substrate ratio 1:100) at 37degreesC for 1 h but some activity remained. Castanopsis TLP exhibited a much more potent inhibitory activity on HIV-1 reverse transcriptase IC50 = 1.6 muM) than kiwi fruit TLP (IC50 greater than or equal to 27 muM). Castanopsis TLP was obtained with a yield of 20 mg from 1 kg chestnut seeds. (C) 2003 Elsevier Science (USA). All rights reserved.