Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis

• Published in: Biochemical and biophysical research communications Vol. 301; no. 2; pp. 364 - 370
• Main Authors: Chu, K.T., Ng, T.B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.02.2003
• Subjects: Amino Acid Sequence; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - metabolism; Antifungal protein; Botrytis cinerea; Castanopsis; Castanopsis chinensis; Chestnut; Fagaceae - chemistry; Fusarium oxysporum; Human immunodeficiency virus 1; Isolation; Molecular Sequence Data; Molecular Weight; Mycosphaerella arachidicola; Physalospora piricola; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Seeds - chemistry; Sequence Alignment; Sweetening Agents - chemistry; Sweetening Agents - isolation & purification; Sweetening Agents - metabolism; Temperature; Thaumatin-like; Isolation; Castanopsis chinensis; Antifungal protein; Thaumatin-like; Chestnut
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/S0006-291X(02)02998-4

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 IC 50 of 0.5 μM against F. 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 40 °C for 10 min, was reduced after incubation at 60 °C, and disappeared after treatment at 80 °C. The antifungal activity underwent a decline after treatment with trypsin (enzyme:substrate ratio 1:100) at 37 °C for 1 h but some activity remained. Castanopsis TLP exhibited a much more potent inhibitory activity on HIV-1 reverse transcriptase ( IC 50=1.6 μM ) than kiwi fruit TLP ( IC 50⩾27 μM ). Castanopsis TLP was obtained with a yield of 20 mg from 1 kg chestnut seeds.