PURIFICATION, N-TERMINAL AMINO-ACID SEQUENCING AND ANTIFUNGAL ACTIVITY OF CHITINASES FROM PEPPER STEMS TREATED WITH MERCURIC-CHLORIDE

Different isoforms of chitinases were purified from pepper (Capsicum a nnuum L. cv. Hanbyul) stems treated with mercuric chloride. The acidic isoform a1 (69 kDa, pI 5.0), basic isoforms b1 (32 kDa, pI 9.0) and b 2 (22 kDa, pI 9.1) were purified by chitin-affinity chromatography, wi th subsequent electroelution from nondenaturing polyacrylamide gel ele ctrophoresis (PAGE) gels. The acidic isoform al has chitin-binding pro perties, but no antifungal activity. The basic isoforms b1 and b2 cont ain high ratios of cysteine and glycine at the N-terminal chitin-bindi ng domain, exhibit chitinase activity, and show antifungal activities against Colletotrichum gloeosporioides, Fusarium oxysporum f. sp. cucu merinum, Magnaporthe grisea, and Trichoderma viride in vitro. Moreover , their antifungal activity shows a high degree of specificity to fila mentous fungi. The chitinases b1 and b2 show a high sequence identity in their N-terminal residues with those from wheat, tobacco, potato, r ice and Arabidopsis thaliana. None of the purified isoforms of chitina ses inhibited hyphal growth of the Oomycete fungus which lacks chitin Phytophthora capsici. In contrast, zoospore germination and germ tube elongation of P. capsici were effectively inhibited by treatment with b1 and b2. (C) 1996 Academic Press Limited