Masanobu Ishihara

Purification, Characterization, and Antifungal Activity of Chitinases from Pineapple (Ananas comosus) Leaf

Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.

Antifungal Activity of Rye (Secale cereale) Seed Chitinases: the Different Binding Manner of Class I and Class II Chitinases to the Fungal Cell Walls

The antifungal activities of rye seed chitinase-a (RSC-a, class I) and -c (RSC-c, class II) were studied in detail using two different bioassays with Trichoderma sp. as well as binding and degradation experiments with the cell walls prepared from its mycelia. RSC-a inhibited more strongly the re-extension of the hyphae, containing mainly mature cells, than RSC-c did. Upon incubation of the fungus with fluorescent chitinases, FITC- labeled RSC-a was found to be located in the hyphal tips, lateral walls, and septa, while FITC-labeled RSC-c was only in the hyphal tip. RSC-a had a greater affinity for the cell walls than RSC-c. RSC-a liberated a larger amount of reducing sugar from the cell walls than RSC-c did. These results inferred that RSC-a first binds to the lateral walls and septa, consisting of the mature cell walls, and degrades mature chitin fiber, while RSC-c binds only to the hyphal tip followed by degradation of only nascent chitin. As a result, RSC-a inhibited fungal growth more effectively than RSC-c. Furthermore, it was suggested that the chitin-binding domain in RSC-a assists the antifungal action of RSC-a by binding to the fungal hypha.

Characterization and Antifungal Activity of Gazyumaru (Ficus microcarpa) Latex Chitinases: Both the Chitin-Binding and the Antifungal Activities of Class I Chitinase Are Reinforced with Increasing Ionic Strength

Three chitinases, designated gazyumaru latex chitinase (GLx Chi)-A, -B, and -C, were purified from the latex of gazyumaru (Ficus microcarpa). GLx Chi-A,-B, and -C are an acidic class III (33 kDa, pI 4.0), a basic class I (32 kDa, pI 9.3), and a basic class II chitinase (27 kDa, pI>10) respectively. GLx Chi-A did not exhibit any antifungal activity. At low ionic strength, GLx Chi-C exhibited strong antifungal activity, to a similar extent as GLx Chi-B. The antifungal activity of GLx Chi-C became weaker with increasing ionic strength, whereas that of GLx Chi-B became slightly stronger. GLx Chi-B and -C bound to the fungal cell-walls at low ionic strength, and then GLx Chi-C was dissociated from them by an escalation of ionic strength, but this was not the case for GLx Chi-B. The chitin-binding activity of GLx Chi-B was enhanced by increasing ionic strength. These results suggest that the chitin-binding domain of basic class I chitinase binds to the chitin in fungal cell walls by hydrophobic interaction and assists the antifungal action of the chitinase.

Localization, accumulation, and antifungal activity of chitinases in rye (Secale cereale) seed.

In order to understand a physiological role of chitinases in rye, the localization and accumulation of rye seed chitinase-a and -c (RSC-a and -c) in the seeds were studied by immunochemical methods. An antiserum specific to the chitin-binding domain (CB-domain), which is an N-terminal part of RSC-a, and an antiserum specific to the catalytic region of RSC-a and RSC-c were used. An immunoblot analysis detected both RSC-a and RSC-c in the endosperm of the rye seed. Immunohistochemical staining indicated that RSC-a was localized in only the aleurone cells, whereas RSC-c existed at least in the starchy endosperm and was also likely to exist in the aleurone cells. It was found by ELISA and an immunoblot analysis that RSC-a and -c accumulated in the seed during the later stage of development. Both chitinases and the Cat-domain exhibited antifungal activity toward Trichoderma species, while the CB-domain did not. Observation of the inhibition of hyphal growth of the T. species suggests that the two chitinases acted in different ways.

Purification and some properties of a thermostable xylanase from thermophilic fungus strain HG-1

Abstract An extracellular xylanase was purified to homogeneity from a wheat bran culture of the thermophilic fungus HG-1, an isolate from a compost heap. The enzyme had a molecular weight of 33,000 by SDS-PAGE and 31,000 by gel filtration; its isoelectric point was 6.8. The optimum temperature and pH for enzyme activity were 70°C and 4.5–5.0. The enzyme was stable in the pH range from 2 to 12 at 30°C. The K m values for birchwood xylan and oat-spelt xylan were 8.3 and 20 mg/ml, respectively. The enzyme produced xylobiose, xylotriose, and a trace of xylose as the endo products from birchwood xylan. The enzyme activity was strongly inhibited by SDS, and partially by Hg 2+ , Mn 2+ , Co 2+ , Ca 2+ , and iodoacetic acid. The enzyme hydrolyzed xylotriose to xylobiose and xylose and showed weak activity toward xylobiose.

Tissue distribution, synthesis stage, and ethylene induction of pineapple (Ananas comosus) chitinases.

We examined the tissue distribution, synthesis stage, and ethylene induction of three types of pineapple chitinase using chitinase activity gel and immunoblot analysis. Type A (acidic class III) exists in all tissues, while type B (weakly basic class I, which has strong antifungal activity) and type C (acidic class I) are localized mainly in the leaf and stem. In a pericarp, type A exists at all stages during fruit development, while type B and type C exist only at the early stage. Synthesis of type A is induced by ethylene, while that of types B and C is not affected by it. These results suggest that the physiological roles of these three types of chitinase in pineapple are different.

Disintegration of Uncooked Rice by Carboxymethyl Cellulase from Sporotrichum sp.HG-I

A thermostable carboxymethyl cellulase (CMCase) was purified to homogeneity from a wheat bran culture of the thermophilic fungus Sporotrichum sp. HG-1, an isolate from a compost heap. The enzyme had a molecular weight (M(r)) of 33,000 by SDS-PAGE. The optimum temperature and pH for enzyme activity were 70 degrees C and 4.5-5.0, and the enzyme was heat stable. Uncooked Thai rice was digested so as to cause its disintegration by the addition of purified CMCase, but not by the addition of xylanase purified from strain HG-1. Ferulic acid conjugated to oligosaccharide was released significantly by the combined action of CMCase and xylanase, but the free form of ferulic acid was not detectable.

Synthesis and Fungicidal Activity of 6-Alkyl Six-membered Cyclic Thiophosphates.

Synthesis and fungicidal activities of new 6-aIkyl six-membered cyclic phosphates were examined. Ten kinds of 6-alkyl six-membered cyclic thiophosphates were synthesized by reaction with 5-alkyl-2-hydroxybenzyl alcohols and phosphoric agents. Among the prepared compounds, 2-ethoxy-6-elhyl-4H-1,3,2-benzodioxaphosphorin 2-sulfide (1) had activity as potent as the commercial fungicide iprobenfos against Pythium sp. and Corticium rolfsii at 10ppm.