The chitinolytic properties of Bacillus pabuli K1 isolated from mouldy grain was studied. Chitinase activity was measured as the release of p-nitrophenol from p-nitrophenyl-N,N'-diacetylchitobiose. Influences of substrate concentration and different environmental variables on growth and chitinase activity were determined. The optimum environmental conditions for chitinase production were: 30 degrees C, initial pH 8, initial oxygen 10% and aw > 0.99. Chitinase production was induced when B. pabuli K1 was grown on colloidal chitin. The smallest chito-oligosaccharide able to induce chitinase production was N,N'-diacetylchitobiose, (GlcNAc)2. Production was also induced by (GlcNAc)3 and (GlcNAc)4. When the bacterium was grown on glucose or N-acetylglucosamine, no chitinases were formed. The highest chitinase production observed was obtained with colloidal chitin as substrate. The production of chitinases by B. pabuli K1 growing on chitin was repressed by high levels (0.6%) of glucose. The production was also repressed by 0.6% starch, laminarin and beta-glucan from barley and by glycerol. The addition of pectin and carboxymethyl cellulose increased chitinase production.

Chitinolytic bacteria are used as biocontrol agents of plant pathogenic fungi. They might also potentially inhibit growth of molds, e.g., Aspergillus spp. and Penicillium spp., in stored plant material. We isolated chitinolytic bacteria from airtight stored cereal grain and evaluated their antifungal capacity. Between 0.01 and 0.5% of the total aerobic counts were chitinolytic bacteria. Gram-negative bacteria, mainly Pseudomonadaceae, constituted approximately 80% of the chitinolytic population. Gram-positive isolates belonged predominantly to the Corynebacterium-Arthrobacter group, Streptomyces, and Bacillus. Chitinolytic activity was evaluated using culture filtrates from chitin-grown isolates as the release of p-nitrophenol from p-nitrophenyl N,N'-diacetylchitobiose and as the formation of clearing zones on chitin agar. No correlation between chitinolytic activity and antifungal effects was found when challenging Penicillium roqueforti Dierckx with bacterial isolates on chitin agar in a dual culture bioassay. Fungal hyphae frequently grew seemingly unaffected through the bacterial colony of a high chitinase producer on colloidal chitin. Only 4% of the chitinolytic isolates had strong effects on fungal growth. Among these, Streptomyces halstedii (K122) and Streptomyces coelicolor (K139) inhibited growth of a broad range of fungi. Streptomyces halstedii affected hyphal morphology and decreased the radial growth rate of all fungi investigated. These effects were not caused by volatile metabolites, polyenes, or N-carbamoyl-D-glucosamine.Key words: antifungal, chitinase, Streptomyces halstedii, Streptomyces coelicolor.

Three methods of quantifying chitinase activity were compared. The activities of crude chitinases of 10 bacterial isolates from different environments were estimated in terms of (1) the release of p-nitrophenol from the chromogenic chito-oligosaccharide analogues, p-nitrophenyl-beta-D-N,N'-diacetylchitobiose, p-nitrophenyl-N-acetyl-beta-D-glucosamine and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, (2) the release of reducing sugars from chitin and (3) the formation of clearing zones on chitin agar. When crude chitinase from Bacillus pabuli was used the hydrolysis of p-nitrophenyl-beta-D-N,N'-diacetylchitobiose correlated well with the release of reducing sugars from chitin and the formation of clearing zones on chitin agar. However, when the activity of crude chitinases from the different bacterial isolates were compared no agreement was found between the hydrolysis of p-nitrophenyl-beta-D-N,N'-diacetylchitobiose and the release of reducing sugars from chitin or the formation of clearing zones on chitin agar. It was concluded that the assay with chromogenic p-nitrophenyl chito-oligosaccharide analogues is not well suited for studies that compare the chitinase activity of different bacteria.

Streptomyces halstedii K122 was previously found to produce antifungal compounds on solid substrates that inhibit radial growth of fungi among Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes, and Zygomycetes, and strongly affected hyphal branching and morphology. During growth of S. halstedii K122 in submerged culture, no antifungal activity could be detected. However, cultivation of S. halstedii in thin (1 mm) liquid substrate layers in large surface-area tissue culture flasks caused intense growth and sporulation of S. halstedii K122, and the biologically active compounds could be extracted from the mycelium with methanol. Antifungal compounds were purified using C18 solid phase extraction and silica gel column chromatography, and identified as bafilomycins B1 and C1, using 2D NMR and FAB MS. Production of bafilomycins, which are specific inhibitors of vacuolar ATPases, has not been reported from S. halstedii previously. Minimum inhibitory concentrations (MIC) of bafilomycins B1 and C1, amphotericin B, and nikkomycin Z were determined at pH 5.5 and 7.0 for the target fungi Aspergillus fumigatus, Mucor hiemalis, Penicillium roqueforti, and Paecilomyces variotii. Penicillium roqueforti was the most sensitive species to all the compounds investigated. The MIC values for amphotericin B were 0.5-4 micrograms.mL-1 for the fungi tested, and pH did not affect the toxicity. The MIC values for nikkomycin Z ranged from < 0.5 microgram.mL-1 for Mucor hiemalis to > 500 micrograms.mL-1 for Aspergillus fumigatus, and pH had no influence on toxicity. Bafilomycins B1 and C1 were equally active against the fungal species tested, with MIC values in the range of < 0.5-64 micrograms.mL-1. All fungi were more sensitive to both bafilomycin B1 and C1 at pH 7.0 than at pH 5.5.

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