Folia Microbiol. 45 (5), 465-468 (2000) http ://www .biomed. cas. cz/mbu/folia/ Chitinolytic Enzymes Produced by Ovine Rumen Bacteria J. KOPE~NY, B. HODROVA Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 104 O0 Prague l O-Uhr'in~ves, Czechia fax +420 2 6771 0803 e-mail kopecny@iapg.cas.cz Received 17 August 2000 ABSTRACT. Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes. Enzyme tests in cultures demonstrated endochitinase, exochitinase, N-acetyl- glucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions In all samples, the highest was the activity of exochitinase (600-1100 nmol mL -1 h-l); the activity of endochitinase (280-500 nmol mL -1 h-1) was also significant. Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was obser- ved. In both strains different isoforms of chitinases of molar mass 36--96kDa were detected. The chitinases from our isolates lyzed cell walls of anaerobic fungi in vitro and inhibited the activity of fungal [3-1,4-endoglucanases. Of the two bacteria examined, one was more effectivein both antifungal effects. Metabolic interactions among different types of microbes are essential for sustaining the microbial community in the rumen. There are many types of interactions, and one - amensalism - is based on the production by one organism of a compound inhibitory to the growth of another (Wolin et al. 1997). This effect has been found in cocultures of anaerobic fungi with rumen cellulolytic bacteria (Bernalier et al. 1993). Simultaneously, it was observed that chitinase produced by the non-rumen microbe Streptomyces griseus significantly inhibited the activity of rumen fungal endoglucanase (Wilson and Wood 1992). Later, it was found that bovine rumen Clostridium tertium strain ChK5 synthesized compounds causing inhibition of cellulose degradation by anaerobic fungi (Kopec~n~ et al. 1996a). Here we characterize the enzymic complex of two chitinolytic rumen bacteria from sheep and their effect on anaerobic fungi and suggest a mechanism ofrumen-fungi inhibition by chitinolytic bacteria. MATERIAL AND METHODS Chemicals. Colloidal chitin was prepared from crab shell chitin (Sigma) according to Shimahara and Yakiguchi (t 988). Microorganisms. Chitinolytic bacteria were isolated from the rumen fluid of sheep and tentatively named Clostridium sp. OV 13 and Clostridium sp. OV 14. These strains were maintained under an anaerobic atmosphere (Nz--COz-H2, 14 : 5 : 1) in modified medium M10 with 0.4 % (W/V) colloidal chitin (Kope~n~ et al. 1996b). Anaerobic fungi were cultivated on the same medium with 20 % of rumen fluid. The polycentric Orpinomyces joyonii strain A4 was isolated from the rumen fluid of a camel (Hodrov6 et al. 1995). The monocentric Neocallimastix frontalis strain OV3 was isolated from the rumen of sheep and fitted the description ofN. frontalis by Orpin (1994). Enzyme assays and analytical methods. Enzyme activities were measured in cellular and exocellu- lar fractions. Bacterial cells were harvested (8500 g, 10 rain, 4 ~ and resuspended in distilled water (lh0 of the original volume). Supematants were concentrated 20• by ultrafiltration (PM. 10, cutoff of M 10 kDa; Amicon). Both enzyme fractions were stored at -30 ~ Exochitinase was asseyd with 4-nitrophenyl-N,N'-diacetylchitobiose according to Roberts and Selitrennikoff (1988). Endochitinase activity was estimated with CM-chitin as substrate and reducing sugars were detected with 4-hydroxybenzhydrazide (Lever 1977). Chitin deacetvlase was determined with colloidal chitin and the produced chitosan was estimated with 3-methyl-2-benzothiazoline hydrazone (Kauss and Bauch 1988). Chitosanase activity was estimated with colloidal chitosan and reducing sugars estimated with 4-hydroxybenzhydrazide (Lever 1977). N-Acetyl-fl-glucosaminidase was assayed with 4-nitrophenyl-N- acetyl-ct-D-glucosaminide (Sigma) according to Kope~n~, et al. (1996b). All enzyme activities were determined at least in triplicate if not otherwise stated and were expres- sed at levels present in the original culture. Zymograms were done by the method for detection of endoglucanases (Flint et al. 1994). Instead of CM-cellulose the separating gel contained 0.1% CM-chitin.