Aims: To characterize the fructose polymer degrading enzymes of rumen bacterium Treponema saccharophilum strain S. Methods and Results: Conventional methods were used to examine bacterial growth and enzyme activities. Electrophoretic zymogram under native conditions, and thin layer chromatography, were applied to identify and characterize the enzymes. Treponema saccharophilum utilized Timothy grass fructan, inulin and sucrose but not free fructose. Timothy grass fructan was degraded at a signi®cantly higher rate than sucrose and inulin. Two fructanolytic enzymes were found in the soluble, and one in the membrane fraction of bacterial cell extract. The ®rst degraded each mentioned carbohydrate to monosaccharides. The second released oligosaccharides only from Timothy grass fructan. Conclusions: The bacterium T. saccharophilum strain S is capable of synthesizing non-speci®c b-fructofuranosidases and 2,6-b-D D-fructan fructanohydrolase. The enzymes are of constitutive character. Signi®cance and Impact of the Study: It has been stated for the ®rst time that the 2,6-b-D Dfructan fructanohydrolase is synthesized by the rumen bacterium T. saccharophilum. This organism appears to be responsible for grass fructan degradation in the rumen.
During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.
P. ruminis strain 3 was isolated from the ovine rumen and identified on the basis of comparison of its 16S rRNA gene with GenBank. The bacterium was able to grow on Timothy grass fructan, inulin, sucrose, fructose and glucose as a sole carbon source, reaching absorbance of population in a range of 0.4-1.2. During 1 d the bacteria exhausted 92-97% of initial dose of saccharides except for inulin (its utilization did not exceed 33%). The bacterial cell extract catalyzed the degradation of Timothy grass fructan, inulin and sucrose in relation to carbon source present in growth medium. Molecular filtration on Sephadex G-150, polyacrylamide gel electrophoresis combined with zymography technique and TLC was used to identify enzymes responsible for the digestion of sucrose and both polymers of fructose. Two specific endolevanases (EC 3.2.1.65), nonspecific beta-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7) were detected in cell-free extract from bacteria grown on Timothy grass fructan.
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