The fructanolytic abilities of the rumen bacterium Butyrivibrio fibrisolvens strain 3071.

AIMS: To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. METHODS AND RESULTS: Strain 3071 utilized 97, 89, 85 and 60% of sucrose, timothy grass fructan, inulin oligosaccharides and inulin, respectively, in the growth medium. A cell extract from timothy grass fructan-grown bacteria was used for identification of fructanolytic enzymes by anion exchange chromatography, gel filtration, zymography and thin-layer chromatography. The bacterium synthesizes a specific endolevanase and a nonspecific ß-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The ß-fructofuranosidase was not able to digest long-chain inulin or timothy grass fructan, but degraded inulin oligosaccharides and sucrose. Addition of 1,4-dithioerythritol to an enzyme solution did not affect the activity of endolevanase(s), but increased the ability of ß-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km  = 2·82 g l(-1) and Vmax  = 4·01 µmoles reducing sugar equivalents × mg(-1)  × min(-1) . CONCLUSION: Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Fructanolytic and saccharolytic enzymes of the rumen bacterium Pseudobutyrivibrio ruminis strain 3--preliminary study.

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.

Phosphorolytic cleavage of sucrose by sucrose-grown ruminal bacterium Pseudobutyrivibrio ruminis strain k3.

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90% sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase (EC 2.4.1.7) of molar mass approximately 54 kDa and maximum activity at pH 6.0 and 45 degrees C. The calculated Michaelis constant (Km) for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction (Vlim) were 1.19 and 0.64 micromol/L per mg protein per min, respectively.

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A.

AIMS: To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose. METHODS AND RESULTS: Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis. Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6.0 and temperature 45 degrees C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K(m) for glucose-1-P formation and fructose release were 3.88 x 10(-3) and 5.56 x 10(-3) mol l(-1) sucrose, respectively - while the V(max) of the reactions were -0.579 and 0.9 mumol mg protein(-1) min(-1). The enzyme also released free glucose from glucose phosphate. CONCLUSION: Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.