Summary auto-generated
This paper describes the purification and characterization of α-D-glucuronidases from two thermophilic anaerobic bacteria: Clostridium stercorarium and Thermoanaerobacterium saccharolyticum. These enzymes catalyze the hydrolysis of side-chain glucuronic acid residues from glucuronoxylan, a complex plant polysaccharide. The researchers developed a novel enzyme assay using 4-O-methyl-α-D-glucuronosyl-xylotriose as substrate and detected glucuronic acid release colorimetrically. Both enzymes were found to be intracellular proteins with dimeric structures composed of ~70 kDa subunits. The α-D-glucuronidases showed pH optima near 6.0 and differed significantly in thermostability and specific activity. These represent the first α-D-glucuronidases purified to homogeneity from bacterial sources, previously characterized only from fungi. The intracellular localization suggests these bacteria employ a coordinated degradation strategy where extracellular enzymes depolymerize high-molecular-mass xylan, and the resulting oligosaccharides are transported into cells for further processing by intracellular α-D-glucuronidase and other enzymes.
Key findings
- α-D-glucuronidases from C. stercorarium and T. saccharolyticum are the first bacterial α-D-glucuronidases purified to homogeneity; previously only fungal enzymes had been purified
- Both enzymes are intracellular dimeric proteins with ~70 kDa subunits, showing pH optimum around 6.0 but differing in thermostability (C. stercorarium half-life 14 h at 60°C versus T. saccharolyticum 2.5 h)
- The T. saccharolyticum enzyme showed higher specific activity (10 U mg⁻¹) compared to C. stercorarium (1.7 U mg⁻¹) toward the oligomeric substrate
- Both enzymes failed to hydrolyze polymeric glucuronoxylan or the synthetic p-nitrophenyl-α-D-glucuronide substrate, indicating substrate specificity for oligomeric substrates
- These bacteria employ a cooperative degradation strategy with extracellular main-chain-cleaving enzymes producing oligosaccharides that are transported intracellularly for side-chain removal by α-D-glucuronidase
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Abstract
alpha-D-Glucuronidases were purified from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum. This enzyme activity was found to be intracellular in each organism, with T. saccharolyticum producing much greater total activity. The specific activities of the purified enzymes (10 U mg-1 T. saccharolyticum; 1.7 U mg-1 C. stercorarium) differed by a factor of approximately 5. For the determination of enzyme activities, 4-O-methyl- alpha-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by alpha-D-glucuronidase action was quantified by a colorimetric procedure. 4-O-Methyl-alpha-D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of 4-O-methyl-alpha-D-glucuronoxylan with xylanases of C. stercorarium. Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected. Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-nitrophenyl-alpha-D- glucuronoside. Both alpha-D-glucuronidases have a dimeric structure, with monomeric molecular masses of 72 and 76 kDa for C. stercorarium and of 71 kDa for T. saccharolyticum. The pI was estimated to be 4.3 for each enzyme. While both enzymes exhibited a similar pH optimum (pH 5.5-6.5) they differed in their thermostabilities. At 60 degrees C, half-lives of 14 and 2.5 h, respectively, were determined for the alpha- D-glucuronidases of C. stercorarium and T. saccharolyticum. This description of alpha-D-glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi.