Purification and characterization of thermostable glucose isomerase from Clostridium thermosulfurogenes and Thermoanaerobacter strain B6A.
Glucose isomerases produced by Thermoanaerobacter strain B6A and Clostridium thermosulfurogenes strain 4B were purified 10-11-fold to homogeneity and their physicochemical and catalytic properties were determined. Both purified enzymes displayed very similar properties (native Mr 200,000, tetrameric subunit composition, and apparent pH optima 7.0-7.5). The enzymes were stable at pH 5.5-12.0, and maintained more than 90% activity after incubation at high temperature (85 degrees C) for 1 h in the presence of metal ions. The N-terminal amino acid sequences of both thermostable glucose isomerases were Met-Asn-Lys-Tyr-Phe-Glu-Asn and were not similar to that of the thermolabile Bacillus subtilis enzyme. The glucose isomerase from C. thermosulfurogenes and Thermoanaerobacter displayed pI values of 4.9 and 4.8, and their kcat. and Km values for D-glucose at 65 degrees C were 1040 and 1260 min-1 and 140 and 120 mM respectively. Both enzymes displayed higher kcat. and lower Km values for D-xylose than for D-glucose. The C. thermosulfurogenes enzyme required Co2+ or Mg2+ for thermal stability and glucose isomerase activity, and Mn2+ or these metals for xylose isomerase activity. Crystals of C. thermosulfurogenes glucose isomerase were formed at room temperature by the hanging-drop method using 16-18% poly(ethylene glycol) (PEG) 4000 in 0.1 M-citrate buffer.
Lee
C Y
CY
Department of Biochemistry, Michigan State University, East Lansing 48824.
Zeikus
J G
JG
D019747:Aldose-Ketose Isomerases; D000596:Amino Acids / Q000032:analysis; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology*; D003460:Crystallization; D004795:Enzyme Stability; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D013816:Thermodynamics
D000596:Amino Acids; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase