Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.
Clostridium thermohydrosulfuricum mutant Z 21-109 produced intracellular thermostable pullulanase and glucoamylase activities. The glucoamylase activity was inactivated by treating C. thermohydrosulfuricum cells with 10% (v/v) propan-1-ol at 85 degrees C in the presence of 5 mM-CaCl2. Pullulanase activity was selectively solubilized from cells by treatment with detergent and lipase. The solubilized pullulanase was purified by treatment with streptomycin sulphate and (NH4)2SO4 and by DEAE-Sephacel, octyl-Sepharose and pullulan-Sepharose chromatography. Pullulanase was purified 3511-fold and displayed homogeneity on SDS/polyacrylamide-gel electrophoresis. The pullulanase was a monomeric glycoprotein with an apparent Mr of about 136,500, and it displayed a pI of 5.9. The enzyme was enriched in both acidic and hydrophobic amino acids. The purified pullulanase was stable and optimally active at 90 degrees C. The optimum pH for activity and pH-stability ranges were 5.0-5.5 and 3.0-5.0 respectively. The enzyme was inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but not by p-chloromercuribenzoate and acarbose. The pullulanase displayed a relative substrate specificity for hydrolysis of pullulan (100%) versus 75% for glycogen and 50% for soluble starch. The apparent Km, Vmax. and Kcat. values for enzyme activity on pullulan at 60 degrees C were 0.675 mg/ml, 122.5 mumol of reducing sugar formed/min per mg of protein and 16,240 min-1 respectively. The novel properties of this extremely thermostable pullulanase are discussed in relation to other purified starch-debranching enzymes.
Saha
B C
BC
Michigan Biotechnology Institute, Lansing 48909.
Mathupala
S P
SP
Zeikus
J G
JG
D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity
D000596:Amino Acids; D006026:Glycoside Hydrolases; C100181:pullulanase