Properties and stabilization of an extracellular alpha-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN1: enzyme activity at 130 degrees C.
An extracellular alpha-glucosidase from the thermophilic archaebacterium Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration and Mono Q chromatography) with a yield of 4%. It is a monomer with a molecular mass of about 60 kDa and a pI around 5. At 98 degrees C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of 1% (w/v) dithiothreitol and 1% (w/v) BSA. Dithiothreitol (1%, w/v) and BSA (0.4%, w/v) also substantially increase the enzyme activity. The Km at 75 degrees C is 0.41 mM with pNP-alpha-D-glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-alpha-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and beta-gentiobiose. A variety of techniques including immobolization (e.g., on epoxy and glass beads), chemical modification (cross- and cocross-linking) and the use of additives (including polyhydroxylic molecules, BSA, salts, etc.) were applied to enhance stability at temperatures above 100 degrees C. The half-life could be increased from about 4 min at 100 degrees C to 30-60 min at 130 degrees C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and 1% (w/v) BSA, and by cross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130 degrees C.
Thermophile Unit, School of Science and Technology, Waikato University, Hamilton, New Zealand.
K
K
Piller
R M
RM
Daniel
H H
HH
Petach
D001105:Archaea / Q000201:enzymology*; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013012:Sorbitol / Q000494:pharmacology; D013379:Substrate Specificity; D014508:Urea / Q000494:pharmacology; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008670:Metals; D012967:Sodium Dodecyl Sulfate; D013012:Sorbitol; D014508:Urea; D000520:alpha-Glucosidases; D004229:Dithiothreitol