Properties and stabilization of an extracellular α-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN 1: enzyme activity at 130°C
Piller, K., Daniel, R.M. & Petach, H.H. (1996). Properties and stabilization of an extracellular α-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN 1: enzyme activity at 130°C. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1292(1), 197-205.
Permanent Research Commons link: https://hdl.handle.net/10289/4471
An extracellular α-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°C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of l% (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°C is 0.41 mM with pNP-α- -glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-α-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and β-gentiobiose. A variety of techniques including immobilization (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°C. The half-life could be increased from about 4 min at 110°C to 30–60 min at 130°C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and l% (w/v) BSA, and by cocross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130°C.
This is an author’s accepted version of an article published in the journal: Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. © 1996 Elsevier Science B.V.