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 archaebacteri um Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration an d Mono Q chromatography) with a yield of 4%. It is a monomer with a mo lecular mass of about 60 ka and a pI around 5. At 98 degrees C, the pu rified enzyme in buffer has a half-life around 35 min, which is increa sed 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 substantial ly increase the enzyme activity. The K-m at 75 degrees C is 0.41 mM wi th pNP-alpha-D-glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-alpha-D-glucoside > nigerose > panose > palatino se > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and beta-gentiobiose. A variety of techniques including im mobilization (e.g., on epoxy and glass beads), chemical modification ( cross- and cocross-linking) and the use of additives (including polyhy droxylic molecules, BSA, salts, etc.) were applied to enhance stabilit y at temperatures above 100 degrees C. The half-life could be increase d from about 4 min at 110 degrees C to 30-60 min at 130 degrees C in p resence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and 1% (w/v) BS A, and by cocross-linking with BSA in the presence of 90% (w/v) sorbit ol. The stabilized enzyme showed good activity at 130 degrees C.