PURIFICATION AND PROPERTIES OF A HYPERTHERMOACTIVE ALPHA-AMYLASE FROM THE ARCHAEOBACTERIUM PYROCOCCUS-WOESEI

The cultivation of the hyperthermophilic archaeobacterium Pyrococcus woesei on starch under continuous gassing (80% H2:20% CO2) caused the formation of 250 U/l of an extremely thermoactive and thermostable alpha-amylase. In a complex medium without elemental sulphur under 80% N2 and 20% CO2 atmosphere enzyme production could be elevated up to 1000 U/l. Pyrococcus woesei grew preferentially on polyand oligosaccharides. The amylolytic enzyme formation was constitutive. Enzyme production was also observed in continuous culture at dilution rates from 0.1 to 0.4 h-1. A 20-fold enrichment of alpha-amylase was achieved after adsorption of the enzyme onto starch and its desorption by preparative gel electrophoresis. The alpha-amylase consisted of a single subunit with a molecular mass of 70000 and was catalytically active at a temperature range between 40-degrees-C and 130-degrees-C. Enzymatic activity was detected even after autoclaving at a pressure of 2 bars at 120-degrees-C for 5 h. The purified enzyme hydrolyzed exclusively alpha-1,4-glycosidic linkages present in glucose polymers of various sizes. Unlike many alpha-amylases from anaerobes the enzyme from P. woesei was unable to attack short chain oligosaccharides with a chain length between 2 and 6 glucose units.