ISOLATION AND CHARACTERIZATION OF A HEAT-STABLE PULLULANASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-WOESEI AFTER CLONING AND EXPRESSION OF ITS GENE IN ESCHERICHIA-COLI

The gene encoding an extremely heat-stable pullulanase from the hypert hermophilic archaeon Pyrococcus woesei was cloned and expressed in Esc herichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chroma tography on a maltotriose-coupled Sepharose 6B column, and anion-excha nge chromatography on Mono Q. The pullulanase, which was purified 90-f old with a final yield of 15%, is composed of a single polypeptide cha in with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzy me activation up to 370% is achieved in the presence of calcium ions a nd reducing agents such as beta-mercaptoethanol and dithiothreitol, wh ereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The hi gh rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sod ium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme se ems to switch from the compact to the unfolded form, which is accompan ied by an apparent shift in the molecular mass from 45 to 90 kDa.