PURIFICATION AND PROPERTIES OF A THERMOSTABLE PULLULANASE FROM A NEWLY ISOLATED THERMOPHILIC ANAEROBIC BACTERIUM, FERVIDOBACTERIUM PENNAVORANS VEN5

Extremely thermophilic anaerobic fermentative bacteria growing at temp eratures between 50 and 80 degrees C (optimum, 65 to 70 degrees C) wer e isolated from mud samples collected at Abano Terme spa (Italy), The cells were gram-negative motile rods, about 1.8 mu m in length and 0.6 mu m in width, occurring singly and in pairs. Cells commonly formed s pheroids at one end similar to Fervidobacterium islandicum and Fervido bacterium nodosum. The new isolate differs from F. nodosum by the 7% h igher G+C content of its DNA (40.6 mol%) but is similar to Fervidobact erium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 bel ongs to the order Thermotogales and shows the highest 16S ribosomal DN A sequence similarity to F. pennavorans, F. islandicum, and F. nodosum , with similarities of 99.0, 98.6, and 96.0%, respectively, During gro wth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed alpha-1,6 glucosidic linkages. The en zyme was purified 65 fold by anion-exchange, gel permeation, and hydro phobic chromatography, The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular ma ss of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stabilit y of the purified pullulanase were pH 6.0 and 85 degrees C, At pH 6.0, the half-life of the enzyme was over 2 h at 80 degrees C and 5 min at 90 degrees C, This is the first report on the presence of pullulanase type I in an anaerobic bacterium.