CHARACTERIZATION OF AMYLOLYTIC ENZYMES, HAVING BOTH ALPHA-1,4 AND ALPHA-1,6 HYDROLYTIC ACTIVITY, FROM THE THERMOPHILIC ARCHAEA PYROCOCCUS-FURIOSUS AND THERMOCOCCUS-LITORALIS

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90-degrees-C) and Pyrococcus furiosus (optimal growth temperature, 98-degrees-C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca2+, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140-degrees-C. The addition of Ca2+ also affected substrate binding, as evidenced by a decrease in K(m) for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the alpha-1,6 linkages in pullulan, alpha-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller alpha-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.