PURIFICATION AND CHARACTERIZATION OF SUCROSE ALPHA-GLUCOHYDROLASE (INVERTASE) FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

Sucrose alpha-glucohydrolase (or invertase) catalyzes the hydrolysis o f sucrose to fructose and glucose. The activity was detected in the cy toplasmic fraction of cell-free extracts of the hyperthermophilic arch aeon (archaebacterium), Pyrococcus furiosus, an organism that grows op timally at 100-degrees-C. The enzyme was purified 570-fold to electrop horetic homogeneity. It is a monomeric protein of M(r) 114,000. It exh ibited optimum activity, as measured by the production of glucose from sucrose, at pH 5.5 and temperatures between 100 and 117-degrees-C. Wh ile maltose was also hydrolyzed by this enzyme, no activity could be d etected towards starch. Kinetic analyses at 95-degrees-C established K (m) and V(max) values for sucrose hydrolysis of 0.12 M, and 130 mumole min-1 mg-1, respectively. Classical competitive inhibition was observ ed in the presence of glucose or fructose with inhibition constants (K (i)) of 0.55 mM and 50 mM, respectively. Nevertheless, the enzyme was capable of partially hydrolyzing sucrose at concentrations up to 2.0 M at 90-degrees-C. However, the P. furiosus enzyme is by far the most t hermostable invertase yet reported, exhibiting a half life of almost 2 days at 95-degrees-C.