CHARACTERIZATION OF A CONIDIAL ALKALINE-PHOSPHATASE FROM THE THERMOPHILIC FUNGUS HUMICOLA-GRISEA VAR. THERMOIDEA

An extracellular alkaline phosphatase (EC 3.1.3.1) was purified from c onidia of Humicola grisea var. thermoidea. The molecular mass of the p urified native enzyme was estimated as 126 kDa by gel filtration. SDS- PAGE revealed a single polypeptide band of 63 kDa, suggesting that the native enzyme was a dimer of identical subunits. The pi of the enzyme was about 3.0. Optima of pH and temperature were 9.0 and 70 degrees C , respectively. In Tris-HCl buffer, pH 9.0, the enzyme was activated b y MgCl2 and strongly inhibited by ZnCl2, CoCl2, Al2Cl3, HgCl2, EDTA an d beta-mercaptoethanol. In contrast, in glycine buffer and at the same pH the enzyme was activated by ZnCl2 and CoCl2, as well as by MgCl2. The enzyme was fully stable when incubated up to one hour at 65 degree s C without magne slum labilized the enzyme incubated at 65 degrees C or 70 degrees C. p-Nitrophenyl phosphate was the best substrate, but A MP, ADP, ATP and beta-glycerophosphate also served as substrates. The remarkable thermostability exhibited by the purified alkaline phosphat ase of H. grisea may be of interest for practical applications.