PURIFICATION AND CHARACTERIZATION OF ALKALINE-PHOSPHATASE FROM THERMOTOGA-NEAPOLITANA

A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100 degrees C in the presence of Co2 followed by ion-exchange and affinity chromatographies. The enzyme wa s purified 2,880-fold with 44% yield. The purified enzyme showed a sin gle protein band of M-r 45,000 on SDS-PAGE and an apparent molecular w eight of 87,000 estimated by gel filtration chromatography. This sugge sted a homogenous dimer structure. The optimal pH and temperature for enzyme activity were 9.9 and 85 degrees C, respectively. Under optimal conditions, T. neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophen yl-phosphate as substrate. The hyperthermostable enzyme had a half-lif e of 238 min at 90 degrees C and K-m and V-max values of 183 mu M and 1,352 U mg(-1), respectively. Co2+ enhanced the enzyme activity, therm ostability, and ligand affinity during column chromatography. The alka line phosphatase was twice as active with Co2+ than with either Zn2+ o r Mn2+ as the metal cofactor. (C) 1997 Elsevier Science Inc.