Purification and Some Properties of a Thermostable Metal Proteinase Produced by Thermomicrobium sp. KN-22 Strain.

Abstract

A extreme thermophile that produces a heat-stable proteinase was isolated from hot-spring water and classified as Thermomicrobium sp. KN-22(growth temperature, 50-83°C; and optimum growth temperature, 70°C). The proteinase was purified from the culture broth of this strain by fractionation with ammonium suitate, chromatography on columns of DEAE-celluloseand CM-SepharoseCL-6B, and HPLCon TSKgel CM-5PW. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and a single peak after HPLC(yield 8.8%). The enzyme had maximumactivity at pH8.5 and at 75°C and it was stable up to 60°C. The molecular weight of the enzymewas 35,000 by SDS-PAGE.Since the enzymatic activity was completely inhibited by EDTA, o-phenanthroline, and phosphoramidon, it appears that the enzyme is a metal proteinase.Generally, enzymes are unstable to heat, and the purification and storage of an enzyme should be done at low temperature. Since enzymes produced by thermophiles are more thermostable than those produced by mesophiles, enzymes from thermophiles are stable at roomtemperature for long periods of time. Therefore, enzymes produced by thermophiles have been developed as reagents for clinical analyses. With respect to proteinases produced by extreme thermophiles, only a few enzymes, namely, caldolysin,1} aqualysin,2) and archaelysin,3) have been reported. We have been searching for novel thermostable proteinases, and wehave already reported a thermostable acid proteinase4) produced by a moderate thermophile.