THERMOPHILIC AMINOPEPTIDASES FROM BACILLUS STEAROTHERMOPHILUS. I. ISOLATION, SPECIFICITY, AND GENERAL PROPERTIES OF THE THERMOSTABLE AMINOPEPTIDASE I *

Abstract

A thermostable aminopeptidase (AP 1) from homogenates of B. stearothermo‐philus was purified by filtration on Sephadex G‐150, by heat treatment, by chromatography on DEAE Sephadex and Sephadex G‐200, and by preparative polyacrylamide gel electrophoresis. The enzyme shows one band in polyacrylamide gel electrophoresis and has a pH optimum between 7.5 and 8 for LNA and between 9.2 and 9.4 for Gly‐Leu‐Tyr. One striking feature in the amino‐acid composition of AP I in comparison with the data of leucinaminopeptidase is the larger proportion of hydrophobic amino‐acid residues and of glycine. As regards its specificity, AP I differs from other amino‐peptidases in certain important respects. The activation energy for the hydrolysis of Leu‐Gly was calculated to be 16,300 cal/M ‐1 . The enzyme remains stable for several hours at 80 C; after 30 min at this temperature the activity increases by about 20 per cent ( V m increases, while K m remains the same). The stability of the enzyme in urea, dodecyl sulphate, and various alcohols was studied. The activity of the enzyme even increases in 10 per cent tertiary butanol, in the same way as after heat treatment ( V m increases, while K m remains the same). The kinetics of the increase in activity following heat treatment or in 10 per cent tertiary butanol are discussed. At 20°C, AP I exhibits a sigmoidal dependence of reaction velocity on the concentration of LNA whereas at 40°C or 80°C the curves show a hyperbolic behaviour. In the metal‐enzyme complex of AP I the Co 2+ ions are strongly bound at pH 8 (up to 10 ‐2 MEDTA). At a pH lower than 6 the stable apo‐enzyme is formed in 10 ‐2 M EDTA. The apo‐enzyme can be reactivated to the holo‐enzyme by adding Co ‐2 . The metal‐enzyme complexes of various metals show different substrate specificities.