Purification and properties of esterase from Bacillus stearothermophilus

An enzyme, which hydrolyzes p-nitrophenyl and m-carboxyphenyl esters of n-fatty acids, is purified from Bacillus stearothermophilus. The enzyme reaction obeys the Michaelis-Menten theory. The Michaelis constant (K_m) decreases with increasing the length of carbon number of the acids, but the maximum velocity (V) is maximum for n-caproate. The enzyme is inhibited by diisopropyl fluorophosphate (DFP),² and 1 mole of DFP reacts with 1 mole of the enzyme of the molecular weight of 42,000–47,000. The enzyme is considered to be carboxylic ester hydrolase (EC 3.1.1.1). The effects of temperature on K_m or V for p-nitrophenyl n-caproate and on the inhibitor constant (K_i) for n-laurate suggest a thermal transition in the conformation of the enzyme protein at 55 °C. The enzyme is strongly inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and 5,5′-dithiobis (2-nitrobenzoic acid) at 65 °C, but less at 30 °C. The relationship between the inhibition of the activity by p-chloromercuribenzoate and temperature may suggest that a thermal transition of the enzyme protein accompanies some structural change around sulfhydryl group.