Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity

Abstract

Thermolysin, a thermophilic metalloproteinase, is markedly activated in the presence of high concentrations (1–5 M) of neutral salts. The activity increases in an exponential fashion with increasing salt concentration, and is enhanced 13–15 times with 4 M NaCl at pH 7.0 and 25°C (K. Inouye, Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy- l-aspartyl- l-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin, J. Biochem. 112 (1992) 335–340). In this study, the effect of NaCl on the thermal stability of thermolysin has been examined at 60–85°C. The activation energy, E a, for the thermal inactivation is 15 kcal/mol at 0 M NaCl, and increases up to 30–33 kcal/mol by the addition of 0.5–1.5 M NaCl. Further increase in [NaCl] decreases the E a value, and at 4 M NaCl it is almost the same as that at 0 M NaCl. Thermolysin at 0.5–1.5 M NaCl is twice as heat-stable as in the absence of NaCl. The NaCl dependence of the stability is different from that of the activity, suggesting that the effects of NaCl on activity and stability are independent. Thermolysin has been demonstrated to be not only a thermophilic enzyme but also a highly halophilic one.