Analysis of Autodegradation Sites of Thermolysin and Enhancement of Its Thermostability by Modifying Leu¹⁵⁵ at an Autodegradation Site

Abstract

The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca²⁺. One of the sites was identified as Gly¹⁵⁴-Leu¹⁵⁵, and Leu¹⁵⁵ was substituted with various amino acids, X=Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80°C increased with the amino acid substitutions in the order of Ala>Phe>Ser>Gly>Leu (WT TLN). An additional autodegradation fragment that was not observed with WT TLN appeared for all mutant TLNs examined. The autodegradation site shifted from the Gly¹⁵⁴-Leu¹⁵⁵ bond to the X¹⁵⁵-Ile¹⁵⁶ one with the mutation at Leu¹⁵⁵. Furthermore, the Ile¹⁶⁴-Asp¹⁶⁵ bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.