Molecular mechanism, protein engineering, and application of a thermophilic and halophilic enzyme, thermolysin

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Effect of chemical modification of Tyr, COOH, and Lys residues of thermolysin (E) on its activity and halophilicity was examined. The activity was decreased by the modification of Tyr and Lys residues, while that was not change by that of carboxyls. The decrease in the halophilicity was observed with decreasing the charges on the surface of E.Lys residues were modified by polyethylene glycol (PEG). The activity of E was increased with the degree of the modification, and was 6-times higher when 6 Lys residues were modified. E was stabilized with the PEG modification. The effect of PEG was similar to that of the addition of salts. Two E varinats (T1 and T2) were isolated from mutants of the E-producing bacteria. As with T1, Val-140 of E was converted to Ala, and with T2, Ala-73 was converted to Val. The protease activity of T1 was 150% in comparison with that of the native E, and the peptidase activity was 35% On the other hand, the protease activity of T2 was 10% of that of the native E … More , but the peptidase activity was increased remarkably, to 1,200%. The both residues were shown to have critical influence in determining of the substrate-specificity of E.Introduction of a bulky group into the place of Asp-73 depressed strongly the protease activity, and turned E to a peptidase. The A73V enzyme has 10-times higher activity of synthesis of the aspartame precurser. The active-site zinc was replaced with cobal, and the Co-E was shown to have 5-times higher activity than the native E.Co-E exhibited halophilicity, as the parent E does, and the activity at 4 M NaCl was 15-times higher than that of the native one. The activity-and-structure of E was compared with that of matrilysin. It showed also strong halophilicity, and higher thermal stability. The structure was found to be similar to that of the catalytic domain of E.It was also observed that the molecular mechanism of of halophilicity of E and matrilysin is different ; namely that the halophilicity of E was derived by the stabilizing the transition-state of the substratre, whereas that of matrilysin was by stabilizing its ground-state of the substrate. Less