SITE-DIRECTED MUTATION OF A LACCASE FROM THERMUS THERMOPHILUS: EFFECT ON THE ACTIVITY PROFILE

A site-directed mutant R453T of a laccase from Thermus thermophilus HB27 (Tth -laccase) was constructed in order to investigate the effect on laccase catalytic properties. The mutated gene was cloned and overexpressed in Escheri- chia coli. Nickel-affinity purification was achieved and followed by copper ion incorporation. The mature mutated enzyme was quantitatively equal to the wild type. A photometric assay based on the oxidation of the substrate 2,2-azino-bis-(3- ethylbenzthiazoline-6-sulfonate) (ABTS) was employed in comparison with the wild-type Tth-laccase on catalytic prop- erties. The R453T mutant exhibited improvement in substrate affinity and specific activity at room temperature, whereas those parameters were not significantly influenced when the temperature increased up to 65°C or higher. The mutant had better catalytic activity than that of the wild type at acidic pH. Investigated by circular dichroism spectroscopy, the mutant Tth-laccase displayed similar profiles at low and high temperatures.