Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation.

To select better performing laccase variants among the 2300 randomly mutated variants of Pleurotus ostreatus POXA1b laccase to develop improved laccase-based biocatalysts. Screening of collections of 2300 randomly mutated variants of POXA1b was performed by assaying activity towards the phenolic substrate 2,6-dimethoxyphenol. Two new variants endowed with higher enzyme activity than the wild-type laccase were characterized, and their ability to decolourize industrial dyes with complex trisazo-, polyazo- and stilbene-type structures, in the absence of mediators, was demonstrated. One of the mutants (2L4A) was also proved to be highly stable at both acidic and alkaline pH values (displaying a half-life of around 1 month at the pH levels of both 5 and 10). In comparison with the wild-type laccase, the new selected 2L4A mutant shows a significant increase in stability at acidic pH, whilst storing its high stability at alkaline pH. This variant also represents a more versatile enzyme with respect to both the variety of xenobiotics degraded and the operative conditions. This work represents the first example of improvement of a basidiomycete laccase for industrial effluents bioremediation by directed evolution.