Wild-Type and mutated laccases for a green industry

In the frame of white biotechnology, enzymes attract an enormous attention both for their potential and for the recent social and economical interest in green chemistry. Thanks to the improved knowledge of production biochemistry, fermentation processes, and recovery methods, an increasing number of enzymes can be affordably produced. The majority of currently used industrial enzymes are the hydrolytic ones, being used for the degradation of various natural substances. Also, various oxidative enzymes, primarily laccases are used in industries such as the starch, textile, detergent and baking industries, and they represent a second important group of enzymes. This project has been focused on the recombinant expression of the industrially attractive laccase POXA1b from Pleurotus ostreatus in Pichia pastoris and on exploiting its industrial application in order to substitute some chemical industrial processes with a better eco-friendly ones. An economical analysis of the recombinant POXA1b laccase production process, in terms of productivity and cost has been evaluated. Furthermore, computational analysis and site-specific mutagenesis of the POXA1b were performed to increase the enzymatic performances towards selected substrates. A POXA1b laccase immobilization process was optimized by statistic methods (Response Surface Methodology) in order to increment the operational stability of the enzyme in specific processes. Applications of the recombinant enzyme were then developed: (i) reduction of the phenol contents inside the fruit juices by immobilized laccase POXA1b; (ii) synthesis of a new eco-friendly dyes for the staining of different textile materials (nylon, cotton and wool) and (iii) synthesis of a new eco-friendly dye for proteins visualization on the polyacrilamide gels; (iv) cotton fiber functionalization with an anti-adhesive and anti-oxidant dye polymer