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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.

In the last years, laccases have become leading biocatalysts in green bioprocesses for dye synthesis. Among fungal laccases, the high-redox potential laccase POXA1b from Pleurotus ostreatus is a valuable candidate since it brings together several key-elements: stability and activity in a wide range of pHs and temperatures and the availability of a cost-effective bioprocess for its production. In this work POXA1b catalyzed the coupling of a p-phenylenediamine and α-naphtol, resulting in the production of a new dyeing product, named SIC-RED. The dye was structurally characterized and its performances in protein gel staining tested. The economic feasibility of the process was demonstrated in the case of an Italian small medium enterprise. The results let envisage a real exploitation of the herein enzymatically synthesized dye for protein gel staining.

Protein heterologous production offers viable opportunities to tailor laccase properties to specific industrial needs. The high redox potential laccase POXA1b from Pleurotus ostreatus was chosen as case study of marketable enzyme, due to its desirable properties in terms of activity/stability profile, and already assessed applicability. POXA1b was heterologously produced in Pichia pastoris by investigating the effect of inducible and constitutive expression systems on both the yield and the cost of its production. System performances were first assessed in shaken-flasks and then scaled-up in bioreactor. The production level obtained in the inducible system is 42 U/mL, while the activity value achieved with the constitutive one is 60 U/mL, the highest obtained in constitutive systems so far. The economic feasibility of recombinant laccase production was simulated, describing the case of an Italian small-medium enterprise. Two scenarios were evaluated: Scenario (I) production based on methanol inducible system; Scenario (II) production based on the constitutive system, fed with glycerol. At all the scales the glycerol-based fermentation is more economic than the methanol-based one. The price forecast for rPOXA1b production is 0.34 € k U−1 for glycerol-based process, and is very competitive with the current price of commercial laccase.

The clarification step represents, in fruit juices industries, a bottleneck process because residual phenols cause severe haze formation affecting juice quality and impairing customers acceptance. An enzymatic step can be efficiently integrated in the process, and use of immobilized enzymes entails an economical advantage. In this work, covalent immobilization of recombinant POXA1b laccase from Pleurotus ostreatus on epoxy activated poly(methacrylate) beads was optimized thanks to a Response Surface Methodologies approach. Through regression analysis the process was well fitted by a quadratic polynomial equation (R(2)=0.9367, adjusted R(2)=0.8226) under which laccase activity reached 2000 ± 100 Ug(-1) of beads, with an immobilization efficiency of 98%. The immobilized biocatalyst was characterized and then tested in fruit juice clarification reaching up to 45% phenol reduction, without affecting health-effective flavanones content. Furthermore, laccase treated juice displays an improved sensory profile, due to the reduction of vinyl guaiacol, a potent off-flavor possessing a peppery/spicy aroma.

Laccase-catalyzed synthesis of dye molecules represents a green choice to reduce the environmental footprint of conventional synthetic processes. Textile industry will benefit from this green technology since the synthesized dyes can be exploited to colour different fabrics. This work describes the application of the Pleurotus ostreatus POXA1b laccase in polymeric dye synthesis using resorcinol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA) as substrates. The potential of the resorcinol/2,5-DABSA coupling route was transferred to a chemical industry, Setas Colour Center, by introducing a greener synthesis step within the process routinely used for textile dyeing. Dye synthesis was performed at different precursor ratios (1:1 and 1:10 2,5-DABSA: resorcinol) and their dyeing properties were compared on different fibres. The two mixtures of synthesized dyes proved to be effective on nylon and wool, with 1:10 ratio displaying the best performances in terms of dyeing efficiency and colour strength. Good and comparable end quality and “performances during use” were observed for nylon and wool coloured with both synthesized dyes.