The history of colour is fascinating from a social and artistic viewpoint because it shows the way; use; and importance acquired. The use of colours date back to the Stone Age (the first news of cave paintings); colour has contributed to the social and symbolic development of civilizations. Colour has been associated with hierarchy; power and leadership in some of them. The advent of synthetic dyes has revolutionized the colour industry; and due to their low cost; their use has spread to different industrial sectors. Although the percentage of coloured wastewater discharged by the textile; food; pharmaceutical; cosmetic; and paper industries; among other productive areas; are unknown; the toxic effect and ecological implications of this discharged into water bodies are harmful. This review briefly shows the social and artistic history surrounding the discovery and use of natural and synthetic dyes. We summarise the environmental impact caused by the discharge of untreated or poorly treated coloured wastewater to water bodies; which has led to physical; chemical and biological treatments to reduce the colour units so as important physicochemical parameters. We also focus on laccase utility (EC 1.10.3.2), for discolouration enzymatic treatment of coloured wastewater, before its discharge into water bodies. Laccases (p-diphenol: oxidoreductase dioxide) are multicopper oxidoreductase enzymes widely distributed in plants, insects, bacteria, and fungi. Fungal laccases have employed for wastewater colour removal due to their high redox potential. This review includes an analysis of the stability of laccases, the factors that influence production at high scales to achieve discolouration of high volumes of contaminated wastewater, the biotechnological impact of laccases, and the degradation routes that some dyes may follow when using the laccase for colour removal

In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2,] substrate concentrations. Plackett-Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L −1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L −1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were V max of 3.163 × 10 −2 mM min −1 and K m of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures. Keywords Plackett-Burman experimental design • One-factor experimental design • Pichia pastoris • Recombinant laccase • Enzyme stability • Enzyme kinetics Abbreviations MCOs Multicopper oxidases ABTS 2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) PBL Pulping black liquor LDPE Low density polyethylene pAOX1 Alcohol oxidase promoter pGAP Glyceraldehyde-3-phosphate dehydrogenase promoter POXA 1B Laccase from P. ostreatus rPOXA 1B Recombinant laccase from P. ostreatus V max Maximum reaction rate K M Michaelis constant YPG Yeast extract, peptone and glucose culture media MCB Master cell bank YPG-Z Yeast extract, peptone, glucose-zeocin culture media EWV Effective work volume PBED Plackett-Burman experimental design OFED One-factor experimental design v.v.m. Air volume per media volume µ x Specific growth rate T d Duplication time r.p.m. Revolutions per minute Y (x/s) Biomass/substrate yield Y (p/s) Enzyme/substrate yield P (x) Biomass productivity P (p) Enzyme productivity SD Standard deviation GFP Green fluorescent protein

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