Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Optimizing enzymatic dyeing of wool and leather

This work reports on the environmental friendly enzymatic dyeing of wool and leather performed at low temperature and mild pH conditions without any dyeing auxiliaries. The substrates have been dyed with “in situ” generated pigment by means of laccase-catalyzed oxidative coupling of dye modifier 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and dye precursor 1,3-benzenediol in a batchwise process. The process reaction variables (laccase, precursor and modifier concentrations, temperature and dyeing time) were optimized by response surface methodology using an appropriate experimental design. The temperature, precursor concentration, interaction between precursor and modifier and time are the most important factors in the dyeing process. The best-optimized wool dyeing conditions (2 h reaction time, 50 μl laccase (0.1 U), 500 mM precursor, 10 mM modifier at 40 °C) were then successfully applied onto leather material. The enzymatic-dyeing optimized process can be successfully performed on wool and leather at low temperature and mild pH obtaining different hues and depths of shades by varying the modifier concentration and time. The colouring enzymatic system has a good reusability (which has a huge advantage in terms of cost reduction) and washing durability and is comparable in terms of fastness properties to the traditional dyeing process for both wool and leather.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for funding the project UID/CTM/00264/2019 and A. Zille contract IF/00071/2015

Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template

The green synthesis of highly conductive polyaniline by using two biological macromolecules, i.e laccase as biocatalyst, and DNA as template/dopant, was achieved in this work. Trametes versicolor laccase B (TvB) was found effective in oxidizing both aniline and its less toxic/mutagenic dimer N-phenyl-p-phenylenediamine (DANI) to conductive polyaniline. Reaction conditions for synthesis of conductive polyanilines were set-up, and structural and electrochemical properties of the two polymers were extensively investigated. When the less toxic aniline dimer was used as substrate, the polymerization reaction was faster and gave less-branched polymer. DNA was proven to work as hard template for both enzymatically synthesized polymers, conferring them a semi-ordered morphology. Moreover, DNA also acts as dopant leading to polymers with extraordinary conductive properties (∼6 S/cm). It can be envisaged that polymer properties are magnified by the concomitant action of DNA as template and dopant. Herein, the developed combination of laccase and DNA represents a breakthrough in the green synthesis of conductive material