The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization

Abstract

Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea. After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris, representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0–7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2′-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications.

Recent Achievements in Dyes Removal Focused on Advanced Oxidation Processes Integrated with Biological Methods

“…Bacterial treatment Isolation of new strains or consortia from activated sludge, oxidation ditch, palm oil mill effluent or desert soil, alkali-, haloand thermophilic strains implementation, consortium with algae, bacteria immobilization, co-substrate addition, proposal of mechanisms, pathways genome and transcriptome analysis [109][110][111][112][113][114][115][116][117][118][119] Fungal treatment Implementation of microbial consortium (e.g., yeast consortium with ability of lignin valorization dye treatment and biodiesel production), fungi immobilization, isolation of new strains from plant roots or effluent site [120][121][122][123][124][125][126][127][128][129][130] Enzyme treatment Optimization of enzyme production, enzyme immobilization, metabolites and toxicity assessment [131][132][133][134][135][136] Algal treatment…”

Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019)

“…Moreover, most laccases known to date have acidic pH optima, which can prohibit their use in systems operating at alkaline conditions. A few examples of such processes are lignocellulose pretreatment for ethanol production, hair coloring, textile dyeing, and human body implants [60]. The two most popular approaches to these issues are the discovery of novel enzymes with desired properties, and genetic engineering for the modification of the properties of known counterparts.…”

“…Thus, they obtained mutants with fivefold higher specific activities at pH 7.5. Yin et al [60] succeeded at shifting the pH optimum of Lcc9 from Coprinopsis cinerea to pH >8.0 by a directed evolution approach. The double mutant was shown to have improved performance in the decolorization of indigo dye.…”

“…Laccases were shown to degrade the textile dye indigo, yielding isatin and subsequently anthranilic acid, enabling both the bleaching of indigo-dyed denim as well as the treatment of indigo-contaminated wastewater [109]. Numerous investigations of textile dye wastewater treatment by laccases were also reported, which is reflected in recent journal publications including immobilized [110][111][112] as well as non-immobilized laccases [60,113]. Apart from wastewater treatment applications, laccases were recently investigated in wool dyeing by the polymerization of syringic acid [114], the antimicrobial coating of textile fibers by polymerization of catechol and p-phenylenediamine [115], the pH-responsive and conducting coating of wool fabrics by the polymerization of diaminobenzenesulfonic acid [116], nylon and wool dyeing by laccase-catalyzed 1,4-dihydroxybenzene, 2,7-dihydroxynapthalene, and 2,5-diaminobenzenesulphonic acid coupling [117].…”

Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea

“…Co-transformation of a selectable vector together with one or more other plasmids is an efficient means to introduce and find non-selectable genes in transformed C. cinerea clones [62]. Because we have a deeper interest in laccase functions and applications [16,[63][64][65][66][67][68], several vectors used here in co-transformations contained either C. cinerea laccase genes for enzyme overexpression or were antisense constructs designed for laccase gene silencing (Tables 2 and 3). Most C. cinerea monokaryons in fungal cultures have some background laccase activities through expression of Lcc1 and Lcc5 and possibly other enzymes, with the exception of the laccase-free strain FA2222 [16,64,65].…”