Laccase

M. oryzae strain Guy11 was used as wild type throughout this work. Both Guy11 and its derivative mutants were cultured on complete medium (CM) [89] (link) for 3–15 days at 28°C to assess the growth and colony characteristics. Fungal mycelia were harvested from liquid CM and used for genomic DNA and RNA extractions. To observe the vegetative growth under the oxidative stress condition, H₂O₂ (Aldrich, 323381, 3 wt. %) was mixed in solid CM, and diameters of fungal colonies were measured after 3 to 5 days as indicated. For the activation of the laccase activity in the Moap1 mutants, copper sulphate was amended in the CM medium for 1 mM at final concentration. Cell wall integrity assay was performed by growing strains in Congo Red (CR, Aldrich, 860956) amended CM medium (200 µg/ml) for 5 days. For conidia collection, strains were normally maintained on corn meal (RDC) medium [43] (link) at 28°C for 10 days and then transferred to constant fluorescent light condition to promote conidiation for another 3–5 days. Conidia were obtained by rubbing mycelia with water followed by filtration through Miracloth (Calbiochem, San Diego, USA). For mycelial growth assay, strains were inoculated in the liquid complete medium for 48 hrs and then transferred to the Petri dish for photograph. To observe conidiophore development and conidiation, strains were inoculated on RDC for 5 days and mycelia were rubbed with a glass rod before transferring to the constant fluorescent light condition to promote conidiation for another 2 days. S. cerevisiae strains were grown in SD medium supplemented with appropriate amino acids and with glucose (3% (w/v)) or galactose (2% (w/v)). All growth assays were repeated for three times, with three replicates each time.

Optimum temperature and pH were determined by performing enzymatic assays at different temperatures (20–80°C) and pH levels (3–8), respectively. The pH level was adjusted using the following buffers: 0.1 M citrate buffer (pH 3–5), 0.1 M phosphate buffer (pH 6–8), and 0.1 M carbonate buffer (pH 9). The stability of the purified laccase at various temperatures was investigated by preincubating the purified laccase at different temperatures between 4 and 70°C for 1 h, followed by determination of the residual activity. The effect of pH on the laccase stability was determined by incubating the purified enzyme at 4°C in different pH levels for 24 h and determining the residual activity. Substrate concentration ranges of 50–500 l M, 500–1500 l M, and 1000–2500 l M were used for kinetic studies against ABTS, DMP, respectively. The effects of metal ions including Cu²⁺_, Ba²⁺_, Mg ²⁺_, Mn²⁺_, Fe²⁺_, and Hg²⁺ and inhibitors (EDTA, 1 and 10 mM; NaN₃, DTT, Urea, and SDS on laccase activity were investigated by incorporating them in to assay mixture prior to determination of residual activity [21 (link)].

Vinyl aniline (30 mg, 0.3 mmol) was dissolved in 1.5 mL of sodium acetate buffer (pH 5, 50 mM) and incubated with T. versicolor laccase (36 U mmol⁻¹_substrate) overnight at 27 °C and 180 rpm in an orbital thermoshaker. After attesting the formation of a more polar UV-visible spot in TLC analysis (petroleum ether–EtOAc = 1:1), an additional aliquot of enzyme was added doubling its concentration and the mixture was left reacting for 6 h. The reaction mixture was then extracted with EtOAc and the combined organic layers were dried over Na₂SO₄ and concentrated in vacuo affording a crude mixture. The more polar spot (compound 3a, R_f = 0.25 in petroleum ether–EtOAc = 7:3) was isolated by means of flash column chromatography on silica gel (petroleum ether–EtOAc = 7:3 → 4:6) and fully characterized (isolated yield = 40%). ¹H-NMR (400 MHz; CDCl₃, r.t.): δ 7.01 (AA’BB’ system, 2H), 6.61 (AA’BB’ system, 2H), 3.42–3.34 (m, 1H), 2.25–2.19 (m, 1H), 2.06–2.02 (m, 2H). ¹³C-NMR (101 MHz; CDCl₃, r.t.): δ 144.3, 135.1, 127.5, 119.5, 115.1, 47.9, 25.9. MS (ESI): calcd for [C₁₆H₁₀N₂]⁺ 239.15428, found 239.15410.

All reagents were of the highest purity grade from commercial suppliers: Merck (St Louis, MO, USA) or VWR (Radnor, PA, USA).
Laccase from Trametes versicolor was from Sigma-Aldrich. The enzyme was used based on their respective activities evaluated according to literature assay based on the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as model substrate. [32 (link)]
Biotransformations were performed in a G24 Environmental Incubator New Brunswick Scientific Shaker (Edison, USA) or in a Thermomixer Comfort (Eppendorf, DE).
Reactions were monitored by thin-layer chromatography (TLC) (precoated silica gel 60 F254 plates (Merck, DE)); development with UV lamp, Komarovsky reagent (1 mL 50% ethanolic H₂SO₄ with 10 mL 2% methanolic 4-hydroxybenzaldehyde), a 20% solution of H₂SO₄ in ethanol or a molybdate reagent ((NH₄)₆Mo₇O₂₄·₄H₂O, 42 g; Ce(SO₄)₂, 2 g; H₂SO₄ conc., 62 mL; made up to 1 L of deionized water). Flash chromatography: silica gel 60 (70–230 mesh, Merck, DE).
NMR spectra were recorded with a Bruker AC spectrometer (400 or 500 MHz) in [D₄]MeOH, [D₆]DMSO or [D₁]CHCl₃. Mass spectra were recorded with a Bruker Esquire 3000 Plus spectrometer.
High-resolution mass spectra (HRMS) were conducted on FT-Orbitrap mass spectrometer in positive electrospray ionization (ESI).