Construction of mutant libraries
Laccases 7A12  and RY2  developed and expressed in S. cerevisiae were fused to the evolved alpha-factor pre-proleader α9H2  and used as starting points of the evolution pathways.
Error-prone PCR was carried out under described conditions , using Mutazyme II DNA polymerase at low mutagenic rate (GeneMorph II Random Mutagenesis kit). Primers Ext-pJRoC30-F sense and RMLC Ext-pJRoC30-R (Additional File 1: Table S3) were designed to generate overhangs of over 20 bp homologous to the ends of the linear vector in the PCR products to facilitate the in vivo cloning in the yeast .
Site directed mutagenesis of F396I was performed at PCR described conditions . Same procedure was used for the directed mutagenesis in V413, S291, D457, A461, T468 and for all the variants developed for increasing the thermal stability of C-LeB laccase (Top-Down and Bottom up mutants). All the primers used in these assays are depicted in Additional File 1: Table S3.
Saturation mutagenesis on Phe454 was carried out using primers 454SM-Fw sense and 454SM-Rv antisense combined with Ext-pJRoC30-F sense and RMLC Ext-pJRoC30-R, respectively (Additional File 1: Table S3). For 50 µL reaction, 5 µL buffer Phusion, 3 µL DMSO, 1 mM dNTPs mix, 2.5 µL each primer (0.25 µm), 1 µL Phusion polymerase and 100 ng of DNA template were added. PCR was carried out under the following conditions: 95 ℃ (2 min), 1 cycle; 94 ℃ (30 s), 55 ℃ (30 s), 74 ℃ (2 min), 28 cycles; and 74 ℃ (10 min), 1 cycle. Same procedure was used for the saturation mutagenesis of F392 and F460 using 392SM-Fw sense and 392SM-Rv antisense, and 460 Li9SM-Fw sense and 460Li9-Rv antisense oligos, respectively. NNK degeneracy of codons was used.
CSM libraries on residues 456–457–458 were performed using mutagenic primers LEA-Fw sense and LEA-Rv antisense (Additional File 1: Table S3). Degeneracy of the codons enabled the replacement of LEA/LDA by the preferred amino acid residues found during the natural evolution of basidiomycete laccases (Additional File 1: Fig. S2), that is NTS VMS VNA. PCR conditions were the same as used for SM.
The optimized αOPT leader  replaced the α9H2 leader for the production of Mol3 variant to improve enzyme secretion in S. cerevisiae.
In each evolution round, PCR products were purified and 400 ng mixed with 100 ng of linearized pJRoC30 vector, and transformed into competent cells of protease-deficient S. cerevisiae BJ5465 strain using a yeast transformation kit (Sigma). Transformed cells were plated on SC dropout plates and incubated for 2 days at 28 °C. Colonies containing the whole autonomously replicating vector were selected and cultivated in microplate fermentations .
Screening of mutant libraries
Up to 2800 S. cerevisiae clones of the epPCR library were screened for laccase activity. For saturation mutagenesis libraries at least 145 clones were screened for 99% coverage of all possible substitutions. In the case of CSM 456–457–458 library, activities of 3450 clones were screened to attain 95% coverage with NTS VMS VNA possible substitutions.
Individual clones from the mutant libraries and the corresponding parent laccases were picked and cultured in 50 µL of minimal medium in sterile 96-well plates . The plates were sealed and incubated at 30 °C in 80% humidity with 180 rpm agitation. After 48 h, 160 µL of expression medium  was added and the plates were incubated for 24 h. Then, plates were centrifuged (6000 rpm, 4 °C, 5 min) and 20 µL supernatant aliquots were transferred to two replica plates using a liquid-handling robot to carry out the enzymatic reactions .
Laccase activities of mutant libraries were screened with DMP and guaiacol. The selective pressure was intensified through the evolution pathway by increasing the pH of the assays from pH 6 to pH 8 and 9. In addition, oxidation of 3 mM ABTS at pH 3 in citrate phosphate buffer was used to monitor the intrinsic acidic activity of laccase along the successive evolution rounds. Enzymatic reactions were started by adding 180 µL of substrate solution (3 mM ABTS in 50 mM citrate phosphate buffer pH 3, 3 mM DMP in 50 mM Britton-Robinson buffer pH 6 and 8, and 9 mM guaiacol in 50 mM Britton-Robinson buffer pH 9). Laccase activity in the wells were measured in a Spectra max plus 384 plate reader (Molecular Devices) by monitoring the oxidation of the substrate at the maximum absorbance of the oxidized product (ABTS: 418 nm, DMP: 469 nm, guaiacol: 470 nm) in kinetic mode. Best variant selected by improved oxidation of lignin phenols in each directed evolution round was used as parent for the next round.
Flask production and purification of selected laccases
The S. cerevisiae clones expressing selected laccase variants were grown in duplicate, in 100 mL or 1L flasks with 30 mL or 300 mL expression medium, respectively, containing 1 M CuSO4 and 3% ethanol . Laccase activity secreted in the liquid cultures was monitored over time by measuring the oxidation of 3 mM ABTS in 50 mM citrate phosphate buffer pH 3, 3 mM DMP 100 mM sodium phosphate buffer (pH 6 and 8) and 9 mM guaiacol in 100 mM Britton-Robinson buffer pH 9 with UV-1900 Shimadzu spectrophotometer. After 3d fermentation, cultures were centrifuged, filtered and concentrated .
Purification of selected enzyme variants by high-pressure liquid chromatography (HPLC) was carried out in 3 chromatographic steps: two anion exchange steps using a HiPrep-QFF 16/10 column in a 100 mL gradient of 0–40% elution buffer, and a MonoQ-HR 5/50 column in a 30 mL gradient of 0–25% elution buffer, followed by size exclusion chromatography with a Superdex75. Fractions containing laccase activity (with 3 mM ABTS in 50 mM citrate phosphate pH 3) were pooled, dialyzed in Tris–HCl pH 7 and concentrated after each chromatographic step. Enzyme purification was confirmed by the electrophoretic mobility of the proteins in SDS-PAGE (12% acrylamide) stained with Coomassie Brilliant blue.
Characterization of pure and non-purified (crude) laccases was carried out in 96-well plates with enzyme aliquots of 0.1 U/mL activity (measured with 3 mM ABTS in 50 mM citrate phosphate buffer at pH 3). Laccase activities were monitored spectrophotometrically in the plate reader by measuring the increase of absorbance of the oxidized products at room temperature.
Optimum pH: reactions of 20 µL enzyme with 180 µL 3 mM ABTS, 3 mM DMP or 9 mM guaiacol solutions (in 100 mM B&R buffer pH 2–10) were carried out by triplicate and the laccase activities monitored in kinetic mode. The relative activities were calculated as a percentage of the maximum activity obtained for each laccase variant.
T50 (10 min): 35 µL enzyme aliquots were transferred to 96-well PCR plates, sealed and incubated at a temperature gradient of 30–80 °C during 10 min in a thermocycler (two assays with temperature ramps of 30–55 °C and 55–80 °C were performed). Then, plates were cooled on ice for 10 min and tempered for 5 min. Then, 20 µL samples were transferred to 96-well plates with 180 µL 3 mM ABTS in 50 mM citrate–phosphate buffer pH 3, and laccase activities were measured.
Medium-term temperature stability assay: enzymes were incubated at different times at 60 and 70 °C with 20 mM Tris–HCl buffer at pH 7.5. Then, 20-µL samples taken at different incubation times were added to 96-well plates filled with 180 µL 3 mM ABTS in 50 mM citrate–phosphate buffer pH 3 to measure laccase activity. Laccase half-life values at 60 and 70 °C and thermal inactivation constants were obtained as described .
Optimum temperature was determined in the spectrophotometer with a Peltier temperature control using 3 mM ABTS in 50 mM citrate phosphate buffer pH 3 (triplicate samples). The oxidation was followed during the first min of reaction with the substrate pre-incubated at the corresponding temperature.
pH medium-term stability assay: Enzymes were incubated for 1 h at 20 and 30 °C with 20 mM Tris–HCl buffer at pH 10 and 10.5. Then, 20 µL-samples taken at different incubation times were added to 96-well plates filled with 180 µL 3 mM ABTS in 50 mM citrate phosphate buffer pH 3 to measure laccase activity.
Kinetic constants for the oxidation of ABTS (ε418 = 36,000 M−1 cm−1) and DMP (ε469 = 27,500 M−1 cm−1) were measured in triplicate with 20 µL enzyme aliquots added to 230 µL solutions of 3 mM ABTS in 50 mM citrate phosphate pH 3, 3 mM DMP in 100 mM Tris HCl pH 8, 9 or 10 mM. To calculate Km and kcat values the average Vmax was represented versus substrate concentration and fitted to a single rectangular hyperbola function in SigmaPlot (version 14.0) software, where parameter “a” was equal to kcat and parameter “b” was equal to Km.
3D protein modelling
The structure models of the mutated laccases were built with Swiss-Model using 7D5 laccase crystal (PDB entry 6H5Y) as a template, and visually inspected using PyMol Molecular Graphics System (Schrödinger, LLC).
Determination of redox potential
The published redox potential (E°) for Fe (2,2′-dipyridyl)2Cl3/Fe(2,2′-dipyridyl)2Cl2 in 8 mM MES buffer (pH 5.3) was 0.76 V/NHE . The E° (1.11 V vs. NHE) in 12.5 mM Tris–HCl (pH 8) was determined by cyclic voltammetry with a glassy carbon working electrode and an Ag/AgCl/KCl (3 M) reference electrode. The redox potential was referenced to NHE by equation E (vs. NHE) = E (vs. Ag/AgCl/KCl, 3 M) + 0.21.
Laccase redox potential was determined by the poised potential method using the redox couple Fe(2,2′-dipyridyl)2Cl2/Fe(2,2′-dipyridyl)2Cl3. Fe(2,2′-dipyridyl)2Cl2 and Fe(2,2′-dipyridyl)2Cl3 solutions were prepared freshly, mixing FeCl2 (or FeCl3) with 2,2′-dipyridyl in the ratio 1:2 in double distilled water. Anaerobicity was achieved by repetitive vacuum-argon cycles, at 4 °C, of solutions, buffers and reaction chamber. All spectrophotometry measurements were carried out under argon atmosphere at 25 °C.
Aliquots of Fe(2,2′-dipyridyl)2Cl3 solution (0.9–18 µM) were introduced to a solution of Fe(2,2′-dipyridyl)2Cl2 (9 µM) in MES (8.8 mM, pH 5.5) or Tris–HCl (12.5 mM, pH 8.0) buffer. After introduction of laccase (0.9 µM), oxidation of Fe(2,2′-dipyridyl)2Cl2 at each titration point was followed by the decrease in absorbance at 522 nm (Fe(2,2′-dipyridyl)2Cl2: ε = 5992 M−1 cm−1 at pH 5.5 and 5836 M−1 cm−1 at pH 8.0; Fe(2,2′-dipyridyl)2Cl3: ε = 260 M−1 cm−1 at pH 5.5 and 225 M−1 cm−1 at pH 8.0) until equilibrium was reached. The concentration of reduced laccase at equilibrium was considered to be 1/4 of the oxidized Fe(2,2′-dipyridyl)2Cl2 concentration.
Kraft lignin oxidation by laccase
Eucalyptus kraft lignin was isolated by the Centre Technique du Papier (Grenoble, France) with LignoBoost from the black liquors of The Navigator Company kraft pulp mill. Kraft lignin (0.5 g/L) was solubilized in 20 mM B&R buffer pH 9–10 and treated with 0.1U/L laccase (100 mL final reaction volume) for 2 and 24 h, at 30 °C and 180 rpm. Same conditions without enzyme served as control lignin. The phenolic and carbonyl contents of laccase-treated and control lignins were spectrophotometrically determined by Folin Ciocalteu Reactive (Abs. 760 nm) and Brady reagent (Abs. 450 nm), respectively  Mw distribution of lignin samples were determined by Size Exclusion Chromatography (SEC) using a Superdex75 column pre-equilibrated with 20 mM Britton-Robinson buffer (pH 11.6). Lignin samples were solubilized in NaOH to pH 11.6, centrifuged (13,400 rpm), and injected in the column and Absorbances at 260 and 280 nm were monitored throughout the chromatographic run.
Heterologous expression in Komagataella pastoris
Expression in Komagataella pastoris was carried out following InvitrogenTM manual. First, laccase CDS was amplified by PCR with “BstBI long target” and “Ext pJRoC30-R” primers (Additional File 1: Table S3). Then, purification of the PCR product was digested with NotI and BstBI to clone the laccase in the pPICZ-B vector (both, laccase and vector present those restriction sites). Linearized pPICZ-B vector with zeocin resistance was ligated with the digested laccase gene by using T4 DNA ligase, in proportion 1:3 (50 ng of vector and 37.5 ng of laccase) and left overnight at 1 °C.
Ligation product was transformed in E. coli and incubated in LB-zeocin plates at 37 °C overnight. Plasmids were isolated with High Pure Plasmid Isolation Kit and digested with SacI in a specific region integrated in AOX (methanol promoter) to linearize the construction and integrate in the genome of K. pastoris.
Komagataella pastoris X33 strain was transformed following the transformation method provided by the Invitrogen™ manual. Transformed cells were plated in YPD-zeocin and incubated for 2–4 days at 28 °C. The obtained colonies were transferred to BMM ABTS agar plates and incubated for 2–4 days at 2 °C until formation of a green halo (due to oxidation of ABTS) indicated the presence of laccase activity.
After cultivation of a fresh culture in YPD-zeocin incubated for 2–4 days at 28 °C, one colony was transferred into a 50 mL BMMY-zeocin flask and cultivated for 20 h at 28 °C. Glycerol (10 mL) was subsequently added, and thus created glycerol cell stock stored at − 70 °C until further use. The larger scale production fermentation trials were conducted using Infors HT Labfors 5 bioreactors (7.5 l volume), monitored by the bioprocess platform software Eve® (Infors HT). The pH of the fermentation was followed by EasyFerm Plus PHI Arc 425 (Hamilton) via automatic addition of 2 M HCl or 15% ammonium hydroxide. Sufficient oxygenation of the cells was maintained by constant aeration (air) and automatic increase of stirring by two Rushton turbines. Dissolved oxygen (DO) was monitored throughout the cultivation with VisiFerm DO Arc 425 (Hamilton). The main fermentation parameters were kept as follow: temperature 30 °C, dissolved oxygen > 20%, pH 5, agitation: 200–900 rpm, air flow aeration, 0.5–2.5 vvm.
The production process of Li10 laccase was performed through a fed-batch fermentation, consisting of three main phases: the seed train carried over in YPD plate and BMGY flasks, the batch phase initiated in the bioreactor and the fed-batch phase where enzyme production occurs. The seed train was started with the plating of a YPD-zeocin incubated for 48 h at 30 °C and inoculated with the –70 °C cell bank glycerol stock. A pair of isolated colonies were then transferred into BMGY medium flasks and cultivated at 30 °C for 16–24 h. After sufficient growth was noted (OD600 nm > 20), 7% of the fermenter initial fermentation volume was transferred from the BMGY flasks to the fermenter to start the fermentation batch phase. The batch phase was carried over in FBSM media supplemented with PTM1 salts at 30 °C during 18–24 h until exhaustion of the carbon source. This media consists of 40 g/L glycerol, 4.13 g/L potassium hydroxide, 14.9 g/L magnesium sulphate heptahydrate; 18.2 g/L potassium sulphate, 0.93 g/L calcium sulphate, 26.7 mL phosphoric acid (85%) and 4.25 mL/L of PTM1 trace salt (6 g/L copper (II) sulphate pentahydrate, 0.08 g/L sodium iodide, 3 g/L manganese sulfate monohydrate, 0.2 g/L sodium molybdate dihydrate, 0.02 g/L boric acid, 0.5 g/L cobalt chloride, 20 g/L zinc chloride, 65 g/L ferrous sulphate heptahydrate and 5 mL/L sulphuric acid). After exhaustion of the glycerol present in the batch, feed phase was initiated by supplemented glycerol for 4–8 h. Methanol feed was subsequently started and supplemented with copper (6 mM–10 mM) during 80–85 h allowing enzyme production. Downstream process of the fermentation was performed by removal of the cell mass by centrifugation (4000 rpm for 30 min) and freezing of the.
Laccase-assisted delignification and bleaching of kraft pulps
An oxygen-delignified hardwood kraft pulp (kappa number: 11.5 and brightness: 52% ISO) was provided by Fibre Excellence’s Saint Gaudens mill (France). Li10 laccase crude expressed in K. pastoris was applied on this pulp with the following conditions: 100 g of kraft pulp, 14 U of laccase (measured with DMP, pH 8) per g of o.d. pulp, 5 mM methylsyringate, pH 8.5, 65 °C 180 min, 10% pulp consistency. Then the pulp was subjected to the Ep D Ep bleaching sequence. The Ep stage was carried out at 72 °C, 120 min, 10% pulp consistency, 5 kg NaOH/t of o.d. pulp and 10 kg H2O2/ton of o.d. pulp. At the end, the pulp was washed with tap water on a funnel and then subjected to the D stage in the following conditions: 73 °C, 300 min, 10% consistency and 23 kg ClO2/ton of o.d. pulp. After the last Ep stage, the pulp was washed with tap water on a funnel to be further characterized. Pulp kappa number was determined according to standard ISO 302:1981. Pulp brightness was evaluated according to standard ISO 2470:1999 and the residual amounts of ClO2 were measured by titration with 0.1 N sodium thiosulfate. The effluents from the different bleaching stages were collected and mixed together. Soluble lignin was determined into the effluent by measurement at 280 nm of an aqueous solution of lignin (10% in water or in 0.25 M NaOH, calibration with vanillin, ɛ = 70 g/L cm−1).
Production of medium-density fibreboards (MDF) assisted by laccase
The wood material utilized for board manufacture was maritime pine. The enzyme applied to the wood chips was laccase Li9. The initial protocol based on previous results comprised the de-structuring of chips with a compression screw prior to enzyme application in order to ease the full impregnation of chips by the liquid solution. De-structured chips were submerged into the enzymatic solution during 1 h at 60 °C with 1625 U/kg wood chips of activity (Li9 laccase activity was measured with pH 6 DMP). In a second simplified protocol the de-structuration step was removed and the immersion of chips into the enzymatic solution was applied for 2 h at same temperature. De-structured chips were steamed for 10 min, whereas non-de-structured ones were steamed for 20 min in order to soften lignin and facilitate fibre separation. Controls were carried out at the same conditions without enzyme addition. Besides, control chips with no water immersion (with and without compression screw) were also processed for MDF production.
The chips were introduced in the refiner for defibering under 6 bar pressure with feeding adapted for flow to be compatible with the equipment. Fibres were produced in a pressurized 12’ Andritz refiner equipped with Durametal plates (Ref. 12SA001) based on the equipment used by the industry for manufacturing MDF fibres. Fibre pressing was performed on a heating hydraulic press with a Plate surface of 60 × 60 cm2, under 200 °C. Wood panels were characterized on equipment delivered by Instron. The fibres were evacuated through the blow line and recovered after separation cyclone. Power consumption was acquired during processing and energy was calculated based on the corresponding flow. Industrial production of MDF comprises several steps that were reproduced in the laboratory (Additional File 1: Fig. S8). Resin was introduced at 12% in mass. Two ranges of densities were aimed at in order to interpolate results. Density of the boards was measured according to EN323, internal bond according to EN319 and water swelling according to EN317 standards.