Ancestral sequence reconstruction (ASR) of LPMO. Fifty-one LPMO from auxiliary activity family 10 (AA10) were downloaded from the NCBI database. Sequences belong to three bacterial phyla: Proteobacteria, Actinobacteria, and Firmicutes. All sequence ID numbers are listed in Supplementary Note 1. The alignment of the sequences was performed using MUSCLE extension on the MEGA platform61. We inferred the best evolutionary model using MEGA, resulting in the Jones-Tylor-Thornton (JTT) with gamma distribution model. The phylogeny was carried out using BEAST v1.10.4 package software, including the BEAGLE library for parallel processing and Bayesian inference using Markov chain Monte Carlo (MCMC). Phyla were well separated with no further action; however, we established monophyletic groups for Proteobacteria, Actinobacteria, and Firmicutes to set priors. We set JTT model with eight gamma categories and invariant distributions, Yule model for speciation, 20 million generations of length chain, and sampling every 1000 generations. The divergence times were estimated by uncorrelated log-normal clock model (UCLN), using molecular information from Time Tree Of Life (TTOL) with default birth and death rates 35. Calculations were run in a multicore server. From the generated trees we discarded the 25% of them as burn-in with the LogCombiner utility from BEAST. We verified the MCMC log file using TRACER and ensured all parameters showed effective sample size (ESS)>100. Posterior probabilities of all nodes were above 0.65, and most of them were near 1. Figtree software v1.4.2 was used to visualize and edit the phylogenetic tree. Finally, ancestral sequence reconstruction was performed by maximum likelihood using PAML 4.8 with a gamma distribution for variable replacement rates across sites and the JTT model 62. Posterior probabilities were calculated for all amino acids, and the residue with the highest posterior probability was chosen for each site. High posterior probabilities ensure the correctness of each bifurcation given the sequence collection, alignment and model used. We selected Last Actinobacteria/Firmicutes common ancestor (LAFCA) for laboratory resurrection.
Protein expression and purification. We used previously described protocol for LPMO expression and purification63. Genes encoding the ancestral and extant LPMO proteins were synthesized, and codon optimized for expression in E. coli cells (Life Technologies). A native signal peptide was included to secure export to the periplasmic space (Supplementary Note 2). This translocation ensures cleavage of the signal peptide leaving a histidine at the N-terminus, essential for copper cofactor binding 64. The genes were cloned into pQE-80L vector and transformed onto E. coli BL21 cells (DE3) (Life Technologies) for protein expression 65. Bacteria were incubated in LB medium at 37 °C until OD600 reached 0.6; IPTG was added to the medium to 1 mM concentration for protein induction overnight at 20 ºC. Bacterial cultures were collected by centrifugation for 10 min at 4000G and 4 ºC, and periplasmic extraction was performed by osmotic shock method. Pellets were resuspended in 30 mL of spheroplast buffer (1 M Tris-HCl pH 7.5, 0.5 M sucrose, 0.5 mM EDTA). Resuspended pellets were incubated on ice and then centrifuge for 10 min at 5000G and 4 ºC. The supernatants were discarded and incubated for 10 min at room temperature, followed by resuspension on 25 mL ice-cold water. After 45 seconds of the resuspension step, we added 1.25 ml of cold 20 mM MgCl2. The mixture was then centrifuged at 15000 G for 10 min at 4 ºC. We collected the supernatant and filter it (0.22 μm pore size). The supernatants were copper saturated by incubation with Cu(II)SO4 at a ratio of 1:3 for 30 min at room temperature 66. The proteins were then further purified, and the non-bonded copper was removed by size exclusion chromatography using a Superdex 200HR column (GE Healthcare). The buffer used was 50mM sodium phosphate pH 7.0. The purified proteins were finally verified by SDS-PAGE with 12% acrylamide gels. The protein concentration was calculated by measuring the absorbance at 280 nm in Nanodrop 2000C, using the equation 280 of x M-1 cm-1 and MW of x g mol-1, with the theoretical extinction coefficient of ε280=39,545 M-1cm-1 and molecular mass of 20 KDa without signal peptide.
LPMO characterization. Reactions were performed in 96-well plates mixing 166 µL of 100 mM phosphate buffer pH 8, 20 µL of 10 mM DMP (final concentration of 1 mM), 38 and 4 µL of 5 mM H2O2 (final concentration of 0.1 mM). Finally, we added 10 µL of enzyme dilution at a suitable concentration to each well in a final volume of 200 µL per well. Plates were orbitally mixed, and the reaction was carried out at 50 ºC. Activity was calculated by measuring the increase of absorbance at 469 nm for 5 minutes using the molar absorption coefficient of coerulignone (ε469=53,200 M-1cm-1) to calculate the peroxidase activity of LPMO. For thermal stability assay, we preincubated the LPMO enzymes to the test temperatures for 5 minutes in a 1.5 mL Eppendorf tube in a thermo mixer and then placed them on ice for 5 minutes. The reaction was carried out at a 96-well plate at 50 ºC, 169.5 µL 100 mM phosphate buffer pH 8, 20 µL10 mM DMP as a chromogenic substrate to a final concentration of 1 mM, 0.5 µL of 5 mM H2O2, and 10 µL of enzyme dissolution with adequate enzyme concentration to a final volume of 200 µL. Absorbance at 469 nm was measured for 30 minutes at Epoch 2 spectrophotometer. For pH assay, different pH buffer dissolutions were prepared from pH 3 to pH 10: 100 mM citric acid for pH 3-5, 100 mM phosphate buffer for pH 6-7, 100 mM carbonate buffer for pH 8 and 100 mM Boric acid for 9-10.
Nanochitin enzymatic isolation and characterization. Commercial α-Chitin from Sigma Aldrich was used as substrate. We used 2.5% α-Chitin suspension in water with a ratio of 10 mg of each enzyme (LFACA-LPMO and BtLPMO) per gram of substrate. As reducing agent, we used 2 mM ascorbic acid from Sigma Aldrich. The oxidative cleavage was carried out at 50 ºC in agitation for 72 hours. Reactions were stopped by placing them on ice, and the mixtures were sonicated with a microtip sonicator UPH 100H Ultrasonic Processor (Hielscher) for 25 min at 75% to separate aggregated nanoentities. Nanochitin was isolated by several centrifugation steps and concentrated by ultracentrifugation at 33,000 G for 30 minutes. Pellets were resuspended in water or 2% acetic acid and lyophilized in a Telstar Lyoquest for physical and chemical characterization by freeze-drying for 24 hours. Nanochitin produced by hydrochloric acid form was donated from “Material and Technology group (GMT)” from UPV/EHU.
Fourier transform infrared (FTIR). The infrared spectra were recorded in attenuated reflection (ATR) mode to analyze functional groups of lyophilized samples of chitin, EnCNCh, and acid CNCh. We used a Perkin-Elmer Frontier FTIR spectrophotometer equipped with an ATR sampling stage within the wavenumber of 4000 and 650 cm-1, with 32 scans and a resolution of 4 cm-1.
Atomic Force Microscopy (AFM). We used a Nanoscope V scanning probe microscope (Multimode 8 Bruker Digital instruments) using an integrated force generated by cantilever/silicon probes. Images were obtained at room temperature, in taping mode, applying 320 kHz resonance frequency and 5-10 nm tip radius and 125 µm long. Sample preparation was made by spin coating using a Spincoater P6700 at 200 rpm for 60 seconds on mica substrate. AFM height and phase images were collected simultaneously in all the samples. The size of the images was 3x3 µm, and nanofibers of different sizes were distinguished. The length and diameter of 100 nanofibers was measured to calculate the average length, diameter, and aspect ratio (Length/Diameter).
X-ray diffraction (XRD). Crystalline structure, crystallinity, and crystallite size of nanochitin was studied using X-ray diffraction (XRD) powder diffraction patterns. Data were collected at room temperature using a Philips X´pert PRO automatic diffractometer from 5 to 50 º and a PIXcel solid state detector (active length in 2θ 3.347º) operating at 40 kV and 40 mA, in theta configuration, a secondary monocromator with Cu-Kα radiation (λ = 1.5418 Å) and a PIXcel solid state detector (active length in 2θ 3.347º). We used an antiscattering slit and a fixed divergence giving a constant volume of sample illumination. From the diffractograms, the crystallinity index (CI%) was calculated by Segal equation 67:
Crystallinity index (%) = (I110 - Iam) /I110 ×100
where I110 is the intensity of the cellulose crystalline peak and Iam is the intensity of the amorphous peak. Crystallite size was measured using the Scherrer's equation:
β = κλ/τcosθ ×100
where λ is the wavelength of the incident X-ray, θ is the angle of the (110) plane, "β” is the full width at half maximum of the (110) peak, τ is the crystallite size, and κ is a constant value.
13C CP/MAS NMR analysis. We used solid-state cross-polarization magic angle spinning 13C nuclear magnetic resonance (13C CP/MAS NMR). 13C CP/MAS NMR spectra were measured using a 400 MHz BRUKER system equipped with a 4 mm MASDVT TRIPLE Resonance HYX MAS probe. 2K scans were taken at Larmor frequencies of 400.17 MHz and 100.63 MHz for 1H and 13C nuclei. Chemical shifts were reported relative to the signals of 13C nuclei in glycine. Sample rotation frequency was 12 kHz, and relaxation delay was 5 s. Polarization transfer was achieved with RAMP cross-polarization (ramp on the proton channel) with a contact time of 5 ms. High-power SPINAL 64 heteronuclear proton decoupling was applied during acquisition.
Thermogravimetric analysis. TGA was used to study the thermal stability of nanochitin and hybrid nanopapers. The data was recorded using TGA/SDTA 851 Mettler Toledo equipment, where 10 mg of the samples were heated from 30 to 800 ºC in a nitrogen atmosphere with a scanning rate of 10 ºC/min. The initial degradation temperature (To) is described as the loss of 5% of the weight of the total sample and the maximum degradation temperature (Td) is the minimum of the degradation peak in the derivative of the thermogravimetric curves (DTG).
Nanochitin EnCNCh/graphene films and bioinks. Nanochitin films were prepared by casting method with an EnCNCh suspension of 1 wt% sonicated for 1 hour. The suspension was placed in a Teflon mold and dried for 2 hours at 50ºC. Conductive films were fabricated by EnCNCh suspension and with graphene oxide (GO) at different final concentrations: 0.3, 1, 2, 5, 10, and 15% wt of GO. GO (4 wt% water suspension) was kindly supplied by Graphenea (San Sebastian, Spain). The mixture was sonicated in a bath to assure homogeneity and cast with the help of a vacuum bomb using an Ultrafiltration disc of 30 KDa from Merck. The wet film was dried at 50ºC for 2 hours, obtaining films with a thickness of approximately 50 µm. GO in the nanopapers was reduced by placing the films in ascorbic acid solution (30 mg/mL) for 2 hours at 95ºC and then washed using miliQ water before drying the films at 50 ºC for 2 hours.
Bioinks for 3D printing were prepared with 10 %wt EnCNCh suspension and 5 %wt graphene oxide. The mixture was sonicated in a bath to ensure homogeneity. After printing and freeze-drying the scaffolds, GO in the scaffold was reduced by placing the films in ascorbic acid solution (30 mg/mL) for 2 hours at 95 ºC and then washed using miliQ water before drying the films at 50 ºC for 2hours.
Scanning Electrical Microscopy. The morphology of the nanopaper surface and graphene was analyzed by SEM using a FEI ESEM Quanta 200 microscope operating at 5–20 kV. Nanopapers were put on carbon tape for adhesion.
Electrical conductivity was measured by a four-point probe method using a Probe Station 4 Everbeing. The specific resistances (q) were calculated with the sheet resistances (Rs, Ω/squares) and the thickness of the nanopapers (t, cm) in Eq.:
q =Rs x t
We used the specific resistance calculated to infer the corresponded conductivity (S/cm) with the following Eq. that was transformed to S/m.
The percolation threshold, ρc, was measured using a power-law equation based on the percolation theory52:
σ= σf x (ρ- ρc) n
where σ is the conductivity of the nanopaper, ρf is the rGO conductivity, ρ is the rGO content expressed at volume fraction, and n is the exponent describing the rapid variation of the conductivity near the percolation threshold (ρc).
3D printing of EnCNCh-based scaffold. Scaffolds were printed using a Voladora 3D printer (Tumaker, S.L. Spain) which has been modified for layer-by-layer syringe extrusion 3D printing. Honeycomb like scaffold and button like scaffolds were printed directly on poly- tetrafluoroethylene slides at room temperature using a needle of 0.8 mm in diameter and speed of 5 mm/s.
2D and 3D cell cultures. HEK293T were maintained in DMEN+10% FBS medium supplemented with 1% (w/v) L-glutamine and penicillin–streptomycin (100 IU/ml). Before growing cells in our material, EnCNCh film and EnCNCh scaffold were sterilized by UV for 3 hours. 20,000 cells were seeded on each substrate and let to growth for 3 days in the incubator at 37 °C in 5% CO2. Cells were fixed in 4% formaldehyde for 30 min and washed with PBS. Films were dehydrated by increasing concentration of ethanol solution until 100% concentration of ethanol was reached. After fixation, cells were dyed with DAPI solution (5 mg/ml) and CellMask Orange Plasma membrane Stain (5 mg/mL) diluted in PBS (1:2000) and after washing each sample three times with PBS. DAPI-Orange Plasma-stained cells were observed after 20 min of incubation by confocal microscopy.