Photocatalyst comprising methane monooxygenase hydroxylase and xanthene-based photo harvester

Abstract

The present invention relates to: a photocatalyst comprising methane monooxygenase (MMO) hydroxylase (MMOH), a halogenated xanthene dye as a light harvester, and MMOB as a catalyst conductivity enhancer; and a use thereof for methanol production, and exhibits catalytic activity superior to that in a natural biocatalytic reaction using MMO-based enzymes.

Description

Photocatalyst comprising methane monooxygenase hydroxylase and xanthen-based light harvester

[0001] The present invention relates to a photocatalyst comprising methane monooxygenase (MMO) hydroxylase (MMOH), a halogenated xanthen dye as a light harvester, and MMOB as a catalyst conductivity enhancer, and the use thereof for methanol production.

[0002] The present invention is a patent created as a result of research conducted by [Ministry Name] Ministry of Science and ICT and [Agreement Institution] National Research Foundation of Korea, [Project No.] RS-2021-NR056597, [Research Project Title] Development of advanced technology for cell factories producing biomaterials for controlling harmful nematodes; [Project No.] RS-2024-00351665, [Research Project Title] Development of a biological reduction system for nitrogen oxides; and [Project No.] RS-2024-00440681, [Research Project Title] Development of customized enzyme-photocatalyst hybrid bioconversion technology for difficult-to-decompose hydrocarbons.

[0003] Methane is a powerful greenhouse gas that causes global warming, and many efforts have been made to stabilize it, including carbon removal, synthesis gas production, and chemical synthesis. Due to its utility as a liquid fuel and a basic component, liquefying methane into methanol is the most promising option; however, this presents problems due to the high energy and cost required for harsh operating conditions (T > 800°C; P > 20 bar) to activate the inert CH bonds of methane. Recently, photocatalytic conversion using photon energy instead of thermal energy has garnered attention. When utilized, methane can be activated by oxygen-containing radicals generated from photogenerated holes in semiconductors, enabling the production of methanol at room temperature. However, oxygen-containing radicals can also peroxidize methanol, generating unwanted carbon dioxide. While some photocatalysts control active radicals to achieve high methanol selectivity (> 90%), their catalytic productivity is unsatisfactory. Therefore, the development of an innovative catalyst platform with high selectivity and productivity is necessary.

[0004] The methane monooxygenase (MMO) of the methanogen is an enzyme that catalyzes O2-dependent methane oxidation with high methanol selectivity under atmospheric conditions, and in the soluble form of the enzyme (sMMO), methane conversion is carried out using three separate protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). MMOH activates methane through a glutamate / histidine-co-acting complex ion center, and MMOR transfers two electrons from nicotinamide adenine dinucleotide cofactor (NADH) to MMOH via its electron transport domain (flavin adenine dinucleotide; [2Fe-2S], ferredoxin) to react with O2 to form methanol. MMOB increases the activity of MMOH, allowing the substrate to access the iron-active site effectively (Fig. 1a) (Schulz, CE et al., J. Am. Chem. Soc. 143, 6560-6577 (2021); Merkx, M. et al. Angew. Chem. Int. Ed 40, 2782-2807 (2001)). However, the application of MMO for methane hydroxylation shows a low conversion rate due to the slow electron transfer from the nicotinamide cofactor to the reductase (MMOR) and further to the hydroxylase (MMOH). Therefore, it is essential to address the issues of cofactor use and slow electron transfer.

[0005] Accordingly, the present invention is designed to solve the above problems and aims to provide a biosolar platform capable of producing methanol through the spontaneous combination of a xanthen-based light harvester and MMOH without NADH activating MMOH for methanol production with high catalytic efficiency.

[0006] To achieve the above objectives, the present invention provides a photocatalyst comprising a methane monooxygenase (MMO) hydroxylase (MMOH); and a xanthen-based light harvester.

[0007] In addition, the present invention provides a composition for methanol production comprising a photocatalyst according to the present invention.

[0008] In addition, the present invention provides a method for producing methanol from methane using a photocatalyst according to the present invention.

[0009] The xanthen-based light harvester of the present invention spontaneously combines with MMOH to directly transfer photo-excited electrons to the enzyme active site, so no cofactor or MMOR is required, and can provide a photocatalytic system exhibiting rapid electron transport and a high methanol conversion rate.

[0010] Figure 1 is a schematic diagram illustrating biocatalytic and biosolar methane conversion to methanol under atmospheric conditions. a, Natural cofactor-dependent MMOH reduction assisted by MMOR and MMOB. b, Biosolar MMOH reduction using XD as a light harvester. FL (X = H, Y = H); EY (X = Br, Y = H); ErB (X = I, Y = H); RB (X = I, Y = Cl). MMOH ox , resting diferric MMOH; MMOH red , diferrous MMOH. CH4+ O2+ NADH + H + + 2e→CH3OH + H2O + NAD + .

[0011] Figure 2 shows the results confirming the direct photoreduction of MMOH by EY: a, Spectral analysis of the interaction between EY (10 μM) and MMOH (2–10 μM) in aqueous solution. b, of EY and EY / MMOH complex solution. 1¹H NMR (400 MHz, 298 K) spectrum. Sample: Deuterium oxide solution containing EY (100 μM) and MMOH (2–10 μM). The af symbol indicates the hydrogen of EY shown in the inset. c, Surface-enhanced Raman spectra of EY and EY / MMOH complexes. Substrate: Hydrophilic Ag nanoparticles. Excitation: 633 nm. Aqueous solutions (5 μL) of EY (10 μM) with or without MMOH (10 μM) were drop-casted onto Ag substrates and dried for 24 hours for Raman experiments. d, Stern-Volmer plot of oxidatively quenched EY (1 μM) with proteins (MMOH, MMOR, and MMOB) in phosphate buffer (100 mM, pH 7). Excitation: 500 nm. K sv represents a Stern-Volmer integer.

[0012] Figure 3 illustrates a computer workflow for understanding the electron transport mechanism from light harvesting to MMOH. a, Blind docking was performed to identify binding sites of EY (dye) on the surface of the MMOH protein. b, The results of the blind docking show clusters of docked EY on the MMOH surface. The largest clusters representing binding sites are highlighted in the inset. c, Potential primary electron acceptors selected based on their proximity to the optimal binding posture of EY. d, Primary electron acceptor residues were identified using Marcus curve analysis, considering EY as a donor and the identified acceptor residues.

[0013] Figure 4 shows the results of the transport analysis of EY on the surface of MMOH: a, Blind docking results of EY on the entire surface of the MMOH protein. The largest EY cluster with the best docking score was analyzed by considering the position of the biiron core in the a-subunit of MMOH. b, Statistical studies identified the largest cluster based on a 2.0 RMSD cutoff. The inset shows the best cluster with the largest form (cluster size = 7) and the lowest average docking score. c, The resulting binding posture of EY on the MMOH structure. This posture highlights the accessibility of EY to neighboring residues as a promising candidate for a primary electron acceptor. d, Spin density figures of residues on the MMOH surface. K323 exhibits the highest spin density.

[0014] Figure 5 illustrates photocatalytic methane hydroxide conversion by EY: a, short-term photocurrent reaction of EY and proteins (MMOH, MMOR, and MMOB) at -0.09 V (vs. RHE). b, Nyquist plot of EY and proteins (MMOH and MMOB) at -0.09 V (vs. RHE). Inset: Equivalent circuit for the plot. The red line indicates the fitted data. Electrolyte in (ab): Methane-saturated phosphate buffer (100 mM, pH 7). Working electrode in (ab): Glassy carbon electrode coated with the EY and protein mixture solution. c, control experiment for methanol time-to-yield of the photocatalytic methane conversion platform. Electrolytes: Methane-saturated TEOA buffer (100 mM, pH 7.5) containing MMOH (2.5 μM), MMOR (5 μM), and MMOB (5 μM) with EY (20 μM) or NADH (50 mM). Methanol time yield was determined after a 1-hour reaction. d, Methanol time yield and quantum yield of EY / MMOH / MMOB by photon flux. Light source in (ad): xenon lamp, I = 0.58 μEcm 2 s -1,λ > 400 nm. e, Energy diagram of photocatalytic methane hydroxide oxidation by EY. f, Energy diagram of conventional methane hydroxide oxidation by multi-protein components (MMOH, MMOR, and MMOB).

[0015] Figure 6 shows the structural effect of XDs in biosolar methane hydroxide: a, dissociation constant (K) of XDs (1 mM) for MMOH (10 μM). d ) was calculated from ITC experiments. b, Fluorescence decay spectra of XDs (1 μM) and MMOH (6 μM) in phosphate buffer (100 mM, pH 7). Excitation: 467 nm. Emission: 518 nm (FL), 538 nm (EY), 555 nm (ErB), and 580 nm (RB). Lifetimes were evaluated by fitting spectra with triplet-exponential functions. c, Energy diagram of DX in direct photoreduction of MMOH. The replacement of atoms for heavier halogens in XD increases inter-spin-crossing (ISC)-induced triplet formation via strong spin-orbit coupling (SOC), thereby increasing the efficiency of photo-induced electron transport (PET) from XD to MMOH. d, XD / MMOH. Methanol time yield and quantum yield of electrolyte: Methane-saturated TEOA buffer (100 mM, pH 7.5) containing XD (20 μM) and MMOH (2.5 μM). Light source: xenon lamp, I = 0.58 μEcm -2 s -1 ,λ> 400 nm. e, Comparison of catalytic activity of methane hydroxide oxidation in RB / MMOH / MMOB complexes with other catalysts. The dashed line indicates catalysis occurring under ground conditions (T= 25 °C; P= 1 atm).

[0016] Figure 7 shows the SDS-gel electrophoresis results of the purified components of sMMO. H: MMOH (subunit α, β, γ), R: MMOR, and B: MMOB.

[0017] Figure 8 shows the amino acid sequences of MMOH, MMOR, and MMOB.

[0018] Figure 9 shows the results of the naphthalene oxidation assay. a, Photographs of the control and MMOH treated with the naphthalene assay. b, Corresponding absorbance of the sample (530 nm).

[0019] Figure 10 shows the results of spectroscopic analysis of the interaction between EY and proteins. ab, (a) absorption spectra of MMOB and (b) MMOR with EY (10 μM). c, absorption spectra of MMOH, MMOB, and MMOR in aqueous solution.

[0020] Figure 11 shows the Fourier-transformed infrared (FT-IR) spectra of EY and EY / MMOH complexes. ab, (a) 1000–1200 cm⁻¹ -1 and (b) 1550 ~ 1800 cm -1 FT-IR spectra of EY and EY / MMOH between wavenumbers. The characteristic peak of EY was suppressed by the addition of MMOH, which means that CO, C=C and C=O stretches were limited by MMOH through dynamic interactions between them (e.g., π-π stacking, hydrogen bonding and ion-dipole interactions).

[0021] Figure 12 shows the photoluminescence (PL) spectra of EY with the addition of proteins. Changes in the PL spectra of EY (1 μM) with the addition of (a) MMOH, (b) MMOR, and (c) MMOB in phosphate buffer (100 mM, pH 7). Excitation: 500 nm.

[0022] Figure 13 shows the PL spectra of EY with and without MMOH under different conditions. Concentration: 1 μM EY, 6 μM MMOH. Excitation: 500 nm.

[0023] Figure 14 shows the electron paramagnetic resonance (EPR) spectrum of the EY / MMOH complex. Temperature: 5 K. Microwave power: 2.31 mW. Microwave frequency: 9.65 GHz. Tuning amplitude (10 G), sweep time (2 min), transition time (20 ms). Concentration: 20 μM EY, 100 μM MMOH. The EPR signals at g = 16, g = 4.3, g = 2.01, and g = 1.83 represent the ferric (Fe₂) in MMOH, respectively. II Fe II ), Iron (Fe Ⅲ ), free radicals, and mixed-form (Fe II Fe III It represents ).

[0024] Fig. 15 shows the dissociation constant (K) of EY for MMOH using isothermal titration calorimetry (ITC). d This shows the estimated results. ITC results of representative unprocessed thermal and integrated isotherms of EY binding to (a) MMOH (WT), (b) MMOH (K323A), (c) MMOH (R320A), and (d) MMOH (R320A / K323A). Concentrations: 1 mM EY, 10 μM MMOH.

[0025] Figure 16 shows photocatalytic methane hydroxide oxidation by hybridization of EY and MMOH mutants. Electrolytes: WT, R320A, K323A, and methane-saturated TEOA buffer containing R320A / K323A and XD (100 mM, pH 7.5). Concentrations: XD (20 μM), MMOH, and mutant (2.5 μM). Light source: xenon lamp, I = 0.58 μEcm -2 s -1 ,λ> 400 nm. The methanol time yield was determined from a 1-hour reaction.

[0026] Figure 17 shows cyclic voltammetry (CV) scans of the protein-modified electrode. CV scans of (a) MMOH and (b) glassy carbon electrodes (GCE) coated with MMOH / MMOR or MMOH / MMOB complexes using ab, DDAB (0.5 wt%) solution. Electrolyte: Methane-saturated phosphate buffer (100 mM, pH 7). Working electrode (WE): Protein-modified GCE. Reference electrode (RE): Ag / AgCl (in 3 M NaCl). Counter electrode (CE): Pt wire. Scan rate: 25 mV s⁻¹ -1 .

[0027] Figure 18 shows the interfacial resistance of the protein-modified electrode estimated from the Nyquist plot at -0.09 V (vs. RHE). Electrolyte: Methane-saturated phosphate buffer (100 mM, pH 7). Light source: xenon lamp, I = 0.58 μEcm -2 s -1 ,λ > 400 nm. WE: Protein modified GCE. RE: Ag / AgCl (in 3 M NaCl). CE: Pt wire. Inset: Equivalent circuit in plot.R b ,R f ,R ct and CPE represent bulk resistance, film resistance, charge transport resistance, and phase resistance, respectively.

[0028] Figure 19 shows the results of methanol quantification by gas chromatography (GC). a, Calibration graph in methanol quantification by GC. [MeOH], Molar concentration of methanol. A MeOH , region of the methanol peak determined from the GC chromatogram. A EtOH , region of the ethanol peak determined in the chromatogram. b, GC chromatogram of methanol produced by the EY / MMOH complex. Electrolyte: Methane-saturated TEOA buffer (100 mM, pH 7.5) containing EY (20 μM) and MMOH (2.5 μM). Ethanol was used as an internal standard.

[0029] Figure 20 shows the effect of an electron donor on photocatalytic methane hydroxide oxidation by an EY / MMOH complex. Electrolyte: Methane-saturated buffer containing EY (20 μM), MMOH (2.5 μM), and an electron donor (100 mM). Electron donors: TEOA, EDTA, ethylenediaminetetraacetic acid, MOPS, 3-(N-morpholino)propanesulfonic acid, and water. Light source: xenon lamp, I = 0.58 μEcm -2 s -1 ,λ> 400 nm. The methanol time yield was determined from a 1-hour reaction.

[0030] Figure 21 shows the results of a methane isotope labeling experiment using an EY / MMOH complex. Photocatalytic activity by EY / MMOH 13 C Detected methanol derived from methane hydroxide 1 ¹H NMR (400 MHz, 298 K) spectrum. Sample: Containing EY (20 μM) and MMOH (2.5 μM) after light irradiation 13 C Methane-saturated D2O. Light source: xenon lamp, I = 0.58 μEcm -2 s -1 ,λ > 400 nm. NMR pulse sequence: zg30. Number of scans: 16. 13 Methyl quantum resonance in CH3OH is 1 H- 13 Split into two resonances by CJ binding. Black circle: MMOH protein.

[0031] Figure 22 shows the results of a binding interaction study using ITC. (a) ITC results of the unprocessed thermal and binding profiles of MMOH:EY, (b) MMOH:MMOR, and (c) MMOH-MMOR:EY. (d) Model showing non-competitive binding of multicomponents (MMOH, MMOR, and EY).

[0032] Figure 23 shows the time profiles of photocatalytic methane hydroxide oxidation by the EY-protein complex and biocatalytic methanol hydroxide oxidation by the natural MMO system. Electrolyte: Methane-saturated TEOA buffer (100 mM, pH 7.5) containing MMOH (2.5 μM), MMOR (5 μM), and MMOB (5 μM) with EY (20 μM) or NADH (50 mM). Light source: xenon lamp, I = 0.58 μEcm -2 s -1 ,λ> 400 nm.

[0033] Figure 24 shows the results of multiple analyses to illustrate the energy level diagram of excited EY. a, CV scan of EY (10 mM), showing the ground-state oxidation potential of EY [E ox (EY ·+ / EY)]. Electrolyte: Phosphate buffer (100 mM, pH 7). WE: GCE. RE: Ag / AgCl (in 3 M NaCl). CE: Pt wire. Scan rate: 25 mV s⁻¹ -1 . b, Normalized absorption and emission spectra of EY (10 μM). First singlet excited state of EY (E 0,0 The excitation-state energy of ) was obtained at the intersection of the normalized absorption and emission spectra. Excitation: 500 nm.

[0034] Fig. 25 shows the XD aqueous solution. 1 This shows the H NMR (400 MHz, D2O, 298 K) spectra. a, (a) FL, (b) EY, (c) ErB and (d) RB 1 ¹H NMR spectrum. Concentration: 100 μM XD.

[0035] Figure 26 shows the results of mass spectrometry (MS) analysis of an aqueous XD solution. MS spectra of (a) FL, (b) EY, (c) ErB, and (d) RB in ad, negative mode. Concentration: 1000 ppm XD.

[0036] Fig. 27 shows the transfer of XD to MMOH using ITC. d This shows the estimated results. ITC results of unprocessed thermal and integrated isotherms for MMOH and (a) FL, (b) EY, (c) ErB, (d), and RB bonds. Concentrations: 1 mM XDs, 10 μM MMOH.

[0037] Figure 28 shows the PL spectra of XD with the addition of MMOH. Changes in the PL spectra of (a) RB, (b) ErB, (c) EY, and (d) FL with the addition of MMOH in phosphate buffer (100 mM, pH 7). Concentration: 1 μM XD. Excitation: 470 nm (FL), 500 nm (EY), 520 nm (ErB), or 530 nm (RB).

[0038] Figure 29 shows the Stern-volmer plot during the oxidative quenching of XD using MMOH. Concentration: 1 μM XD. Excitation: 470 nm (FL), 500 nm (EY), 520 nm (ErB), or 530 nm (RB).

[0039] Figure 30 shows the current-voltage analysis results of XD at the ground-state oxidation potential of XD. CV scans of (a) FL, (b) EY, (c) ErB, and (d) RB, representing the ground-state oxidation potential of XD [E ox (XD ·+ / XD)]. Electrolyte: Phosphate buffer containing XD (10 mM) (100 mM, pH 7). WE: GCE. RE: Ag / AgCl (in 3 M NaCl). CE: Pt wire. Scan rate: 25 mV s⁻¹ -1 .

[0040] Figure 31 shows the spectroscopic analysis results of XD at the excitation-state energies of the initial singlet filtered state of XD. Normalized absorption and emission spectra of (a) FL, (b) EY, (c) ErB, and (d) RB. Initial singlet excited state of XD (E0,0 The excitation state energy of ) was obtained at the intersection of the normalized absorption and emission spectra. Concentration: 10 μM XD. Excitation: 470 nm (FL), 500 nm (EY), 520 nm (ErB), or 530 nm (RB).

[0041] Figure 32 shows the energy diagram of photocatalytic methane hydroxide oxidation by XDs. The oxidation potentials of ground-state and excited-state XDs were obtained from current-voltage / spectroscopic analysis.

[0042] FIG. 33 shows the variant redeployment energy (λ), driving force (△G°), and activation energy (△G°) required for electron transport from EY to K323. ‡ This shows the Marcus curve of EY having surface residue K323 representing ).

[0043] Figure 34 shows the results of a calibration analysis of electron transport velocity and redistribution energy with a donor-acceptor distance. a, logarithm of electron transport velocity (log(k ET Plot showing the relationship between )) and the donor-acceptor distance. Correlation coefficient (r 2 ) indicates a strong negative association (r²=0.86), suggesting that electron transport velocity decreases with increasing distance. b, Plot illustrating the relationship between redistribution energy (λ) and donor-acceptor distance. Strong positive association (r 2 =0.85) suggests that the redeployment energy increases with increasing distance.

[0044] Figure 35 shows the time profile of residue activity of XD / MMOH. Electrolyte: Methane-saturated TEOA buffer (100 mM, pH 7.5) containing XD (20 μM) and MMOH (2.5 μM). Light source: xenon lamp, I = 0.58 μEcm -2 s -1,λ > 400 nm. Slope: Relative inactivation ratio. The relative inactivation ratio was estimated through residue activity toward methane during the reaction. Activation was estimated based on the amount of methanol produced by irradiating 0-h, 1-h, 2-h, 4-h, and 6-h reaction samples with visible light for 3 minutes.

[0045] Figure 36 shows the time profiles of sun-mediated biocatalytic methane hydroxide oxidation by an RB / MMOH / MMOB complex and biocatalytic methanol hydroxide oxidation by a natural MMO system. Electrolyte: Methane-saturated TEOA buffer (100 mM, pH 7.5) containing MMOH (2.5 μM), MMOR (5 μM), and MMOB (5 μM) with RB (20 μM) or NADH (50 mM). Light source: Artificial sun, I = 100 mW cm⁻¹ -2 ,λ: 260 to 900 nm.

[0046] Figure 37 shows the results of methane isotope labeling experiments using the RB / MMOH / MMOB complex. Photocatalytic activity by RB / MMOH / MMOB 13 C Detected methanol derived from methane hydroxide 1 ¹H NMR (400 MHz, 298 K) spectrum. Sample: Containing RB (20 μM), MMOH (2.5 μM), and MMOB (5 μM) after light irradiation 13 C Methane-saturated D2O. Light source: Artificial sun, I = 100 mW cm⁻¹ -2 ,λ: 260 to 900 nm. NMR pulse sequence: zg30. Number of scans: 16. 13 Methyl quantum resonance in CH3OH is 1 H- 13 Split into two resonances by CJ binding. Black circle: MMOH protein. Blue circle: MMOB protein.

[0047] The present invention will be described in more detail below.

[0048] The present invention relates to a photocatalyst comprising a methane monooxygenase (MMO) hydroxylase (MMOH); and a xanthen-based light harvester, a composition for methanol production comprising the same, and a method for methanol production.

[0049] The above photocatalyst may additionally include a methane monooxygenase (MMO) regulatory protein (MMOB), and may be, for example, in the form of an MMOH / xanthen-based light harvester / MMOB complex, but is not limited thereto.

[0050] The xanthen-based light harvester may comprise one or more xanthen dyes (XD) selected from the group consisting of fluorescein (FL), eosin (EY), erythrosine B (ErB), and rhoese Bengal (RB), and may, for example, comprise one or more halogenated xanthen dyes selected from the group consisting of eosin (EY), erythrosine B (ErB), and rhoese Bengal (RB), but is not limited thereto. The xanthen-based light harvester may comprise rhoese Bengal (RB), but is not limited thereto.

[0051] The above photocatalyst can produce methanol through the hydroxylation of methane, but is not limited thereto.

[0052]

[0053] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0054] Example 1.

[0055] Chemicals and materials

[0056] FL, Eosin Y disodium salt (EY), ErB, RB, β-Nicotinamide adenine dinucleotide (NADH), potassium phosphate monobasic, potassium phosphate dibasic, triethanolamine (TEOA), ethylenediaminetetraacetic acid, 3-(N-morpholino)propanesulfonic acid, methanol, ethanol, and deuterium oxide (D2O) were purchased from Sigma-Aldrich. Ar, CH4, 13 CH4 and O2 gas (99.999% purity) were purchased from Special Gas Co. (Korea). Premium silver and hydrophilic surface-enhanced Raman spectroscopy substrates were purchased from SERSitive (Poland). All chemicals were purchased at the highest possible purity and used exactly as received.

[0057]

[0058] Enzyme expression and purification of sMMOs

[0059] Recombinant sMMO was expressed in the Ms. trichosporium SMDM strain in which the entire sMMO-coding operon was deleted. Plasmid pT2MKL was used for the cloning and expression of the wild-type strain and inserted into Ms. Trichosporium OB3b-SMDM via conjugation. The recombinant sMMO strain was initially treated with various antibiotics (ampicillin (50 μg / mL)). -1 ), gentamycin, streptomycin, and spectinomycin (20 μg mL -1They were grown on NMS plates containing each). MMOH was grown in NMS medium containing 1 mM Fe. The MMOH component of sMMO was purified by fast protein liquid chromatography (FPLC) at 4°C using a BioRad Biologic Duo-flow. Soluble cell proteins were loaded onto HiPrep 16 / 100 DEAE FF. The column was equilibrated with 25 mM Tris-HCl buffer (pH 7.2) containing 5 mM sodium thioglycolate (Buffer A). The flow rate was 0.5 mL min -1 ...under these conditions, the sMMO component bound to the column. MMOH was eluted with 0.15 M NaCl in Buffer A (pH 7.2), concentrated, desalted using an ultrafiltration membrane concentrator (exclusion size: 100 kDa; Sartorius), and reloaded onto a HiLoad 16 / 600 Superdex 200 pg column (flow rate: 1 mL min -1 The protein was eluted using the same buffer. The active fraction was collected and concentrated using an ultrafiltration membrane concentrator of the same size. Finally, the expression level and subunit molecular weight of sMMO were evaluated using 12% SDS-PAGE and visualized by Coomasiv blue staining. K323A mutants were prepared using the same method as the wild type.

[0060]

[0061] Expression and Purification of MMOB and MMOR

[0062] MMOB and MMOR were expressed in the vector pET28a. Then, the recombinant plasmids pET28a-MMOB and pET28a-MMOR were transfected into E. coli BL21 (DE3) competent cells, and 50 μg ml -1Positive clones were selected from LB agar plates containing kanamycin. MMOB and MMOR transformants were treated with 50 μg / ml until an OD of 600 nm was reached. -1 The cells were cultured in LB broth containing kanamycin. Then, protein expression of MMOB and MMOR was induced by adding 0.1 mM IPTG at 16°C for 18 hours. Cells were harvested by centrifugation at 4000 rpm for 15 minutes and resuspended in elution buffer (10 mM imidazole, 300 mM sodium chloride, and 50 mM sodium phosphate; pH 7). Cells were pulverized by sonication and centrifuged at 12000 rpm for 10 minutes to obtain soluble enzymes. Gravity-flow affinity chromatography was used for His-labeled enzyme purification using a Ni-NTA agarose column. The bound enzymes were eluted using elution buffer (500 mM imidazole, 300 mM sodium chloride, and 50 mM sodium phosphate; pH 7). Desalting and concentration of enzyme analytes were performed using Vivaspin®500 centrifugal concentrators (10,000 MWCO, Sartorius). Protein molecular weight was measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0063]

[0064] Example 2.

[0065] Spectral characterization

[0066] Absorption spectra of XD with and without proteins (MMOH, MMOR, and MMOB) were performed using a V-650 spectrophotometer (JASCO). 1H NMR spectra were obtained using a 400 MHz NMR spectrometer (Bruker) equipped with water suppression technology to increase the signal-to-noise ratio. Mass spectra were recorded using an XEVO G2-XS QTof (Waters Corporation). Raman spectra (excitation wavelength: 633 nm) were collected using an ARAMIS (Horiba Jobin Yvon) instrument equipped with hydrophilic Ag nanoparticles to increase the signal-to-noise ratio. Infrared peaks of functional groups in DX with and without protein were identified using a Nicolet iS50 (Thermo Fisher Scientific Instrument). PL spectra (excitation: 470 nm (FL), 500 nm (EY), 520 nm (ErB), or 530 nm (RB)) were recorded using an RF-5301PC spectrofluorophotometer (Shimadzu). Time-resolved PL decay profiles (excitation: 467 nm) were obtained using a Fluorolog3 spectrofluorometer (HORIBA). Fluorescence decay curves were obtained using a time-interrelated single-photon calculation approach with a time resolution of 10 ps. Fluorescence lifetimes were evaluated using the following triplet-exponential function (Equation (1)):

[0067] (1)

[0068] In the above equation, α and τ are the amplitude and decay time, respectively, and I(t) represents the fluorescence intensity at a given time t. avg The average lifespan of ) was evaluated using the following equation (2).

[0069] (2)

[0070]

[0071] Simulation of charge transport

[0072] The computer workflow in Fig. 3 illustrates the electron transport mechanism between the light harvester and MMOH. Starting with blind docking (Fig. 4a), the workflow identified binding sites of the EY dye on the MMOH surface and yielded multiple clusters of the docked molecules. The inset in Fig. 4b shows the largest cluster, indicating the most promising binding site. Then, promising first electron acceptor residues that approximate the optimal binding posture of EY and are potentially involved in initial electron transport were selected (Fig. 4c). Subsequently, the first electron acceptor residues were identified using Marcus's curve analysis (Tables 1 and 2). This computer workflow highlighted the complex electron transport from the light harvester to MMOH, involving binding site identification and electron acceptor selection.

[0073]

[0074]

[0075]

[0076] Blind Docking Analysis

[0077] The crystal structure in sMMO (PDB ID: 6VK5) was obtained and analyzed by selecting a chain (MMOB) containing D and H chains for additional calculations. Although only chain A is essential for the calculation, the selection of additional chains prevents the docked EY from binding to the chain-bonding region of the MMO, thereby allowing for a more accurate analysis. The EY structure was retrieved from the PubChem database (PubChem ID: 11048). AutoDock Tools 1.5.7 was used for all EY-to-MMO molecular docking experiments. All residues in the active site were included in a blind grid calculation with a spacing of 0.5 Å between atoms, and the box was fixed at 126 × 126 × 126 Å. One thousand runs of the Lamarckian Genetic Algorithm (LGA) were performed, and the calculation was a maximum of 25 × 10 6The process was stopped after energy evaluation. A crossover rate of 0.8 was used, and the population size was limited to 150 based on GA. The morphology with the lowest docking score from the largest cluster was selected and evaluated using MD simulation. Hydrophobic interactions and hydrogen bonding between ligands and proteins were visualized using Maestro (Schrodinger 2022 suite).

[0078]

[0079] Molecular dynamics simulation and molecular dynamics-generalized native surface region analysis

[0080] MD simulations for interaction analysis and evaluation of binding free energy were performed using Desmond (Schrodinger 2022 suite). All systems were organized using “System Builder” in Maestro. Selected docking results were placed in an orthorhombic box with a buffer distance of 10 Å to generate a hydration model for energy minimization. The hydration model was developed using the SCP water model. Cutoff radii for van der Waals forces, time step, initial temperature, and system pressure were set to 9 Å, 2.0 fs, 300 K, and 1.01 bar, respectively. Electrostatic forces were evaluated separately for near and far ranges, with the boundary between the near and far ranges determined to be 9 Å. The sampling interval during the simulation was set to 2 ps. MD simulations were performed using Desmond with an NPT ensemble for 100 ns. The stability of surface bonds was further verified using MMPBSA calculations with 250 evenly spaced structures derived from the stable portion of 100 ns simulations run in Desmond with an OPLS force field. These structures were converted to PDB format and analyzed using the “MMPBSA.py” script. To identify major residue contributions, bond free energies were evaluated by considering van der Waals, electrostatic, desolvation, and internal strain energies according to free energy decomposition. The calculations used default parameters recommended by AMBER.

[0081]

[0082] Quantum mechanics / molecular dynamics analysis

[0083] In QM / MM minimization, the 8 Å layer of water molecules surrounding the protein and the active site region was ionized (Fe) into the QSite (Schrodinger 2022 suite) using unrestricted density functional theory (DFT) calculations. 3+It was optimized for the oxidation form. Additionally, using QM / MM-based spin-density calculations in MMOH, the transport region was located and the initial electron acceptor residue was identified. These residues initially receive electrons from the dye located on the protein surface. In the QM / MM calculations, the transport region was included in the QM region, and the rest of the protein was considered the MM region, where the electron donor / acceptor was frozen (previously optimized QM region). Additional electrons were injected into the QM region and the doublet state. Singlet-point calculations using the QM / MM method were performed with QSITE, which combines unrestricted DFT with the def2-TZVP basis set (def2-TZVP for heavy atoms and 6-311++G** for lighter atoms) and residues, to identify regions containing additional spin densities. This method was used to select the initial electron acceptor from the dye and identify the residue with the highest spin density.

[0084]

[0085] Calculation of electronic transport speed

[0086] Based on a spin-density-based method, residues were obtained from QM / MM calculations that provide high density when free electrons are introduced into the system. Calculations using the QM / MM approach yielded residues produced using a spin-density-based method. These calculations yielded high spin densities when free electrons are injected into the system. The next step in the calculation involves transporting the residues with the highest spin density to an MM column and recalculating the spin density using the parameters described above. Here, the driving force (△G°), the relocation energy (λ), and the activation energy (△G) derived from the driving force and relocation energy are given using the complete Marcus equation below. ‡ ) was calculated (Equation (3)):

[0087] (3)

[0088] In the above equation, H DA Is It refers to the electronic bonding between the donor and the acceptor. Assuming that the electronic bonding remains constant and the solvent does not participate in the reaction, the reliability was predicted using a Moser-Dutton ruler, and the donor-acceptor distance (R) was calculated using the following equation (Equation (4)):

[0089] (4)

[0090] Marcus curves are a useful tool for determining the relative efficiency of different competing electron transport pathways. The most promising first acceptor residues in terms of energy efficiency were determined by plotting the electron transport rate as a function of the free energy difference. Additionally, the curves were used to identify the activation energies required for electron transport, which provides additional information regarding the reaction mechanism. In the simulations, sample frames were obtained from structures with the lowest energy. The top 50 lowest energy structures were selected for the calculations. Marcus curves were plotted on 50 randomly selected distinctive structures obtained through MD simulations. For EY and K323 electron exchange, Marcus curves were plotted in the natural state and in the production state with an electron-containing acceptor, as well as in the reaction state with electrons containing a donor, dye, and acceptor. Both states were fitted using polynomial curves.

[0091]

[0092] Photocatalytic Reaction and Analysis

[0093] For the photocatalytic conversion of methane to methanol, TEOA buffer (100 mM, pH 7.5, 1.5 mL) containing MMOH (2.5 μM), MMOR (5 μM), and MMOB (5 μM) along with XD (20 μM) or NADH (50 mM) was prepared. The electrolyte was foamed with CH4 and O2 gases for 20 minutes and then added to a diaphragm-sealed borosilicate glass vial using a magnetic stirrer. Visible light (I = 0.58 μEcm⁻¹) -2 s -1 The system was stirred at 500 rpm while adding CH4 (2 mL) and O2 (2 mL) every hour under λ > 400 nm. Methanol was quantified using a 7890A gas chromatograph (Agilent Technologies) equipped with a DB-1 column (length: 60 m, diameter: 0.320 μm) and a flame ionization detector. Oven temperatures are shown in Table 3. Methanol was quantified using a predetermined calculation graph with ethanol as an internal standard (Fig. 19). The methanol reaction time and quantum yield were determined using the following equations (5) and (6).

[0094] (5)

[0095] (6)

[0096]

[0097]

[0098] Protein quantification and enzyme analysis

[0099] sMMO protein concentrations were determined by the Bradford method using bovine serum albumin as the standard protein. The activity of sMMO in recombinant wild-type (WT) sMMO was confirmed using methane as a substrate in a 20-mL sealed reaction vial with a final volume of 500 μL, while shaking at 30°C. The analysis was performed using a specific molar ratio of MMOH:MMOR:MMOB (1:2:2). Gas was removed from the large upper empty space and replaced with an equal amount of methane. The aqueous concentration of methane was determined using Henry's integers. The reaction was initiated upon the addition of NADH (5 mM). Methanol formation was measured using gas chromatography (DB-1 Column; Agilent 7890A). A naphthalene assay was performed on the entire cell. 1 mL of cell suspension was incubated with 5 mg of naphthalene crystals at 30°C for 1 hour with moderate stirring (200 rpm). After centrifuging the reaction solution, 0.1 mL of o-dianisidine tetrazotized dye (1%) was added to 1 mL of the suspension to determine the naphthol content. Then, the purple color of the diazo dye was observed as an absorption peak at 530 nm on a V650 spectrophotometer (JASCO).

[0100]

[0101] Electron paramagnetic resonance (EPR)

[0102] EPR spectra were collected using an EMX / Plus spectrometer equipped with a dual-mode cavity (Bruker). EPR measurements were performed at a temperature of 5 K with an external magnetic field (from 10 to 590 mT), a sweep time (2 min), a transition time (20 ms), a microwave frequency (9.65 GHz), a microwave power (2.31 mW), and a tuned amplitude (10 G). EPR samples were prepared in TEOA buffer (100 mM, pH 7.5) containing EY (20 μM) and MMOH (100 μM). MMOH was reduced by EY under visible light (λ > 400 nm) during EPR measurements. All samples were deoxygenated with high-purity Ar gas (99.999%) before use at sufficient EPR intensity.

[0103]

[0104] Electrochemical characterization

[0105] Voltage-current experiments were performed using a potentiostat / galvanostat (WBCS 3000, WonAtech) with a 3-electrode configuration (glassy carbon working electrode, Ag / AgCl (in 3 M NaCl) reference electrode, and platinum wire counter electrode). The glassy carbon was modified by drop-casting 20 μL of DDAB (0.5 wt%) followed by coating with a mixed solution of 20 μL of EY (20 μM) and protein (20 μM). The protein was represented as vs. RHE (reversible hydrogen electrode) according to the following formula:

[0106]

[0107] Electrochemical impedance spectroscopy (EIS) analysis was performed in a 3-electrode configuration using a ZIVE SP1 (WonAtech) at frequencies (from 100 kHz to 0.1 Hz) and amplitudes (10 mV). Photocurrent and EIS measurements were taken in visible light (I = 0.58 μEcm⁻¹). -2 s -1 It was carried out at a voltage of -0.09 V (vs. RHE) under λ > 400 nm.

[0108]

[0109] Gibbs energy of photo-induced electron transport

[0110] Photo-induced electron transport (ΔG PET The change in Gibbs energy of ) is the excited-state XD (E ox (XD ·+ / XD * Oxidation potential of )) ground-state XD (E ox (XD ·+ Oxidation potential of / XD)), and singlet excited state energy of XD (E 0,0 E was evaluated using ).E 0,0는 It was determined from the intersection of the normalized absorption and emission spectra. ox (XD ·+ / XD * ) was calculated according to the following formula:

[0111]

[0112] XD photo-excited by the substrate (sub) (XD * ΔG in the oxidation of ) PET It was obtained using the following formula:

[0113]

[0114]

[0115] Isothermal titration calorimetry

[0116] ITC experiments were performed using the MCS ITC system (Microcal Inc., Northampton, MA, USA). The experiments were conducted at 30°C in 100 mM phosphate buffer (pH 7.0). The protein concentration in the calorimetric cells was 10 μM, and the ligand concentration in the syringe was 0.1–1 mM. A highly concentrated stock solution of the ligand was prepared in purified water and adjusted to pH 7.0. Prior to titration, the stock solution was diluted tenfold with 100 mM phosphate buffer. The heat of protein dilution was negligible. The heat of ligand dilution was corrected by subtracting the average heat of the injection after saturation. For ITC multi-component measurements, K was used as a basic parameter to evaluate k and Δh by adding the ligand MMOR solution titrated with MMOH+EY solution. A app and ΔH A app It was conducted to obtain and determine the interaction type (independent, positive or negative cooperation, and competitive combination). K A app is required to estimate the cooperativeness of k or an integer. If the calculated value of k is 1, the interaction is non-competitive. The value of k was calculated according to the following equation:

[0117]

[0118] In the above equation, K A app The value is 0.043 μM (Table 4), which is the definitive binding constant in A (EY) in the presence of B (MMOR) at concentrations [B or MMOR]. The cooperative binding enthalpy was calculated using the following equation:

[0119]

[0120] In addition to the cooperability constant (k), other parameters such as coupling entropy can also be determined in coupling studies. ΔH A The value of is the binding entropy at A (EY), and Δ Bis the binding entropy in B (MMOR), and ΔH A app ΔH is the distinct bond enthalpy at A (EY) in the presence of B (MMOR) at a concentration [B or MMOR]. A app The value is -622.83 (Table 4) and Δh is the enthalpy of cooperative binding. When the estimated value of the enthalpy of cooperative binding is less than 1 and greater than 0, the ligand binds non-competitively.

[0121]

[0122]

[0123] Example 3.

[0124] Direct photoreduction of MMOH

[0125] Spectroscopic analysis confirmed the binding of EY to three sMMO components (MMOH, MMOR, and MMOB) (Figs. 2 and 6 to 11). The absorption spectrum of EY showed that the peak at 517 nm underwent a stepwise red-shift (~3 nm) with increasing MMOH concentration, while MMOR and MMOB exhibited only minor changes (Figs. 2a and 10). These changes in the absorption spectra are attributed to the association between EY and MMOH. The EY-MMOH binding 1 This was confirmed using nuclear magnetic resonance (NMR) spectroscopy (Fig. 2b). As the concentration of MMOH increased, xanthene (H) was identified due to the shielding effect of functional groups (e.g., carboxylates, amides, and imidazoles) in MMOH. a and H b ) and benzoate (H c , H d , H e , and H fA high field shift was observed at the outer edge quantum of ). Specific interactions between EY and MMOH were confirmed using surface-enhanced Raman spectroscopy (Fig. 2c). In the Raman spectrum, the aromatic ring stretch of the carboxylate in EY (~645 cm⁻¹) -1 ) / Variation (~712 cm -1 ), COC loop stretch (~1180 cm -1 ), and asymmetrical stretch (~1560 cm -1 The characteristic peak of ) disappeared after binding with MMOH, which is due to non-covalent interactions (e.g., π-π stacking, hydrogen bonding, and ion-dipole interactions) between the dye molecule and the protein. In the EY / MMOH complex, CO (~1090 cm⁻¹) -1 ), C=C (~1605 cm -1 ), and C=O (~1730 cm -1 The reduced infrared peak intensity of the stretch demonstrated the self-bonding of EY and MMOH through non-covalent interactions (Fig. 11).

[0126] The possibility of MMOH reduction by photoexcited EY molecules was investigated using photoluminescence (PL) spectroscopy. Oxidative fluorescence quenching of EY was observed in MMOH and MMOR, but not in MMOB (Figs. 2d and 12). MMOB, lacking a redox component, cannot accept photo-induced electrons from EY. MMOM (1.95×10⁻⁶ 4 M -1 The Stern-Volmer constant of ) was twice as high as MMOR. These results are due to the easy binding of EY and MMOH and rapid photo-induced electron transport to the iron active center.

[0127] PL spectroscopy was used to further confirm the photo-induced electron pathway for photocatalytic methane hydroxide oxidation (Fig. 13). The oxidative fluorescence quenching of EY by MMOH is enhanced by O2 and further increased with methane, indicating photo-induced electron transport from EY to MMOH, and further to O2 for methane activation, and methane activation. To confirm the detailed electron transport mechanism in MMOH, the electron paramagnetic resonance (EPR) of the protein was measured (Fig. 14). The diferric state (Fe) at g = 16 II Fe II The EPR signal of MMOH in ) appeared as light in the presence of EY, and g = 4.3 [ferric ion (Fe Ⅲ )] and g = 1.83 [mixed-valent form (Fe II Fe III It disappeared in )]. These results indicate EY-sensitive reduction of the iron-active site in MMOH. Taken together, studies using absorption, NMR, Raman, PL, and EPR spectroscopy confirmed that EY binds to MMOH and transports its photoinduced electrons to the iron site for methane activation.

[0128]

[0129] Electron transport from EY to MMOH

[0130] In the computer workflow of the present invention (Fig. 3), the initial electron acceptor on the MMOH surface was highlighted to confirm its role in electron transport from EY to MMOH. Blind docking simulations were performed to verify the binding mechanism of EY to the MMOH surface. The binding mechanism with the lowest docking score and the largest cluster with seven conformations was found to have a docking score of -6.83 kcal / mol with an average binding energy of -6.76 kcal / mol (Figs. 4a,b). To minimize energy, conformations were selected, and molecular dynamics (MD) simulations were performed for 100 ns using an isothermal-isobaric ensemble (NPT). The relative binding energy of EY to the surface of MMOH (ΔG bind ) was calculated using molecular dynamics-generalized born surface area (MM-GBSA). The calculated bond free energy (ΔΔG MM-GBSA ) is -3.51 kcal / mol associated with a suitable non-covalent bond between the dye and MMOH (Table 5). The docked EY molecule is surrounded by residues K323, R320, W317, M247, Y251, Y324, and V255 (Fig. 4c).

[0131]

[0132] To verify the initial electron acceptor, spin density and electron transport rate (k) ET ) was evaluated. Based on the analysis, K323 was 0.78 e / Å 3 It exhibited the highest spin density, followed by R320, W317, M247, Y251, Y324, and V255 (Fig. 4d). k from EY to K323 ET (k ET = 1.61×10 6 s -1 ) was the highest, which is R320 (1.68×10 5 s -1), W317 (1.46×10 1 s -1 ), M247 (3.83×10 2 s -1 ), Y251 (3.01×10 4 s -1 ), Y324 (1.64×10 4 s -1 ), and V255 (2.80×10 4 s -1 It is a speed exceeding ) (Table 1). High k due to low redistribution energy (λ = 2.84 eV). ET This was possible, implying that minimal molecular structure control is required for electron transport. Furthermore, K323 possesses a negative free energy of reaction (△G° = -0.40 eV), indicating that the electron transport process is spontaneously favorable. Additionally, their spatial layout was confirmed by measuring the shortest distance between the electron-radiating oxide ions of the EY and residues on the tricyclic ring. Computer analysis revealed a complex electron transport mechanism from EY to MMOH, specifically to the MMOH surface. High k ET Computer simulations and analyses, evidenced by several key factors including minimum redistribution energy, negative free energy suggesting spontaneous transport, the lowest activation energy barrier for rapid electron transport, and proximity to EY, clearly confirmed that lysine residue K323 is the primary electron acceptor. Experimental results showing reduced binding affinity and methanol productivity of K323A (lysine at K323 changed to alanine), R320A (arginine at R320 changed to alanine), and R320A / K323A mutants compared to the wild-type MMOH (WT) support the computer analysis results, highlighting that lysine residue K323 plays an essential role in maintaining the functional integrity of MMOH (Figs. 15 and 16).

[0133]

[0134] Photocatalytic methane hydroxide hydroxide

[0135] The roles of MMOH, MMOR, and MMOB in methane hydroxide oxidation were investigated using protein membrane voltammetry. In the cyclic voltammetry results, the cathodic current between 0 and -0.2 V (vs. reversible hydrogen electrode (RHE)) increased with the addition of MMOH and methane, suggesting that electrons are transported from the glassy carbon electrode (GCE) to MMOH during methane activation (Fig. 17a). The cathodic current increased further when MMOH was combined with MMOB within the methane-saturated buffer (Fig. 17b), indicating that MMOH utilizes electrons from the GCE to activate methane, and MMOB facilitates the methane's access to MMOH. Conversely, the addition of MMOR to MMOH reduces the cathodic current of MMOH due to electron annihilation by MMOH. Additionally, the short-term photocurrent of EY-stained proteins was measured to confirm possible electron transport between EY and proteins under visible light. Figure 5a shows a significantly high photocurrent in the presence of MMOH, which increased further with the addition of methane and MMOB. The photocurrent reaction indicates that EY transports its photo-induced electrons to MMOH for methane activation, while MMOB promotes the catalytic conduction of methane. Conversely, MMOR reduces the photocurrent of the EY / MMOH complex by extinguishing photo-excited electrons that MMOH could use for methane activation. This hypothesis is supported by the PL quenching of EY by MMOR and the photocurrent of EY / MMOR, and was demonstrated by the EY-sensitive reduction of MMOR (Figures 2d and 5a). The charge transport resistance between EY and the MMOH / MMOB complex decreased 1.75-fold after light irradiation (Figures 5b and 18), indicating that protein reduction by photo-excited EY occurs more rapidly than by GCE.

[0136] Atmospheric conditions (T= 25℃; P= 1 atm) and visible light irradiation (I= 0.58 μEcm) -2 s -1 The photocatalytic performance of the EY-protein complex was evaluated under λ > 400 nm. According to the results (Figs. 19 and 20), triethanolamine (TEOA) acted as the optimal electron donor to sustain photocatalytic methane hydroxide oxidation. Control experiments including each reaction component (TEOA, light harvester, sMMO, methane, and light) produced negligible methanol, confirming that photobioreduction by EY is a critical step in methane hydroxide oxidation (Fig. 5c). 13 Methane isotope testing using C methane confirmed the production of methanol originating from photocatalytic methane hydroxide (Fig. 21). When combined with MMOB or MMOR, the EY / MMOH complex showed an increase (1.36-fold) and a decrease (1.22-fold) in methanol hour yield, respectively. MMOB accelerated methane activation in MMOH, whereas MMOR inhibited activation by consuming photoexcited electrons from EY. As a result, EY / MMOH / MMOB exhibited the highest methanol hour yield (4.13 mmol g⁻¹) among EY-protein complexes. cat -1 h -1 ) and methanol productivity (22.59 mmol g cat -1 ; 6 h reaction) was shown (Fig. 5c and Fig. 21). Isothermal titration calorimetry (ITC) results indicated binding interactions between the ligands (EY and MMOR) and MMOH were non-competitive (Fig. 22 and Table 4). An increase in photon flux further increased the initial production rate, showing a high quantum yield of 0.13 (Fig. 5d). The catalytic performance of the protein complex was demonstrated in the NADH-dependent MMO series (0.13 mmol g). cat -1 h -1) and reported sMMO systems (0.02-0.4 mmol g cat -1 h -1 It was superior to (Fig. 23). To explain the superior performance of the photocatalytic system, two pathways of MMOH reduction were compared: (1) the excited states of EY and (2) NADH / MMOR (Figs. 5e,f, Fig. 24, and Table 6). From a thermodynamic perspective, the oxidation potential of excited EY (-0.58 V vs. RHE) has a higher driving force for reducing MMOH (reduction potential of 0.33 V vs. RHE) than that of NADH (0.09 V vs. RHE). In addition to strong redox power, EY was able to activate MMOH faster than NADH / MMOR through its shorter electron pathway, achieving the highest methanol production yield (Fig. 5e,f).

[0137]

[0138]

[0139] Biosolar methane hydroxide by xanthen derivatives (XD)

[0140] Based on the experimental results of the present invention, the structural effects of XD molecules in photocatalytic methane hydroxide oxidation were evaluated. Due to the influence of halogen atoms on the physicochemical and photophysical properties of XD, non-halogenated [fluorescein (FL)] and halogenated [EY, Erythrosine B (ErB), and RB] dye molecules were selected for the experiment (Fig. 1b and Figs. 25 and 26). The binding affinity of each XD to MMOH was determined using ITC (Fig. 6a, Fig. 27, and Table 7) to confirm that it is enthalpically driven. RB exhibited the highest binding affinity to MMOH compared to ErB, EY, and FL (with a lowest K dIt showed (of 9.23 ± 1.3 μM). The high polarization of RB due to heavy halogen atoms increases hydrophilic interactions between xanthene halide molecules and proteins, thereby increasing binding affinity for MMOH. The favorable binding of RB to MMOH is highly suitable for photocatalytic methane hydroxide oxidation. XD-sensitive reduction of MMOH was further investigated using PL spectroscopy. All XDs exhibited oxidative fluorescence quenching in the presence of MMOH, indicating photoreduction of MMOH by XD (Figs. 28 and 29). Additionally, the Stern-Volmer constant increased with increasing XD halogenation. For heavier halogen atoms, the average PL lifetime decreased in the following order: RB (0.83 ns) < ErB (1.30 ns) < EY (2.01 ns) < FL (4.33 ns) (Fig. 6b). The heavy halogen atoms of xanthen increase spin-orbit coupling between the singlet and triplet states, processing inter-term electron crossing more rapidly. Consequently, the fluorescence lifetime and PL intensity from the singlet state decreased as triplet formation was increased by strong spin-orbit coupling (Fig. 6c). Taken together, among the XDs, RBs containing heavy iodine and chlorine atoms exhibited the fastest decay lifetime (0.83 ns) and the highest Stern-Volmer constant (4.15 × 10⁻⁶). 4 M -1This indicated rapid photo-induced electron transport from RB to MMOH. To compare with the driving force of the redox reaction, kinetic indices such as the oxidation potentials of the ground and excited states in electron transport related to the reduction of MMOH by XDs were further evaluated. Following the excitation of XD by visible light, photo-induced electrons in the excited state of XD exhibited a potential sufficient to drive MMOH reduction (reduction potential of 0.33 V vs. RHE) (FL: -0.55 V vs. RHE, EY: -0.58 V, ErB: -0.62 V, and RB: -0.69 V), while TEOA oxidation (oxidation potential of 1.46 V vs. RHE) stabilized XD to maintain photocatalytic conduction (Figs. 30-32 and Table 7). The excited RB molecule exhibits the highest oxidation potential (-0.69 V vs. RHE) and a mostly negative Gibbs energy (-1.02 eV) for reducing MMOH, indicating that RB can act as the most effective light-harvester for reducing MMOH among xanthen derivatives (Tables 6 and 8).

[0141]

[0142]

[0143] Blind docking simulations were performed on XD / MMOH complexes to investigate the binding of XD to MMOH and subsequent electron transport between them. RB exhibited the lowest free energy (-7.65 kcal / mol) among the light harvesters, indicating the highest binding affinity to the MMOH surface (Table 5). MM-GBSA simulations provided more accurate binding free energies by considering MM interactions and solvation effects. Following docking analysis, the MM-GBSA results further supported the higher binding interactions between RB and the MMOH surface; RB showed the lowest binding free energy (-5.26 kcal / mol) in the MM-PBSA simulations, indicating more favorable and stable binding interactions compared to other XDs. Additionally, RB (k ET = 1.46×10 11 s -1 ) is another dye ErB (k ET = 1.91×10 8 s -1 ), EY (k ET = 1.61×10 6 s -1 ), and FL (k ET = 2.77×10 4 s -1 It exhibited a significantly faster electron transport rate to the initial acceptor residue K323 than ) (Fig. 33 and Table 2). This is mainly due to the lowest rearrangement energy of RB (λ = 1.57 eV), indicating minimal structural rearrangement during electron transport. The lower rearrangement energy acts as an energy barrier (△G) for electron transport. ‡ Reduce ) to k ET It increases. In addition, among all XDs, RB has the highest driving force (△G° = -0.53 eV) for the fastest and most proficient electron transport and the shortest donor-acceptor distance (R = 3.1 Å), resulting in the highest k ET achieved (Table 2). These results are kET This supports a strong correlation between redeployment energy, and the distance between the donor and acceptor (Fig. 34). The catalytic productivity of various XD-sensitive MMOHs under visible light was evaluated and compared. ErB (3.93 mmol g⁻¹) cat -1 h -1 , 0.12, and 9.9% h -1 ), EY (3.04 mmol g cat -1 h -1 , 0.09, and 10% h -1 ), and FL (1.20 mmol g cat -1 h -1 , 0.04, and 9.49% h -1 Compared to ), the RB / MMOH complex was 4.93 mmol g cat -1 h -1 methanol hour yield of 0.15, quantum yield of 0.15, and 8.73% h -1 It exhibited the highest photocatalytic performance with an inactivation rate (Figs. 6d and 35). RB is the most promising light harvester for activating MMOH due to its favorable binding to proteins and strong redox power for reducing diiron active sites. Under atmospheric conditions (T= 25 °C; P= 1 atm) and sunlight (at an air mass of 1.5 global intensity, 100 mW cm⁻¹). -2 To demonstrate comprehensive biosolar methane hydroxide oxidation under ), the catalytic performance of MMOH was evaluated using RB as a light harvester and MMOB as a catalytic conductivity enhancer. The RB / MMOH / MMOB complex was 7.52 mmol g cat -1 h -1 39.8 mmol g for 6 hours at an initial rate cat -1Methanol was produced, which is 60 times faster than the natural biocatalytic reaction by the MMO series (MMOH, MMOB, and MMOR with excess NADH) (Fig. 36).

[0144] The methanol productivity of the BioSolar platform is compared to reported sMMO systems (0.02-0.4 mmol g⁻¹). cat -1 h -1 It is superior to ). In addition, the catalytic activity of RB / MMOH / MMOB in methane hydroxide oxidation of methanol is 13 This was confirmed by a methane isotope experiment using C methane (Fig. 37).

[0145] The RB-MMO platform outperformed state-of-the-art thermocatalysts, photocatalysts, and biocatalysts in methane hydroxide (Fig. 6e and Table 9). Reported thermocatalysts, such as metal-organic frameworks and zeolites, showed significant catalytic conductivity (0.001–3.8 mmol g⁻¹). cat -1 h -1 To achieve this, harsh operating conditions (T > 200°C; P > 6 atm) are required, and it exhibits high energy intensity. Photoactive metal oxides and methane-oxidation photocatalysts trigger the conversion of methane to methanol under mild conditions, but their conversion rates are very low (< 0.4 mmol g⁻¹) due to slow electron transport between the catalyst and the substrate. cat -1 h -1 The biosolar platform of the present invention accelerates electron transport by directly activating MMOH using a powerful RB light harvester. Therefore, RB-MMO exhibits the highest catalytic performance (7.52 mmol g⁻¹) under atmospheric conditions (T= 25°C; P= 1 atm) among the latest catalysts for the conversion of methanol to methane. cat -1 h -1 Achieved ).

[0146]

[0147] As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily propose other inventions that are inferior or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the same spirit. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

1. A photocatalyst comprising methane monooxygenase (MMO) hydroxylase (MMOH); and a xanthen-based light harvester.

2. In Paragraph 1, A photocatalyst that additionally contains a methane monooxygenase (MMO) regulatory protein (MMOB).

3. In Paragraph 1, The above xanthen-based light harvester is a photocatalyst comprising one or more xanthen dyes (XD) selected from the group consisting of fluorescein (FL), eosin (EY), erythrosine B (ErB), and rhoese Bengal (RB).

4. In Paragraph 1, The above xanthen-based light harvester is a photocatalyst comprising a halogenated xanthen dye.

5. In Paragraph 1, The above xanthen-based light harvester is a photocatalyst comprising one or more xanthen dyes (XD) selected from the group consisting of eosin (EY), erythrosine B (ErB), and rhoz's bengal (RB).

6. A composition for producing methanol comprising a photocatalyst according to any one of claims 1 to 5.

7. In Paragraph 6, A composition that produces methanol through the hydroxylation of methane.

8. A method for producing methanol from methane using a photocatalyst according to any one of claims 1 to 5.

Images