USE OF UV-ACTIVATED ENZYMES TO IMPLEMENT OXIDATION REACTIONS AND THE CORRESPONDING PROCESSES
20230340546 · 2023-10-26
Assignee
- INSTITUT NATIONAL DE LA RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT (Paris, FR)
- Centre National De La Recherche Scientifique (Paris, FR)
- UNIVERSITE D'AIX MARSEILLE (Marseille Cedex 07, FR)
- ECOLE CENTRALE DE MARSEILLE (Marseille, FR)
Inventors
Cpc classification
International classification
Abstract
The use of UV-activated Copper Radical Oxidase (CRO) enzymes in the implementation of oxidation reactions. Also, a process for oxidizing organic compounds using enzymes which are activated by UV light. The process also leads to concomitant formation of hydrogen peroxide, that can optionally be used in hydrogen peroxide mediated processes. Further, the process relates to the oxidation of alcohols in aldehydes.
Claims
1-17.(canceled)
18. A process for the chemical oxidation of an organic compound, said process comprising the step of contacting an organic compound bearing an oxidizable function with at least one Copper-Radical Oxidase (CRO) enzyme in the presence of molecular oxygen, wherein said at least one CRO enzyme is activated by a step of exposing said at least one CRO enzyme to UV light, to obtain a UV-activated CRO enzyme, whereby the organic compound is oxidized into an oxidized organic product, and whereby hydrogen peroxide is generated.
19. The process for chemical oxidation according to claim 18, wherein the step of exposing said at least one CRO enzyme to UV light is carried out during the step of contacting, before the step of contacting, or both before and during the step of contacting.
20. The process for chemical oxidation according to claim 18, wherein during the contact of said organic compound with said enzyme, the exposure to UV light is continuous or intermittent.
21. The process for chemical oxidation according to claim 18, wherein the UV light has a wavelength comprised from 240 to 320 nm, preferably from 270 to 290 nm, more preferably has a wavelength of about 280 nm.
22. The process for chemical oxidation according to claim 18, wherein, during the step of exposing said at least one CRO enzyme to UV light, the enzyme is exposed to UV light having a light intensity comprised from 0.01 to 1000 mW/cm.sup.2, in particular from 1 to 100 mW/cm.sup.2. or wherein, during the step of exposing said at least one CRO enzyme to UV light, the enzyme is exposed to of from 1 to 100 μmol photon.Math.s.sup.−1.Math.m.sup.−2; in particular of from 10 to 30 μmol photons.sup.−1.Math.m.sup.−2.
23. The process for chemical oxidation according to claim 18, wherein said process comprises between the step exposing said at least one CRO enzyme to UV light before the step of contacting and the step of contacting said UV-activated enzyme with said organic compound, a step of transfer whereby the organic compound and the UV-activated enzyme are mixed.
24. The process for chemical oxidation according to claim 18, wherein said process is carried out in an aqueous medium, preferably in a buffered aqueous medium, preferably at a temperature comprised between 20 and 50° C. preferably at a temperature of about 23° C.
25. The process for chemical oxidation according to claim 18, wherein the at least one CRO enzyme belongs to the AA5 family, in particular to the AA5_1 or the AA5_2 subfamilies.
26. The process for chemical oxidation according to claim 18, wherein the at least one CRO enzyme belongs to the AA5_2 subfamily and is an alcohol oxidase (AlcOx), preferably is an alcohol oxidase extracted from Colletotrichum graminicola, in particular having SEQ ID NO: 1, or having at least 60%, in particular at least 70% identity with SEQ ID NO: 1, or wherein the at least one CRO enzyme belongs to the AA5_2 subfamily and is a galactose oxidase (GalOx) and more preferably is a galactose oxidase extracted from Fusarium graminearum, in particular having SEQ ID NO: 2, or having at least 60%, in particular at least 70% identity with SEQ ID NO: 2, or wherein the at least one CRO enzyme belongs to the AA5_2 subfamily and is an aryl alcohol oxidase (AAO) and is preferably extracted from Colletotrichum graminicola, in particular having SEQ ID NO: 3, or having at least 60%, in particular at least 70% identity with SEQ ID NO: 3, or wherein the at least one CRO enzyme belongs to the AA5_1 subfamily and is a glyoxal oxidase (GLOx) and more preferably is a glyoxal oxidase extracted from Pycnoporus cinnabarinus, in particular having SEQ ID NO: 4, or having at least 60%, in particular at least 70% identity with SEQ ID NO: 4, or wherein the at least one CRO enzyme is a GlxA-type enzyme which is preferably extracted from the bacterium Streptomyces lividans, in particular having SEQ ID NO: 5, or having at least 60%, in particular at least 70% identity with SEQ ID NO: 5.
27. The process for chemical oxidation according to claim 18, wherein said organic compound selected from the group consisting of: saturated (C.sub.1 to C.sub.20) primary alcohols, unsaturated (C.sub.1 to C.sub.20) primary alcohols, saturated (C.sub.1 to C.sub.20) secondary alcohols, unsaturated (C.sub.1 to C.sub.20) secondary alcohols, (C3 to C.sub.10) cyclic alcohols, aryl alcohols, heteroaryl alcohols, and geminal diols, in particular selected from the group consisting of: saturated (C.sub.1 to C.sub.20) primary alcohols, allylic alcohols, aryl alcohols comprising a primary hydroxyl group linked to the aryl group by a Ci alkyl group, and geminal diols.
28. The process for chemical oxidation according to claim 18, wherein said enzyme is an alcohol oxidase (AlcOx), and wherein said organic compound is: a primary alcohol and the obtained oxidized organic product is an aldehyde, an aryl alcohol comprising a primary hydroxyl group linked to the aryl group by a C1 alkyl group, in particular selected from benzyl alcohol, 4-nitrobenzyl alcohol, anisyl alcohol, veratryl alcohol and 4-hydroxybenzyl alcohol, or aryl alcohols comprising an allylic alcohol attached to the aryl group, in particular cinnamyl alcohol, a saturated, or unsaturated (C1 to C20) primary alcohol, linear or branched, in particular chosen from n-butanol, n-pentanol, n-hexanol or 2,4-hexadiene-1-ol, or a naturally-occurring polymer comprising long aliphatic chains bearing hydroxyl functions or a sugar, in particular polymers chosen from waxes, cutins and hemicellulose.
29. The process for chemical oxidation according to claim 18, wherein said enzyme is a glyoxal oxidase (GLOx), and wherein said organic compound is: 5-hydroxymethylfurfuryl alcohol, or lignocellulose derived compounds, in particular glyoxal, methyl glyoxal, glyoxylic acid, formaldehyde or glycerol.
30. The process for chemical oxidation according to claim 18, wherein said enzyme is a galactose oxidase (GalOx), and wherein said organic compound is: forest and agricultural biomass, in particular fibres, and hemicelluloses, in particular compounds comprising a galactopyranose moiety, more in particular xyloglucan.
31. A method for implementing hydrogen peroxide-driven enzymatic reactions, comprising a step of generating hydrogen peroxide by contacting an organic compound bearing an oxidizable function with at least one Copper-Radical Oxidase (CRO) enzyme in the presence of molecular oxygen, wherein said at least one CRO enzyme is activated by a step of exposing said at least one CRO enzyme to UV light, to obtain a UV-activated CRO enzyme, whereby the organic compound is oxidized into an oxidized organic product, and whereby hydrogen peroxide is generated.
32. The method according to claim 31, wherein the hydrogen peroxide-driven enzymatic reaction is selected from the group consisting of decarboxylations, hydroxylations, halogenations, epoxidations, sulfoxidations and Baeyer-Villiger oxidations, or wherein the hydrogen peroxide-driven enzymatic reaction consists in the enzymatic conversion of said hydrogen peroxide into oxygen and water, in particular using a catalase enzyme, or wherein the hydrogen peroxide-driven enzymatic reactions consists in the degradation of a polysaccharide, said reaction comprising contacting said polysaccharide with one or more lytic polysaccharide monooxygenase (LPMO), in the presence of an external source of electrons, said source of electrons being in particular a reducing agent.
Description
FIGURES
[0238]
[0239]
[0240] .square-solid. represents a reaction without activation of the enzyme (control reaction), .box-tangle-solidup. represents a reaction wherein the enzyme is activated with horseradish peroxidase, .circle-solid. represents a reaction according to the Invention, the enzyme being pre-activated with UV light, and .diamond-solid. represents a reaction wherein the buffer is exposed to UV light, in the absence of enzyme (negative control)
[0241]
[0242] .square-solid. represents a reaction in the dark, without UV light (control reaction), .box-tangle-solidup. represents a reaction with intermittent UV exposure, but in the absence of a CRO enzyme (negative control), .circle-solid. represents a reaction according to the Invention, wherein the enzyme was pre-activated by UV, and .diamond-solid. represents a reaction according to the Invention, wherein the enzyme intermittently activated by UV light, the grey vertical bars represent the periods of light exposure.
[0243]
[0244] □ represents a reactions using UV light.
[0245] ⋄ represents a reaction using broad spectrum UV-Vis light.
[0246]
[0247]
[0248]
[0249] x represents continuous UV light.
[0250] □ represents discontinuous UV light.
[0251] ⋄ represents discontinuous broad spectrum UV-Vis light.
[0252] .circle-solid. represents non light exposure (dark).
[0253]
[0254]
[0255] □ represents on/off cycles of 2/30 min
[0256] ⋄ represents on/off cycles of 10/30 min.
[0257]
[0258] The error bars show the standard deviation for 2 independent experiments.
[0259] The y-axes correspond to the aldehyde product concentrations, expressed in mM, and the x-axes correspond to the reaction time (min).
[0260] □ represents absence of activation (reaction in the dark).
[0261] ⋄ represents activation by discontinuous UV light.
[0262]
[0263] Δ represents the absence of catalase.
[0264] x represents the presence of catalase.
[0265]
[0266]
[0267]
[0268] Δrepresents absence of activation (reaction in the dark).
[0269] ⋄ represents activation by UV light.
[0270]
EXAMPLES
[0271]
TABLE-US-00007 Abbreviations and definitions BMGY medium Buffered Glycerol-complex Medium BMMY medium Buffered Methanol-complex. Medium EPR Electron Paramagnetic Resonance HRP Horseradish peroxidase OD.sub.600 nm Optical Density measured at 600 nm Pluronic E8100 Non-ionic surfactant (anti-foam) PTM1 trace salts Salt-metals mix solution Rpm Revolutions per minute YPD agar Yeast Extract Peptone Dextrose Agar g relative centrifugal force
General Remarks
[0272] Most chemicals were purchased from Sigma-Aldrich (Darmstadt, Germany) or VWR. HRP type II (MW 33.89 kDa) and catalase from bovine liver (monomer MW 62.5 kDa) were purchased from Sigma-Aldrich.
[0273] Molar concentrations of HRP was estimated by Bradford assay.
[0274] All alcohol substrates stock solutions were prepared in H.sub.2O whenever possible, aliquoted and stored at −20° C. The concentration of H.sub.2O.sub.2 stock solution was verified at 240 nm (ε.sup.240=43.6 M.sup.−1.Math.cm.sup.−1).
Example 1: DNA Cloning and strain production
[0275] The DNA cloning and strain production was performed according to methods described in the literature: [0276] DNA cloning and strain production of the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx, Genbank ID XM_008096275.1, Uniprot ID E3QHV8) was performed according to D. (Tyler) Yin et al., “Structure—function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family,” Nat. Commun., vol. 6, p. 10197, December 2015. [0277] DNA cloning and strain production of the galactose oxidase from Fusarium graminearum (FgrGalOx, Genbank ID XM_011327027.1, Uniprot ID I1S2N3) was performed according to O. Spadiut et al., “A comparative summary of expression systems for the recombinant production of galactose oxidase,” Microb. Cell Fact., vol. 9, pp. 1-13, 2010. [0278] DNA cloning and strain production of the glyoxal oxidase from Pycnoporus cinnabarinus (GLOx, PciGLOx-2, ORF ID BN946_scf184747.g42, Uniprot ID A0A060SYB0) was performed according to M. Daou et al., “Heterologous production and characterization of two glyoxal oxidases from Pycnoporus cinnabarinus,” Appl. Environ. Microbiol., vol. 82, no. 16, pp. 4867-4875, 2016. [0279] DNA cloning and strain production of the aromatic alcohol oxidase from Colletotrichum graminicola (CgrAAO, Genbank ID EFQ27661, Uniprot ID E3Q9X3) was performed according to Y. Mathieu et al., “Discovery of a Fungal Copper Radical Oxidase with High Catalytic Efficiency toward 5-Hydroxymethylfurfural and Benzyl Alcohols for Bioprocessing,” ACS Catal., vol. 10, no. 5, pp. 3042-3058, 2020.
Example 2: Heterologous Enzyme Production
[0280] For preliminary tests, all proteins were first produced in 2 L flasks. To this end, single colonies of P. pastoris X33 expressing the gene of interest were individually streaked on a YPD agar plate containing Zeocin (100 μg.Math.mL.sup.−1) and incubated 3 days at 30° C. A single colony was then used to inoculate 10 mL of YPD, in a 50 mL sterile Falcon tube, incubated during 5 h (30° C., 160 rpm). This pre-culture was used to inoculate at 0.2% (vol/vol), 500 mL of BMGY medium in a 2 L baffled flask, incubated during approximately 16 h (30° C., 200 rpm) until the OD.sub.600 nm reached 4-6. The produced cellular biomass was then harvested by centrifugation (5 min, 16° C., 3,000 g). The cell pellet was resuspended in 100 mL BMMY medium in a 500 mL flask supplemented with CuSO.sub.4 (500 μM) and methanol (1%, vol/vol) and incubated for 3 days (16° C., 200 rpm), with daily additions of methanol (1% added, vol/vol). Then, the extracellular medium was recovered by centrifugation (10 min, 4° C., 3,000 g) and the supernatant filtered on 0.45 μm membrane (Millipore, Massachusetts, USA) and stored at 4° C. prior to purification.
[0281] CgrAAO and PciGLOx-2 were produced according to known procedures: Y. Mathieu et al., “Discovery of a Fungal Copper Radical Oxidase with High Catalytic Efficiency toward 5-Hydroxymethylfurfural and Benzyl Alcohols for Bioprocessing,” ACS Catal., vol. 10, no. 5, pp. 3042-3058, 2020 and M. Daou et al., “Heterologous production and characterization of two glyoxal oxidases from Pycnoporus cinnabarinus,” Appl. Environ. Microbiol., vol. 82, no. 16, pp. 4867-4875, 2016, respectively.
Example 3: Heterologous Enzyme Bioreactor Production
[0282] CgrAlcOx and FgrGalOx were also produced at larger scale, in bioreactors. Bioreactor production was carried out in a 1.3-L New Brunswick BioFlo 115 fermentor (Eppendorf, Germany) Precultures were prepared as described above for flask production and were used to inoculate at 0.2% 100 mL of BMGY medium, in a 500 mL flask, incubated (30° C., 200 rpm) until the OD.sub.600 nm reached 2-6. Four hundred mL of basal salt medium containing 1.8 mL PTM1 trace salts (both made according to the P. pastoris fermentation process guidelines—Invitrogen—version B 053002) were inoculated with 10% (vol/vol) of the BMGY culture. Temperature was set to 30° C. One hundred μL of Pluronic E8100 (BASF, Germany) were added after 6 h of culture to prevent foam. After full consumption of glycerol (as indicated by a return of dissolve oxygen (DO) level at 100%), sorbitol-methanol transition phase was initiated by addition of 80 mL sorbitol (250 g.Math.L.sup.−1 stock solution), 1.6 mL PTM1 traces salts and 2 mL methanol. After full consumption of carbon sources, the temperature was lowered to 20° C. and a methanol fed-batch was initiated with a feeding rate of 3.9 mL/h/L (mL per hour per liter of initial fermentation volume) of a methanol solution complemented with PTM1 trace salts (12 mL/L). New additions of 100 μL Pluronic E8100 were made after 30 h and 53 h of fermentation. Methanol feeding rate was increased to 7.8 mL/h/L after 53 h of fermentation. Throughout the fermentation, pH was maintained at 5 by automated adjustment with NH.sub.3 base. Air flow was maintained at 0.5 slpm (standard liter per minute). A cascade with a set point of 20% dissolved oxygen is maintained through agitation between 400 to 900 rpm and the percentage of pure oxygen addition between 0 to 50%. Fermentation was ceased after 118 hours. The harvested biomass was centrifuged (10 min, 5500 g, 4° C.). The supernatant was filtered through a 0.45-μm membrane (Millipore), flash-frozen in liquid nitrogen and stored at −80° C.
[0283] Flash-freezing did not cause any enzyme activity loss, for both CgrAlcOx and FgrGalOx.
Example 4: Protein Purification
[0284] The filtered culture broth was buffer exchanged by ultrafiltration through a 10 kDa cut-off polyethersulfone membrane (Vivacell 250, Sartorius Stedim Biotech GmbH, Germany) with Tris-HCl(50 mM pH 8.7) or Tris-HCl (50 mM pH 8.0) for CRO-AlcOx and CRO-GalOx, respectively. CRO-AlcOx was purified by anion exchange chromatography by loading the crude protein sample on a DEAE-20 mL HiPrep FF 16/10 column (GE Healthcare, Illinois, USA), equilibrated with buffer A1 (Tris-HCl, 50 mM, pH 8.7) and connected to an Äkta Express system (GE Healthcare). Elution was performed by applying a linear gradient from 0 to 50% of buffer B1 (Tris-HCl, 50 mM, pH 8.7+1 M NaCl) over 15 column volumes (CV) at a flow rate of 3 mL.Math.min.sup.−1.
[0285] FgrGalOx was purified by ionic metal affinity chromatography by loading the crude protein sample on a His-Trap HP 5-mL column (GE Healthcare, Buc, France) and connected to an Äkta Xpress system (GE Healthcare). Prior to loading, the column was equilibrated with buffer A2 (50 mM Tris-HCl, pH 7.8, 150 mM NaCl). After loading, non-specific proteins were eluted by applying a first washing step of 5 CV at 2% of buffer B2 (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 500 mM imidazole) and the target protein was eluted during a second step of 5 CV at 30% of buffer B2. Loading and elution were carried out at a flow rate of 3 mL.Math.min.sup.−1. In all cases, the collected fractions were analyzed by SDS-PAGE in 10% polyacrylamide precast gel (Bio-Rad) stained with Coomassie blue. Fractions containing the recombinant enzyme were pooled, concentrated and buffer exchanged in sodium phosphate (50 mM, pH 7.0) or sodium acetate (50 mM, pH 5.2), for CgrAlcOx and FgrGalOx, respectively. The protein concentration was determined by UV absorption at 280 nm using a Nanodrop ND-200 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) for CgrAlcOx (ε=101215 M.sup.−1.Math.cm.sup.−1), FgrGalOx (ε=124,135 M.sup.−1.Math.cm.sup.−1) , PciGLOx-1 (ε=47,690 M.sup.−1.Math.cm.sup.−1), CgrAAO (ε=107,760 M.sup.−1.Math.cm.sup.−1).
Example 5: Spectrophotometric Monitoring of Benzyl Alcohol Oxidation
[0286] Benzyl alcohol can be oxidized by several CROs, including CgrAlcOx, FgrGalOx and CgrAAO. The oxidation of benzyl alcohol into benzaldehyde was monitored by developing a simple absorbance assay consisting in measuring changes in absorbance at 254 nm upon oxidation of benzyl alcohol (1.5 mM) by the CRO (10 nM final concentration). Reactions were carried out in sodium-phosphate buffer (50 mM, pH 7.0), at 23° C., in UV-transparent cuvettes (1 mL reaction volume). In positive controls (carried out in the dark), horseradish peroxidase (HRP, 50 μg mL.sup.−1) was added to the mixture. The reactions were initiated by adding the CRO (100 μL of 100 nM stock solution), which was either stored in the dark or UV-exposed (vide infra). The reaction was mixed by vigorously pipetting up and down. In negative controls, the enzyme stock solution was replaced by sodium phosphate buffer (50 mM, pH 7.0), kept in the dark or UV-exposed. The absorbance (pathlength=1 cm) was measured using an Evolution 201 UV-Vis spectrophotometer (Thermo-Fisher). Given a 1:1 stoichiometry of the reaction, one can calculate the concentration of benzaldehyde according to Equation 1, where the molecular extinction coefficient of benzaldehyde at 254 nm is 57-fold higher (ε.sup.254.sub.benzaldehyde=8,497 M.sup.−1.Math.cm.sup.−1) than its alcohol counterpart (ε.sup.254.sub.BnoH=149 M.sup.−1.Math.cm.sup.−1).
[Benzaldehyde].sub.t=(Abs.sup.254 nm.sub.t−Abs.sup.254 nm.sub.t0)/(ε.sup.254 .sub.benzaldehyde−ε.sup.254.sub.BnOH) (equation 1)
Example 6: CRO Pre-Photoactivation
[0287] Pre-activation experiments consisted in exposing the enzyme stock solution (100 nM, 1 mL) to UV light during a given amount of time (usually 10 min) before transferring (ca. 30 sec step) a fraction (100 μL) to the reaction mixture. The reaction was then monitored as described above. In preliminary tests, a UV-lamp model EF-180/F (Spectroline, Spectronics Corporation, Westbury, USA) was used, delivering 254 nm UV light at a maximum power of 1350 μW/cm.sup.2, equivalent to 29 μmoles of photon.Math.s.sup.−1.Math.m.sup.−2), positioned side-wise (relatively to the reaction cuvette) at a 3 cm distance from the cuvette.
[0288]
Example 7: CRO Intermittent Illumination
[0289] In intermittent illumination experiments, the full reaction mixture, i.e. containing buffer, CRO and benzyl alcohol, was submitted to cycles of UV exposure (2 min) followed by on-line spectrophotometric monitoring (2 min). In such experiments, the illumination set-up was the same as described in example 6.
[0290]
Example 8: Electron Paramagnetic Resonance
[0291] CgrAlcOx (50 μM), prepared in sodium phosphate buffer (50 mM, pH 7.0), was flash-frozen in liquid nitrogen and continuous wave EPR spectrum was recorded. Then, the sample was thawed, exposed to UV light (280 nm +/−10 nm, sample positioned side wise and at 40 cm from lamp source, 400 mW received by the sample) during 10 min and flash-frozen again before recording a new EPR spectrum. Controls containing only buffer were also carried out. The illumination system was an Arc Lamp Housing equipped with a xenon-mercury bulb (Newport, USA, model 67005) and connected to an OPS-A150 Arc Lamp power supply (50-500 W) (Newport, USA). The power was set to 350 W. EPR spectra were recorded on a Bruker Elexsys E500 spectrometer operating at X-band at 110 K (BVT 3000 digital temperature controller) with the following acquisition parameters: number of scans, 3; modulation frequency, 100 kHz; modulation amplitude, 5 G; attenuation, 10 dB and microwave power, 20 mW.
Example 9: Optimized small-scale photobiocatalytic reaction
[0292] Optimal illumination conditions are determined by exposing the full reaction mixture to different light intensities (by varying the distance between the light source and the reaction vessel and/or varying the power supply) and different light wavelengths (by using different bandpass filters). Continuous versus intermittent light exposure is also probed, where the duration of intermittent light and “dark” time are varied (from 0 sec to 10 min). An optimal reaction condition is defined as a reaction yielding a stable reaction kinetic and/or a high final product yield (e.g., >70%). The quantity of reaction products is measured off-line by sampling regularly the reaction mixture and stopping the reaction (by either adding EDTA (10 mM final) or acidifying the mixture with HCl (1N final)) prior to quantification. Different CRO/substrate couples are tested, including: [0293] CRO-AlcOx/benzyl alcohol or fatty alcohols (e.g., hexanol) [0294] CRO-GalOx/galactose [0295] CRO-GLOx/glyoxal or methyl glyoxal or glyoxylic acid [0296] CRO-AAO/5-hydroxymethylfurfural (HMF)
[0297] In standard reaction conditions, the CRO (10 nM-1 μM) is mixed with the substrate (3-30 mM) in buffered aqueous medium (sodium phosphate (50 mM, pH 8.0) for CRO-AlcOx and CRO-GalOx, sodium 2,2′-dimethylsuccinate (50 mM, pH 6.0) for CRO-GLOx, sodium phosphate (50 mM, pH 7.0) for CRO-AAO). The reaction is carried out at 23° C., under magnetic stirring, in Quartz cuvettes (Hellma, France). The effect of the supply of O.sub.2 is also tested by bubbling through a syringe either air or a mixture of N.sub.2/O.sub.2 (various ratio of O.sub.2 between 0 and 100% v/v), with a gas flow rate of 100 mL/min.
[0298] The illumination system is an Arc Lamp Housing equipped with a xenon-mercury bulb (Newport, USA, model 67005) and connected to an OPS-A150 Arc Lamp power supply (50-500 W) (Newport, USA). The power is set to 350 W. A bandpass filter (280±10 nm) with 50 mm optical diameter (Edmund Optics, Lyon) is mounted on the lamp. The light intensity received by the sample is measured outside of the reaction vessel, on the front side facing the light beam, with an optical power meter (Newport, USA).
Example 10: Photobiocatalytic Reactions Coupled to a Secondary Enzymatic System
[0299] Using optimized illumination conditions, we evaluate the effect of secondary enzymatic reactions on the primary photobiocatalytic CRO reaction. A first secondary reaction is the conversion of H.sub.2O.sub.2 produced in situ into H.sub.2O and O.sub.2 by a catalase. Various concentrations of catalase are tested (1 nM-1 μM). Another secondary reaction aims at using H.sub.2O.sub.2 produced in situ as co-substrate of a peroxygenase reaction. In this order, to the photobiocatalytic CRO reaction is added a mixture containing an LPMO (1 μM), cellulose (10 g.Math.L.sup.−1Avicel or 0.1% w/v phosphoric acid swollen cellulose) and a reducing agent (100 μM ascorbic acid or cellobiose (3 mM)/cellobiose dehydrogenase (CDH, 0.1-1 μM)). The LPMO is the AA9E from Podospora anserina (PaLPM09E) and the CDH is the CDH from Podospora anserina (PaCDHB), produced and purified as previously described by C. Bennati Granier et al. “Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina,” Biotechnol. Biofuels, vol. 8, no. 1, p. 90, December 2015.
Example 11: Photobioreactor Upscaling
[0300] After establishing the proof-of-concept in 1 mL reactions, the photobiocatalytic reactions is upscaled to 100 mL, using the same illumination system as described above but with a top-wise illumination, with the light source placed at optimal distance from the reaction mixture surface. The reaction is carried out in a 250 mL beaker without spout, under magnetic stirring, at 23° C. The top of the beaker is closed with a home-made Quartz window, equipped with a rubber ring, to allow UV light to reach the solution while minimizing losses by evaporation.
Example 12: Reaction Mixture Analysis
[0301] The CRO-AlcOx activity on benzyl alcohol is determined by quantifying benzaldehyde spectrophotometrically at 254 nm as described above.
[0302] The CRO-AlcOx activity on fatty alcohol is determined as follows: 500 μL of the reaction mixture is mixed with 500 μL of cyclohexane/ethyl acetate mixture (1:1), followed by shaking and centrifugation (5 min, 2300 rpm). The organic layer is transferred into a new vial by pipetting and injected in an Optima-σ-3 GC capillary column (30 m×0.25 mm×0.25 μm, Machery-Nagel GmbH&Co KG, Germany) mounted on a gas chromatography (GC)-2014 apparatus (Shimadzu, Japan) equipped with a flame ionization detector (FID). Nitrogen is used as carrier gas, under constant pressure (200 kPa). The inlet and detector temperature are set at 250° C. The temperature gradients of the GC method are described in Table 1. Heptan-1-al (1 mM) is added as internal standard.
[0303] The CRO-GalOx activity on galactose is determined in two ways: [0304] For initial qualitative assessment of galactose consumption, we use thin layer chromatography (TLC), where the chromatograms are developed on silica gel plates (Sigma-Aldrich, L'Isle d'Abeau Chesnes, France) with butan-1-ol/acetic acid/H.sub.2O (4:1:1) as the solvent. The plates are dried before immersion in an acidic solution of orcinol (0.1% orcinol (w/v) dissolved in H.sub.2O/ethanol/H.sub.2SO.sub.4 (22:75:3, vol/vol/vol) and visualized by heating at 105° C. for 5 min. [0305] For quantitation, we use high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) (ICS-6000 system, ThermoFisher Scientific, Villebon sur Yvette, France). Samples are injected on a CarboPac PA1 (2×50 mm) column operated with 0.1M NaOH (eluent A) at a flow rate of 0.25 ml.Math.min.sup.−1 and a column temperature of 30° C. Elution is achieved using a stepwise gradient with increasing amounts of eluent B (0.1 M NaOH, 1 M NaOAc), as follows: 0-10% B over 10 min; 10-30% B over 25 min; 30-100% B over 5 min; 100-0% B over 1 min; and 0% B (reconditioning) for 9 min Chromatograms are recorded using Chromeleon 7.0 software.
[0306] The activities of CRO-GLOx on glyoxal/methylglyoxal/glyoxylic acid and CRO-AAO on HMF are analyzed as previously described by M. Daou et al., “Heterologous production and characterization of two glyoxal oxidases from Pycnoporus cinnabarinus,” Appl. Environ. Microbiol., vol. 82, no. 16, pp. 4867-4875, 2016. Briefly, the reaction products are separated on an Aminex HPX-87H column (300×7.8 mm; Bio-Rad) connected to a high pressure liquid chromatography (HPLC) apparatus (Agilent 1260) coupled to a diode-array detector (DAD, monitored wavelengths: 210, 280, 290, 330 and 350 nm). The column temperature is set to 45° C., sulfuric acid (2.5 mM) is used as isocratic eluant with a flow rate of 0.5 ml.Math.min.sup.−1. All samples are filtered through 10-kDa-molecular-mass-cutoff Nanosep polyethersulfone membrane columns (Pall Corporation, Saint-Germainen-Laye, France) and 0.45-μm pore-size polyvinylidene difluoride syringe filters (Restek, Lisses, France) before injection in the column.
TABLE-US-00008 TABLE 1 GC method Rate T Time Compounds (° C. .Math. min.sup.−1) (° C.) (min) Hexanol — 40 4 8 100 0 25 220 3
Example 13: Light Intensity-Dependent Activity of CgrAlcOx
[0307] A solution containing BnOH (3 mM) and CgrAlcOx (10 nM) in aqueous sodium phosphate (50 mM, pH 7.0), at 23° C., was exposed to varying intensities of either UV light (λ=280±10 nm) or UV-Vis broad spectrum light (λ=200-800 nm), under magnetic stirring in air.
[0308] Benzyl alcohol was thus oxidized to benzaldehyde. The enzyme activity as a function of the light intensities was measured resulting in the data as shown in
[0309] Thus, 40% intensity, for example, corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0310] It can be seen from
Example 14: Effect of Duration of Light Exposure on CgrAlcOx Activity
[0311] A solution containing benzyl alcohol (3 mM) and CgrAlcOx (10 nM) in aqueous sodium phosphate (50 mM, pH 7.0), at 23° C., was exposed to varying illumination modes under magnetic stirring in air: [0312] continuous UV light (λ=280±10 nm), [0313] discontinuous UV light (λ=280±10 nm), or [0314] discontinuous UV-Vis light broad spectrum (200-800 nm).
[0315] The discontinuous mode consisted in on/off illumination cycles of 1/30 min, repeated over 180 min.
[0316] A control of CgrAlcOx-catalyzed reaction (in the absence of any activator) gave the residual activity measured in the dark.
[0317] Experiments were carried out at 40% or 100% of I.sub.max for UV light and UV-Vis light, respectively. I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0318] Thus, 40% intensity, for example, corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0319] The time-course of benzaldehyde (PhCHO) production was observed as shown in
[0320] It can be seen from
Example 15: Effect of Pre-Photoactivation on CgrAlcOx Activity
[0321] A solution containing benzyl alcohol (3 mM) and CgrAlcOx (10 nM) in aqueous sodium phosphate (50 mM, pH 7.0), at 23° C. was left in the dark under magnetic stirring in air.
[0322] The enzyme had been pre-exposed beforehand to UV light (λ=280±10 nm; 40% of I.sub.max) for varying amounts of time (0 to 1800 s). I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0323] Thus, 40% intensity corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0324] The results obtained are as shown in
[0325] It can be seen from
Example 16: Effect of Discontinuous Illumination Mode on CgrAlcOx Activity
[0326] A solution containing BnOH (3 mM) and CgrAlcOx (10 nM) in aqueous sodium phosphate (50 mM, pH 7.0), at 23° C., was subjected to benzyl alcohol oxidation to benzaldehyde, by exposure, under magnetic stirring, in air, to UV light (λ=280±10 nm; 40% of I.sub.max) with two different discontinuous illumination modes: on/off cycles of either 2/30 min or 10/30 min, repeated over 180 min. I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0327] Thus, 40% intensity corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0328] The results obtained are as shown in
[0329] It can be seen from
Example 17: Photo-Activation of CgrAAO
[0330] CgrAAO-catalyzed oxidation reactions were tested for: [0331] (A) benzyl alcohol (BnOH, 3 mM) into benzaldehyde (PhCHO), [0332] (B) 5-hydroxymethylfurfural (HMF; 3 mM) to 2,5-diformylfuran (DFF), and [0333] (C) 5-hydroxymethyl-2-furan carboxylic acid (HMFCA, 3 mM) to 5-formyl-2-furan carboxylic acid (FFCA).
[0334] CgrAAOx (50 nM) was activated by discontinuous UV light (λ=280±10 nm). A control in the dark was also carried out. The discontinuous mode consisted in on/off illumination cycles of 1/30 min, repeated over 180 min.
[0335] Experiments were carried out at 40% of I.sub.max. I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2. Thus, 40% intensity corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0336] All reactions were carried out in an aqueous sodium phosphate solution (50 mM, pH 7.0), at 23° C., under magnetic stirring, in air.
[0337] The results obtained for benzyl alcohol oxidation are as shown in
[0338] The results obtained for 5-hydroxymethylfurfural oxidation are as shown in
[0339] The results obtained for 5-hydroxymethyl-2-furan carboxylic acid oxidation are as shown in
[0340] Overall, the results displayed in
Example 18: Synergy Between CgrAlcOx and Catalase
[0341] The time-course of benzaldehyde (PhCHO) production upon BnOH (3 mM) oxidation by CgrAlcOx (10 nM) was determined. Reaction mixture was exposed to discontinuous UV light (λ=280±10 nm), in the absence or presence of catalase (5 nM final). The discontinuous mode consisted in on/off illumination cycles of 2/30 min, repeated over 180 min.
[0342] Experiments were carried out at 40% of I.sub.max (λ=280±10 nm). I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0343] Thus, 40% intensity corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0344] All reactions were carried out in an aqueous sodium phosphate solution (50 mM, pH 7.0), at 23° C., under magnetic stirring, in air.
[0345] The results obtained are as shown in
Example 19: Stability of Benzaldehyde and Benzyl Alcohol Under Light Exposure
[0346] Benzaldehyde (PhCHO, 1.2 mM) or BnOH (3 mM) were exposed, in the absence of enzyme, to discontinuous UV light (λ=280±10 nm). The discontinuous mode consisted in on/off illumination cycles of 2/30 min, repeated over 180 min.
[0347] Experiments were carried out at 40% of I.sub.max (λ=280±10 nm). I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0348] Thus, 40% intensity, corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0349] All reactions were carried out in an aqueous sodium phosphate solution (50 mM, pH 7.0), at 23° C., under magnetic stirring.
[0350] The results obtained are as shown in
Example 20: Photoactivation of FgrGalOx
[0351] The oxidation of benzyl alcohol (3 mM) to benzaldehyde (PhCHO), and the oxidation of lactose (3 mM) to lactonic acid (LacOx), catalyzed by FgrGalOx (50 nM) were studied.
[0352] The reaction mixtures were exposed to discontinuous UV light (λ=280±10 nm), consisting in on/off illumination cycles of 1/30 min.
[0353] Experiments were carried out at 40% of I.sub.max (λ=280±10 nm). I.sub.max (at 4 cm, λ=280±10 nm)=1.6 mW.Math.cm.sup.−2, i.e. 36.5 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0354] Thus, 40% intensity, corresponds to a light flux (at 4 cm distance) of 0.64 mW.Math.cm.sup.−2, i.e. 14.6 μmol photon.Math.s.sup.−1.Math.m.sup.−2.
[0355] All reactions were carried out in an aqueous sodium phosphate solution (50 mM, pH 7.0), at 23° C., under magnetic stirring, in air.
[0356] The results obtained for the benzyl alcohol oxidation are as shown in
[0357] The results obtained for the lactose oxidation are as shown in