Chemoenzymatic processes for the cogeneration of disulfides and sulfoxides or sulfones.

Abstract

The present invention relates to a chemoenzymatic process for the co-production of disulfides and sulfoxides or sulfones from a composition M comprising: (1) a sulfide, (2) an optional oxidizing agent, (3) an organic compound having at least one thiol group, (4) an enzyme E that catalyzes the oxidation of sulfide to sulfoxide or sulfone, (5) an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of an organic compound having at least one thiol group to form a dimer, and (6) a cofactor common to enzymes E and D. The present invention also relates to a composition that makes it possible to carry out this process. It also relates to the use of a mercaptan to reduce a disulfide bridge formed between two equivalents of an organic compound having at least one thiol group, more particularly, as a regenerated substrate for the above process.

Description

[Technical field]

[0001] The present invention relates to a chemoenzymatic process for the co-production of disulfides and sulfoxides or sulfones from mercaptans and sulfides, respectively, and in particular to compositions allowing the implementation of this process. The invention also relates to the use of mercaptans for the reduction of disulfide bridges formed between two equivalents of organic compounds bearing at least one thiol group, and more particularly to the use of mercaptans as regenerated substrates for an enzyme cascade allowing the oxidation of sulfides. [Background technology]

[0002] Mercaptans are substances of great industrial interest and are currently widely used in the chemical industry, especially as starting materials for the synthesis of more complex organic molecules. For example, methyl mercaptan (CH 3 Methyl mercaptan (SH) is used as a starting material for the synthesis of methionine, an essential amino acid used in animal nutrition. Methyl mercaptan is also used in the synthesis of dialkyl disulfides, in particular dimethyl disulfide (DMDS), a sulfurized additive in hydrotreating catalysts for petroleum fractions.

[0003] Mercaptans (particularly methyl mercaptan) are generally industrially synthesized by a known method starting from alcohols and hydrogen sulfide in the presence of a catalyst at high temperatures according to the following formula (1). [ka]

[0004] However, this reaction produces by-products such as sulfides, as shown in the following formula (2). [ka]

[0005] Mercaptans can also be synthesized from halogenated derivatives and alkali metals, alkaline earth metals or ammonium hydrogen sulfide according to formula (3) below (for example, using chlorinated derivatives and sodium hydrogen sulfide). [ka]

[0006] This second synthetic route also results in the presence of unwanted sulfides.

[0007] Finally, mercaptans can be synthesized from olefins and hydrogen sulfide by acid catalysis or photochemically, depending on whether branched or unbranched mercaptans are desired, according to equation (4) below. [ka]

[0008] During this synthesis, sulfides are also produced as by-products.

[0009] These sulfides are obtained industrially in large quantities and are mainly sent for destruction, which is an efficiency loss in the process of producing mercaptans and an increased cost for disposal. The generation of such waste is an industrial problem for mercaptan producers, which calls for deriving value from the by-products. There are various ways to achieve this.

[0010] First, dimethyl sulfide can be used as a food flavoring agent or an anti-coking agent in the steam cracking of petroleum raw materials, and the sulfides themselves have market value, but the demand in these markets is very small compared to the production volume of sulfides.

[0011] Sulfides can also be converted to the corresponding mercaptans by sulfur hydrolysis reactions, but the conditions for this reaction are relatively harsh and involve additional parasitic reactions, limiting its industrial application.

[0012] Finally, another way to obtain value from the sulfides produced is by their oxidation to sulfoxides and / or sulfones. Chemical oxidation reactions of this type are well known. They can be carried out with or without a catalyst using sodium hypochlorite, hydrogen peroxide, oxygen, ozone or N 2 O 4 These use various types of oxidizing agents such as nitrogen oxides.

[0013] As well as chemical oxidation, the oxidation of sulfide can be catalysed in biological processes by enzymatic catalysis in solution or in organisms, generally microorganisms.

[0014] French Patent Applications FR1906488 and FR1906489 each describe a process for the selective preparation of sulfoxides or sulfones from organic sulfides by enzyme catalysis, the contents of which are incorporated herein by reference in their entirety.

[0015] Thus, in the processes described in these two patent applications, it is possible to selectively obtain sulfoxides or sulfones by using enzymes that catalyze the oxidation of sulfide to sulfoxides or sulfones, depending on the amount of sulfide present in the enzymatic reaction. If sulfide is not completely consumed during the step of performing the enzymatic sulfide oxidation reaction, sulfoxides are selectively obtained. If sulfide is completely consumed during the enzymatic sulfide oxidation reaction, sulfones are selectively obtained. Summary of the Invention [Problem to be solved by the invention]

[0016] However, the enzymes used may require a cofactor, which must be able to be reused (or regenerated) so that the enzymatic oxidation reaction can occur again.

[0017] This is because cofactors are generally not added stoichiometrically in industrial enzymatic processes due to their complexity and economics, so various approaches have been developed to recycle cofactors and regenerate enzyme cascades: Use of molecules similar to the cofactor. This approach is less costly because it uses simpler molecules. However, the affinity between the enzyme and the cofactor is not high enough for industrial applications, and the costs are unacceptable for industrial processes producing low value-added products. Use of enzymatic redox systems that use a sacrificial substrate. This technique generally requires the use of a second enzyme so that the cofactor used can be recycled. However, the cost of the sacrificial substrate strongly impacts the economics of such processes, especially when sulfides are oxidized to sulfoxides or sulfones with low added value. Use of whole cells. In this case, it is the cellular machinery that regenerates the cofactors used. The process is therefore closer to a fermentation process. To increase the performance characteristics of the system, it is common to add carbon / hydrogen containing molecules (such as glucose or glycerol), which comes at an additional cost.

[0018] Therefore, there is a need for an industrially viable route to regenerate the cofactors used in enzymatic processes for oxidizing sulfide.

[0019] There is also a need for a commercially and economically viable process for extracting value from sulfides, particularly those resulting from mercaptan synthesis. [Means for solving the problem]

[0020] One object of the present invention is to meet all or part of the above needs.

[0021] A more specific object of the present invention is to provide a pathway for regenerating (or recycling) the cofactor used in the enzymatic oxidation of sulfides to sulfoxides or to sulfones.

[0022] Another object of the present invention is to provide a process for the chemoenzymatic co-generation of sulfoxides or sulfones and disulfides.

[0023] It is a further object of the present invention to provide an industrially viable chemoenzymatic co-production process of sulfoxides or sulfones with disulfides, especially one that incorporates a simple, efficient and economical cofactor recycling pathway.

[0024] Another object of the present invention is to propose a process which makes it possible to obtain value from the sulfides which arise during the manufacture of mercaptans, and more particularly methyl mercaptan. [Brief description of the drawings]

[0025] [Figure 1] Figure 1 shows the concentrations (in mM) of diethyl sulfide (DES), diethyl sulfoxide (DESO) and bis(2-hydroxyethyl) disulfide (disulfide) present in the reaction as a function of time (expressed in hours) when the oxidation reaction was catalyzed by the enzyme CHMO. An element of the cascade allowing the recycling of the cofactor NADP was also added: cells overexpressing glutathione reductase (GR) from E. coli were added at the same concentration as CHMO-expressing cells, oxidized glutathione (GSSG) was introduced in catalytic amounts, and 2-mercaptoethanol was introduced at twice the concentration relative to DES. [Diagram 2]Figure 2 shows the concentrations (in mM) of diethyl sulfoxide (DESO), diethyl sulfone (DESO2), and bis(2-hydroxyethyl) disulfide (disulfide) present in the reaction as a function of time (expressed in hours) when the oxidation reaction was catalyzed by the enzyme CHMO. An element of the cascade allowing the recycling of the cofactor NADP was also added: cells overexpressing glutathione reductase (GR) from Escherichia coli were added at the same concentration as CHMO-expressing cells, oxidized glutathione (GSSG) was introduced in catalytic amounts, and 2-mercaptoethanol was introduced at twice the concentration relative to DESO. [Diagram 3] Figure 3 shows the concentration (in mM) of diethyl sulfoxide (DESO) present in the reaction as a function of time (expressed in hours) when the reaction is catalyzed by the enzyme CHMO. In this example, 2-mercaptoethanol was replaced by methyl mercaptan (MeSH) as the proton donor. A cascade element allowing the recycling of the cofactor NADP was also added: cells overexpressing glutathione reductase (GR) from E. coli were added at the same concentration as the cells expressing CHMO, oxidized glutathione (GSSG) was introduced in catalytic amounts, and MeSH was gradually added to the reaction at a flow rate of about 4 mmol / L / h. [Figure 4] FIG. 4 is a diagram illustrating the enzyme cascade according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present inventors have discovered a method for producing sulfoxides or sulfones by a (preferably selective) chemoenzymatic method incorporating a system for recycling a cofactor of an enzyme that catalyzes the oxidation of sulfide, thereby adding added value.

[0027] The inventors have surprisingly found a chemoenzymatic cascade that is not only compatible with the processes for the production of sulfoxides or sulfones, but also allows for the co-generation of a second target, a disulfide, instead of using a sacrificial substrate, thus allowing the recycling of the cofactor used in the sulfoxidation by oxidizing a mercaptan to a disulfide at the end of the cascade.

[0028] For example, if the oxidant is oxygen, the overall equation for the cascade can be written as: RSR'+O 2 +2R”SH⇒RS(O)R'+H 2 O+R”SSR”(for sulfoxide) RSR'+2O 2 +4R”SH→RS(O)2R'+2H 2 O+2R” (for sulfone)

[0029] Thus, mercaptans can be considered to be hydrogen donors.

[0030] More specifically, the enzyme cascade according to the present invention proceeds as follows.

[0031] In the presence of an enzyme that catalyzes the oxidation of sulfide to sulfoxide (hereinafter enzyme E), its cofactor, and a suitable oxidizing agent, the sulfide is converted to a sulfoxide or sulfone and the reduced cofactor is converted back to its oxidized form.

[0032] Next, in the presence of an organic compound having at least one thiol group and an enzyme (hereinafter enzyme D) that catalyzes the formation of disulfide bridges between two equivalents of this organic compound: The oxidized cofactor is reduced (recycled by hydrogen atoms donated by organic compounds), A disulfide bridge between two equivalents of the above organic compound results in the formation of a dimer (the organic compound goes from a reduced "monomeric" form to an oxidized "dimeric" form).

[0033] Thus, according to the present invention, an "organic compound" is a compound of formula R-SH, and a "dimer" of this compound is understood to be a compound of formula RSSR (a well-known example is the dimer of glutathione G-SH, GSSG, also called glutathione disulfide).

[0034] A chemical equilibrium then terminates the cascade: the oxidized dimer is reduced by reaction with two equivalents of a mercaptan and converted to the corresponding disulfide.

[0035] Thus, the presence of mercaptans allows, via chemical equilibrium governed by Le Chatelier's principle, that: reduction of the dimer to two equivalents of the organic compound (the latter is thus also recycled by the hydrogen atom provided by the mercaptan); and Forms disulfides.

[0036] It is the enzymatic reaction that shifts the chemical equilibrium in favor of disulfide formation.

[0037] The mercaptan may be used in a stoichiometric amount relative to the sulfide.

[0038] More specifically, two equivalents of mercaptan are used to generate one equivalent of sulfoxide, and four equivalents of mercaptan are used to generate one equivalent of sulfone.

[0039] The enzyme cascade operates until a substrate, reactant, or cofactor, etc. is depleted, or by inhibition, inactivation, or destruction of enzymes E and / or D.

[0040] This cascade allows the co-production of a sulfoxide or sulfone with a disulfide while regenerating the cofactor and organic compound used.

[0041] It is also very possible for this cascade to go in the opposite direction, so that all of the above reactions are reversed.

[0042] In this way, one of the advantages of the present invention is realized: it allows the insertion into an overall process for obtaining value from by-products resulting from the production of mercaptans, more particularly methyl mercaptan. In the production of mercaptans, sulfides are by-products, as indicated above. According to the present invention, these sulfides are preferably selectively oxidized to sulfoxides or sulfones, and the mercaptans formed are used in part to regenerate the cofactors used in the sulfoxidation, while they themselves can be converted into disulfides, which are other products of interest.

[0043] Thus, the present invention relates to a process for co-producing a disulfide and a sulfoxide or sulfone, comprising the steps of: (a) (1) Sulfide, (2) optionally an oxidizing agent; (3) an organic compound having at least one thiol group; (4) an enzyme E that catalyzes the oxidation of the sulfide to a sulfoxide or sulfone; (5) an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) A cofactor common to the above two enzymes E and D, providing a composition M comprising: (b) carrying out the (preferably simultaneous) enzymatic reactions of oxidation of sulfides and formation of disulfide bridges to give sulfoxides or sulfones and dimers (of said organic compounds); (c) reducing the dimer obtained in step (b) by reacting (in particular by chemical reaction) with a mercaptan to obtain the corresponding disulfide corresponding to said mercaptan and said organic compound having at least one thiol group; (d) optionally recovering the disulfide obtained in step (c) and / or the sulfoxide or sulfone obtained in step (b); Here, the mercaptan can be added in step (a), (b) or (c), and it is preferable to add the mercaptan in step (a).

[0044] [Definition] Carbon number 1 to 20 (C 1 -C 20 The term "alkyl" (N-alkyl) refers to a saturated aliphatic hydrocarbon of 1 to 20 carbon atoms, which may be linear or branched. Preferably, alkyl consists of 1 to 12 carbon atoms, or 1 to 4 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. The term "branched" is understood to mean that the alkyl group is substituted along the alkyl backbone.

[0045] Carbon number 2-20 (C 2 -C 20 The term "alkenyl" refers to an alkyl, as defined above, containing at least one carbon-carbon double bond.

[0046] Carbon number 2-20 (C 2 -C 20 The term "alkynyl" refers to an alkyl, as defined above, containing at least one carbon-carbon triple bond.

[0047] Carbon number 6-10 (C 6 -C 10 The term "aryl" refers to monocyclic, bicyclic, or tricyclic aromatic hydrocarbon compounds, more particularly phenyl and naphthyl.

[0048] "Carbon number (C 3 -C 10The term "cycloalkyl" refers to a mono- or bicyclic saturated aliphatic hydrocarbon group of 3 to 10 carbon atoms (eg, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, etc.).

[0049] Carbon number 3 to 10 (C 3 -C 10 ) is understood to be a cycloalkane containing 3 to 10 carbon atoms and containing at least one sulfur atom (preferably tetrahydrothiophene) and optionally at least one other heteroatom.

[0050] Carbon number 4-10 (C 4 -C 10 ) heteroarene" is understood to be an arene containing 4 to 10 carbon atoms and containing at least one sulfur atom (e.g. thiophene) and optionally at least one other heteroatom.

[0051] Heteroatoms are understood to be in particular atoms selected from O, N, S, Si, P and the halogens.

[0052] A "catalyst" is generally understood to be a substance that promotes a reaction and does not change at the end of this reaction. According to one embodiment, the enzyme E catalyzes the oxidation of sulfides to sulfoxides or to sulfones. According to one embodiment, the enzyme D catalyzes the formation of disulfide bridges between two equivalents of the organic compound bearing at least one thiol group to form dimers.

[0053] A "catalytic amount" is understood in particular to be an amount sufficient to catalyze the reaction, more particularly an amount sufficient to catalyze the oxidation of sulfides to sulfoxides or to sulfones and / or to form disulfide bridges. More particularly, a reactant used in a catalytic amount is used in a smaller amount (for example between about 0.01% and 20% by weight) with respect to the amount by weight of the reactant used in the stoichiometric proportion.

[0054] The selectivity of a reaction generally refers to the number of moles of product produced relative to the number of moles of reactant consumed after the reaction.

[0055] The usual definitions of conversion, selectivity, and yield are as follows:

[0056] Conversion rate = (moles of initial reactants - moles of reactants remaining after reaction) / (moles of initial reactants) Selectivity = moles of reactant converted to desired product / (moles of initial reactant - moles of reactant remaining after reaction) Yield = Conversion rate x Selectivity

[0057] Thus, a "selective process for preparing sulfoxides" is understood to be in particular a process which consumes sulfides to produce sulfoxides without the formation of sulfones (or with negligible amounts of sulfones formed). According to one embodiment, the oxidation reaction of sulfides to sulfoxides is chemoselective.

[0058] By "selective process for the preparation of sulfones" is understood in particular a process which consumes sulfides to produce sulfones without the formation of sulfoxides (or with negligible amounts of sulfoxides formed). According to one embodiment, the oxidation reaction of sulfides to sulfones is chemoselective.

[0059] For example, the process according to the invention, more particularly step (b), results in a selectivity of 95% to 100%, preferably 99% to 100%, for the sulfoxide or sulfone.

[0060] The formation of a disulfide bridge between two equivalents of an organic compound having at least one thiol group to form a dimer is considered in particular to correspond to the formation of a disulfide bridge between two molecules of said organic compound (i.e. 2R-SH becomes RSSR).

[0061] Process for co-producing disulfides and sulfoxides or sulfones The present invention more specifically relates to a process for co-producing a disulfide and a sulfoxide or a sulfone, the process comprising the steps of: (a) (1) Formula R 1 -SR 2 Sulfides of (2) optionally an oxidizing agent; (3) an organic compound having at least one thiol group; (4) an enzyme E that catalyzes the acidification of the sulfide to a sulfoxide or sulfone; (5) an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) A cofactor common to the above two enzymes E and D, providing a composition M comprising: (b) carrying out the (preferably simultaneous) enzymatic reactions of oxidation of sulfides and formation of disulfide bridges, ·Formula R 1 -S(O) n -R 2 where n=1 or 2, and ·Dimer to obtain; (c) reacting the dimer obtained in step (b) with a compound of formula R 3 By reaction of -SH with mercaptan, ·Formula R3 -SSR 3 and The above organic compound having at least one thiol group to obtain; (d) optionally, the disulfide obtained in step (c), and / or The sulfoxide or sulfone obtained in step (b) to recover; wherein said mercaptan can be added in any one of steps (a), (b) or (c), preferably said mercaptan can be added in step (a); R 1 , R 2 and R 3 is defined as follows:

[0062] According to a preferred embodiment, The sulfide is dimethyl sulfide, The organic compound having at least one thiol group is glutathione, Enzyme E is a Baeyer-Villiger monooxygenase (BVMO), preferably a cyclohexanone monooxygenase (CHMO); Enzyme D is glutathione reductase, The common cofactor for the two enzymes E and D is NADP.

[0063] <Production of sulfoxide or sulfone> The process according to the invention in particular comprises a step of carrying out an enzymatic oxidation reaction of sulfides to sulfoxides or sulfones, said reaction being preferably selective.

[0064] In a first embodiment, if the sulfide is not completely consumed during the step of carrying out the enzymatic sulfide oxidation reaction, sulfoxide is obtained selectively. Thus, according to one embodiment, said composition M still contains a sufficient amount of sulfide for the enzyme E to convert it to sulfoxide, and preferably no sulfone is formed. According to one embodiment, said sulfide is provided in excess in said composition M.

[0065] In this case, the amount of sulfide remaining after step (b) of carrying out the enzymatic reaction E may be, in weight ratio relative to the starting amount of sulfide (i.e., the amount in step (a)), 0.0001% by weight to 99.9% by weight, preferably 0.1% by weight to 99% by weight, preferably 1% by weight to 50% by weight, for example 1% by weight to 10% by weight.

[0066] In a second embodiment, if the sulfide is completely consumed during the step of carrying out the enzymatic sulfide oxidation reaction, the sulfone is selectively obtained. Thus, the step of carrying out the enzymatic oxidation reaction may in particular comprise the following two steps: (b1) completing the oxidation of sulfides to sulfoxides; (b2) Oxidation of sulfoxides to sulfones.

[0067] "Complete oxidation of sulfide" means that sulfide is completely consumed during step (b1). According to one embodiment, sulfide is the limiting reactant in composition M (i.e. the reactant present in deficit). "Completely consumed" means in particular that the amount of sulfide remaining after step (b) of carrying out the enzymatic reaction may be 0% to 20% by weight, preferably 0% to 5% by weight, for example 0% to 1% by weight, more preferably even 0% to 0.01% by weight, relative to the starting amount of sulfide (i.e. the amount of step (a)).

[0068] This reaction is described in French Patent Application No. FR1906488 for the production of sulfoxides and in French Patent Application No. FR1906489 for the production of sulfones.

[0069] <Co-production of disulfide with sulfoxide or sulfone> Preferably, steps (b) and (c), or steps (a), (b) and (c), are carried out in one and the same reactor, and preferably steps (b) and (c) proceed simultaneously.

[0070] In step (a), the various components of composition M may be added in any order, for example by simple mixing of the various components. Composition M may be prepared before being introduced into the reactor or directly in the reactor (wherein step (b) and, optionally, step (c) proceed).

[0071] In step (b), the enzymatic sulfide oxidation reaction may be carried out before the enzymatic disulfide bridge formation reaction or may be carried out simultaneously with the enzymatic disulfide bridge formation reaction.

[0072] More specifically, in step (b) the dimer of the organic compound obtained is in oxidized form, whereas in step (c) the organic compound is in reduced form.

[0073] Step (B) can in particular be divided into a number of steps as follows: (i) carrying out an enzymatic sulfide oxidation reaction with enzyme E; Sulfoxides or sulfones, and A cofactor common to the oxidized enzymes E and D to obtain; (ii) carrying out an enzymatic disulfide bridge formation reaction with enzyme D; A cofactor common to the reduced forms of enzymes E and D, and Dimers of the above organic compounds To obtain.

[0074] Step (i) may be carried out prior to or simultaneously with step (ii).

[0075] The enzymatic reaction in step (b) may be carried out at a pH of 4 to 10, preferably 6 to 8, more preferably 7 to 8, for example 7.

[0076] The enzymatic reaction in step (b) may be carried out at a temperature of 5°C to 100°C, preferably 20°C to 80°C, and more preferably 25°C to 40°C.

[0077] The cells as defined below can be used directly in step (b) without any treatment.

[0078] Step (c) may be carried out under the same conditions as step (b), more particularly at the same pH and at the same temperature.

[0079] The pressure used in the enzymatic reaction and / or in step (c) may range from reduced pressure to several bars (several hundred kPa) relative to atmospheric pressure, depending on the reactants and the equipment used.

[0080] According to one embodiment, the process according to the invention comprises, between steps (b) and (c), a step (b') of stopping the enzymatic reaction by inactivation of the enzyme and / or the enzymes E and / or D. This step (b') can be carried out by known means such as heat shock (for example at a temperature of about 100° C.) or osmotic shock, application of high pressure, addition of a solvent allowing the disruption and / or precipitation of the cells and / or the enzymes E and / or D, or a pH change (either a low pH of about 2, or a high pH of about 10).

[0081] The sulfide and / or mercaptan and / or optionally the oxidizing agent may be added continuously, preferably in step (a).

[0082] In step (d), the sulfoxides, or sulfones and / or disulfides may be recovered in liquid or solid form. Depending on their solubility, the sulfoxides, or sulfones and / or disulfides may be recovered in aqueous solution, in liquid form by decantation, or in solid form by precipitation. The disulfides may be extracted into the organic phase or separated from the reaction mixture by techniques well known to those skilled in the art (e.g. ultrafiltration followed by distillation or centrifugation).

[0083] The product obtained may then be optionally purified by conventional methods. For example, after separation of the cells (containing enzymes E and D) by ultrafiltration or centrifugation, distillation may allow the separation of the sulfoxide or sulfone and the disulfide. This distillation may be carried out at atmospheric pressure, at reduced pressure (e.g. under vacuum), or at elevated pressure, if the skilled person judges it to have any advantage. Membrane separation may also be envisaged with the aim of reducing the water content of the mixture for distillation or with the aim of accelerating the crystallization process. If the sulfoxide or sulfone and / or sulfide are recovered by decantation from the aqueous reaction mixture, drying over molecular sieves (or any other drying method) may be carried out.

[0084] The process may be carried out batchwise or continuously. There are many advantages offered by the process of the present invention. These advantages include the ability to operate in aqueous solutions, under very mild temperature and pressure conditions, and under near-neutral pH conditions. All of these conditions are typical of biocatalytic processes that are termed "green" or "sustainable".

[0085] Composition M may contain an organic compound, an enzyme E, an enzyme D, and a cofactor in catalytic amounts. Composition M may include: (1) Stoichiometric amounts of sulfide; (2) a stoichiometric amount of an oxidizing agent; (3) a catalytic amount of an organic compound having at least one thiol group; (4) a catalytic amount of an enzyme E that catalyzes the oxidation of the sulfide to a sulfoxide or to a sulfone; (5) a catalytic amount of an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) a catalytic amount of a cofactor common to both enzymes E and D; and (7) Stoichiometric amounts of mercaptans.

[0086] More specifically, the molar ratio of mercaptan / sulfide is from 0.1 to 100, more preferably from 1 to 5, preferably from 2 to 4, for example 2.

[0087] <Sulfide> The sulfides are in particular organic sulfides, which are any organic compounds containing at least one -CSC- functional group.

[0088] According to one embodiment, composition M comprises at least one sulfide. It may, for example, comprise one, two or several different sulfides.

[0089] The sulfides may be symmetrical, meaning that the sulfur atom represents the center of symmetry for the compound.

[0090] According to one embodiment, the sulfide has the general formula: R 1 -SR 2 Here, R 1 and R 2 may be the same or different, and each independently represents (C 1 -C 20 ) alkyl, (C 2 -C 20 ) alkenyl, (C 2 -C 20 ) alkynyl, (C 3 -C 10 ) cycloalkyl, and (C 6 -C 10 ) aryl; or R1 and R 2 together with the sulfur atom to which they are attached form a ring, preferably (C 3 -C 10 ) Heterocycloalkane or (C 4 -C 10 ) forming a heteroarene group, The above alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkane, and heteroarene groups may be optionally substituted with one or more substituents, The above alkyl, alkenyl, alkynyl, cycloalkyl, and aryl groups may contain one or more heteroatoms.

[0091] The above alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkane, and heteroarene groups may be optionally substituted with one or more substituents selected from the group consisting of: (C 1 -C 20 ) alkyl, (C 3 -C 10 ) cycloalkyl, and (C 6 -C 10 )aryl; By way of example and not limitation, it may be optionally functionalized with one or more functional groups selected from alcohol, aldehyde, ketone, acid, amide, nitrile, and ester functional groups, or other functional groups containing sulfur, phosphorus, and silicon.

[0092] According to one embodiment, the above alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkane, and heteroarene groups may be optionally substituted with one or more substituents selected from the group consisting of: 1 -C 20 ) alkyl, (C 3 -C 10 ) cycloalkyl, (C6 -C 10 )aryl, -OH, -C(O)OH, -C(O)H, -C(O)-NH 2 , -NH 2 , -NHR, -NRR', -C(O)-, -C(O)-NHR', -C(O)-NRR', -COOR, and -CN; Here, R and R′ are each independently represented by the formula (C 1 -C 20 ) represents an alkyl group.

[0093] According to a preferred embodiment, R 1 and R 2 may be the same or different, and may be independently 1 -C 20 ) alkyl, (C 2 -C 20 ) alkenyl, (C 2 -C 20 ) alkynyl, and (C 3 -C 10 ) cycloalkyl; or R 1 and R 2 together with the sulfur atom to which they are attached (C 3 -C 10 ) forming a heterocycloalkane group.

[0094] R 1 and R 2 is (C 1 -C 20 ) alkyl, or R 1 and R 2 with their associated sulfur atoms (C 3 -C 10 ) heterocycloalkane. The radical R of the sulfide is preferably 1 and R 2 are preferably the same (ie, thus forming a symmetric sulfide).

[0095] More preferably, the sulfides are selected from dimethyl sulfide, diethyl sulfide, dipropyl sulfide, dibutyl sulfide, dioctyl sulfide, didodecyl sulfide, and tetrahydrothiophene. The sulfides may be selected from dimethyl sulfide, diethyl sulfide, di-n-propyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diisobutyl sulfide, di-sec-butyl sulfide, di-tert-butyl sulfide, di-n-octyl sulfide, di-n-dodecyl sulfide, and tetrahydrothiophene. According to the invention, dimethyl sulfide is particularly preferred. According to one embodiment, the sulfides are symmetrical.

[0096] <Mercaptan> By mercaptans is meant in particular organic mercaptans, ie organic compounds which have at least one functional group of the type --C--SH.

[0097] According to one embodiment, the mercaptan has the general formula R 3 -SH, where R 3 is a hydrocarbon radical which is optionally substituted, saturated, straight chain, branched or cyclic.

[0098] More specifically, mercaptans have the general formula R 3 -SH, where R 3 is a hydrocarbon radical which may be saturated, linear, branched or cyclic, and which may be any of the following: -OH, -C(O)OH, -NH 2 , -OR a , -C(O)OR a , -N(R a )H, -NR a R b , and (C 6 -C 10 ) aryl, where R a and R b are each independently 1 -C 20 ) alkyl.

[0099] The mercaptan preferably has the general formula R 3 -SH, where R 3 is a hydrocarbon radical which may be saturated, linear, branched or cyclic, and which may be any of the following: -OH, -C(O)OH, -NH 2 and (C6-C10)aryl (more preferably, -OH, -C(O)OH, and -NH 2 Optionally substituted with at least one (e.g., one or two) groups selected from the group consisting of:

[0100] R 3 is more particularly selected from the group consisting of methyl, ethyl, octyl and dodecyl, preferably R 3 is methyl.

[0101] The mercaptan may be selected from the group consisting of mercaptoethanol, methyl mercaptan, ethyl mercaptan, propyl mercaptan, butyl mercaptan, octyl mercaptan, dodecyl mercaptan, benzyl mercaptan, thioglycolic acid, 3-mercaptopropionic acid, cysteine, and homocysteine.

[0102] The mercaptan is in particular selected from the group consisting of mercaptoethanol, methyl mercaptan, ethyl mercaptan, n-propyl mercaptan, isopropyl mercaptan (or 2-propanethiol), n-butyl mercaptan, sec-butyl mercaptan, tert-butyl mercaptan, n-octyl mercaptan, tert-octyl mercaptan, n-dodecyl mercaptan, tert-dodecyl mercaptan, benzyl mercaptan, thioglycolic acid, 3-mercaptopropionic acid, cysteine, and homocysteine. Preferably, the mercaptan is methyl mercaptan.

[0103] <Oxidizing agent> By oxidizing agent is understood a compound capable of oxidizing sulfides to sulfoxides or sulfones.

[0104] The oxidant may be selected from the group consisting of air, oxygen-depleted air, oxygen-enriched air, and pure oxygen.

[0105] When air (which may be oxygen-deficient or oxygen-rich) is used, it is of course the oxygen in the air that is consumed during the enzymatic oxidation reaction carried out in step (b) as the oxidizing agent.

[0106] If the oxidizing agent is in gaseous form, it is present in the form of a dissolved gas in composition M. The proportion of oxygen in the oxygen-enriched or oxygen-depleted air is selected in a manner known to those skilled in the art depending on the reaction rate and compatibility with the enzyme system.

[0107] The oxidizing agent may be present in composition M in a stoichiometric amount or in excess. If in excess, the sulfide present is completely consumed together with the oxidizing agent (to form a sulfone) in the enzymatic sulfide oxidation reaction carried out in step (b).

[0108] The oxidizing agent may be present in composition M in a stoichiometric amount. In this case, the sulfides present are partially but not completely consumed (formation of sulfoxides) together with the oxidizing agent in the enzymatic sulfide oxidation reaction carried out in step (b).

[0109] At the end of the oxidation reaction, oxygen is generally converted into water if the enzyme E used is a monooxygenase, whereas oxygen is completely consumed if the enzyme E is a dioxygenase. The process according to the invention is therefore particularly advantageous in terms of emissions and environmental considerations.

[0110] <Enzyme E> "Oxidizing enzyme" is understood to mean in particular enzyme E, i.e. an enzyme which allows the oxidation of sulfides to sulfoxides and / or sulfones and which requires the use of a cofactor.

[0111] The enzyme E may be an oxidoreductase, preferably an oxidoreductase selected from the group consisting of monooxygenases and dioxygenases, more preferably an oxidoreductase selected from monooxygenases.

[0112] The enzyme E is preferably Baeyer-Villiger monooxygenase (BVMO).

[0113] More preferably, among the BVMOs, enzyme E may be cyclohexanone monooxygenase (CHMO), more particularly cyclohexanone 1,2-monooxygenase, cyclopentanone monooxygenase (CPMO), more particularly cyclopentanone 1,2-monooxygenase, or hydroxyacetophenone monooxygenase (HAPMO), more particularly 4-hydroxyacetophenone monooxygenase.

[0114] Cyclohexanone 1,2-monooxygenases in particular belong to the class EC 1.14.13.22.

[0115] According to certain embodiments, the CHMO is a CHMO from Acinetobacter sp. (eg, strain NCIMB 9871) and / or a CHMO encoded by the gene chnB belonging to cluster AB006902.

[0116] Cyclopentanone 1,2-monooxygenases in particular belong to the class EC 1.14.13.16.

[0117] In certain embodiments, the CPMO is a CPMO from Comamonas sp. (eg, strain NCIMB 9872) and / or a CPMO encoded by the gene cpnB.

[0118] Hydroxyacetophenone monooxygenases are in particular those belonging to the class EC 1.14.13.84. According to one particular embodiment, the HAPMO is a HAPMO from Pseudomonas fluorescens, encoded by the gene hapE.

[0119] <Enzyme D and organic compounds having a thiol group> An organic compound having a thiol group is understood to be a hydrocarbon compound with or without heteroatoms and / or any type of known chemical functional group (hereinafter organic compound) having at least one -SH group. The organic compound according to the invention may contain one or two thiol groups (-SH groups). The compound may exist as a dimer, which is formed by a disulfide bridge between two equivalents of said organic compound (2R-SH being RSSR).

[0120] The organic compound may be selected from the group consisting of an amino acid having a thiol group, a peptide having a thiol group, mycothiol (CAS number 192126-76-4), and dihydrolipoic acid (CAS number 462-20-4). The organic compound is preferably an amino acid having a thiol group or a peptide having a thiol group. More specifically, the organic compound is selected from the group consisting of cysteine, homocysteine, glutathione, thioredoxin, mycothiol, and dihydrolipoic acid.

[0121] According to one embodiment, when the mercaptan according to the invention is cysteine ​​or homocysteine, the organic compound is different from cysteine ​​or homocysteine ​​(respectively). According to one embodiment, the organic compound and the mercaptan are different.

[0122] More specifically, the organic compound is selected from the group consisting of cysteine, homocysteine, glutathione, and thioredoxin.

[0123] It is particularly preferred that said organic compound is glutathione. The glutathione (GSH) / glutathione disulfide (GSSG) couple is widely known in biology. In the reduced form (glutathione) or in the oxidized form (glutathione disulfide), this entity forms an important redox couple within the cell.

[0124] Enzyme D catalyzes the formation of disulfide bridges between two equivalents of the above organic compounds to form dimers (herein referred to as dimers or dimers). In particular, it catalyzes the formation of disulfide bridges between amino acids having two equivalents of thiol groups or peptides having two equivalents of thiol groups to form diamino acids or dipeptides. Enzyme D can be defined as a "recycling enzyme" of reduced or oxidized common cofactors, preferably recycling the oxidized common cofactor to the reduced common cofactor.

[0125] The enzyme D may be a reductase or a dehydrogenase, and is preferably selected from the group consisting of glutathione reductase, thioredoxin reductase, cysteine ​​reductase, homocysteine ​​reductase, mycothiol disulfide reductase, and dihydrolipoyl dehydrogenase. The enzyme D is more particularly selected from the group consisting of glutathione reductase, thioredoxin reductase, cysteine ​​reductase, and homocysteine ​​dehydrogenase.

[0126] Glutathione reductase may be designated by the enzyme classification number EC 1.8.1.7 or EC 1.6.4.2, cysteine ​​reductase by EC 1.8.1.6, thioredoxin reductase by EC 1.8.1.9 or EC 1.6.4.5, and mycothione reductase by EC 1.8.1.15 (this enzyme is also called mycothione disulfide reductase). Particularly preferably, enzyme D is glutathione reductase.

[0127] More specifically, each organic compound is capable of forming a corresponding dimer by binding with a corresponding enzyme D. Thus, the following organic compound / enzyme D enzyme pairs or complexes are particularly useful for carrying out the process according to the invention: Glutathione / glutathione reductase, Thioredoxin / thioredoxin reductase, Cysteine / cysteine ​​reductase, homocysteine / homocysteine ​​reductase, Mycothiol / mycothiol disulfide reductase, and ·Dihydrolipoic acid / dihydrolipoyl dehydrogenase.

[0128] <Common cofactor(s)> By "common cofactor" is understood in particular a cofactor which is necessary for the catalytic activity of the enzymes E and D defined above and / or which makes it possible to enhance their catalytic activity. By "common cofactor" of the enzymes E and D is preferably understood a cofactor which can be reduced and / or oxidized by the action of these enzymes.

[0129] According to one embodiment, one or more cofactors are present in composition M. For example, it is possible to incorporate into composition M a cofactor that is already naturally present in enzyme E and / or enzyme D in addition to another cofactor.

[0130] The cofactor may be selected from nicotinic cofactor and flavin cofactor. More particularly, the cofactor may be nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and / or their corresponding reduced forms (i.e., NADH,H+, NADPH,H+, FMNH 2 , F.A.D.H. 2 ).

[0131] The above cofactors are advantageously used in their reduced form (e.g. NADPH,H+) and / or in their oxidized form (e.g. NADP+), i.e. they may be added to composition M in reduced and / or oxidized form, preferably in reduced form.

[0132] Preferably, the enzyme E used is a cyclohexanone monooxygenase (e.g. from Acinetobacter sp.), the cofactor used is NADP (optionally supplemented by FAD) and the enzyme D is glutathione reductase.

[0133] Composition M according to the invention may also comprise: optionally, one or more solvents selected from water, phosphate buffer, Tris-HCl, Tris base, buffers such as ammonium bicarbonate, ammonium acetate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), CHES (N-cyclohexyl-2-aminoethasulfonic acid), salts such as sodium chloride, potassium chloride, or mixtures thereof; · Optionally, additives such as surfactants, especially to facilitate solubility of one or more reactants or substrates of the enzymatic reaction.

[0134] Composition M is preferably an aqueous solution. For example, composition M contains 50% by weight to 99% by weight of water, preferably 80% by weight to 97% by weight or less of water, based on the total weight of composition M.

[0135] According to one embodiment, composition M may be said to comprise a reaction mixture.

[0136] The various components of composition M prepared in step (a) above are readily commercially available or may be prepared by techniques well known to those skilled in the art. These different elements may be in solid, liquid or gaseous form and may, very advantageously, be solubilized or dissolved in water or any other solvent for use in the process of the invention. The enzymes used may also be grafted onto a support (in the case of supported enzymes).

[0137] According to one embodiment, the enzymes E and / or D, the optional organic compound, and the optional common cofactor are as follows: in isolated and / or purified form (e.g. in aqueous solution); or in a crude extract (i.e. an extract of disrupted cells); or ·In whole cells.

[0138] Whole cells are preferentially used. [Sulfide] (unit: mmol / L) / [cell] (unit: g cdw. L -1 ) ratio is preferably 0.01 to 10 mmol / g in the step (b) of carrying out the enzyme reaction. cdw , preferably 0.01 to 3 mmol / g cdw The mass concentration of dry cells in grams (gCDW for cell dry weight) is determined by conventional techniques.

[0139] The enzymes E and / or D may or may not be overexpressed in the above cells (hereinafter referred to as host cells).

[0140] The host cell may be any host suitable for producing the enzymes E and / or D from the expression of the corresponding coding genes. This gene may then either be located in the genome of the host or be carried by an expression vector such as those defined below.

[0141] For the purposes of the present invention, a "host cell" is understood to be, in particular, a prokaryotic or eukaryotic cell. Host cells commonly used for the expression of recombinant or non-recombinant proteins are, in particular, bacterial cells such as Escherichia coli or Bacillus sp. or Pseudomonas, yeast cells such as Saccharomyces cerevis or Pichia pastoris, fungal cells such as Aspergillus niger, Penicillium funiculosum or Trichoderma reesei, insect cells such as Sf9 cells or other mammalian (in particular human) cells, such as, for example, HEK 293, PER-C6 or CHO cell lines.

[0142] The host cells may, for example, be in stationary growth phase and removed from culture medium.

[0143] Preferably, the enzymes E and / or D, the optional organic compound, and the optional common cofactor are expressed in the bacterium E. coli. The CHMO and / or HAPMO are preferably expressed in a strain of E. coli, such as, for example, E. coli BL21(DE3).

[0144] <Incorporation of an expression vector containing the coding sequence of enzymes E and / or D into a host cell> When using expression vectors such as plasmids, transformation of prokaryotic and eukaryotic cells is a technique well known to those skilled in the art, for example by lipofection, electroporation, heat shock, or chemical methods. The expression vector and the method of introducing the expression vector into the host cell are selected depending on the host cell selected. This transformation step results in transformed cells expressing genes encoding recombinant enzymes E and / or D. The cells can be cultured in a culture / incubation step to produce enzymes E and / or D.

[0145] Incubation / cultivation of prokaryotic and eukaryotic cells is a technique well known to those skilled in the art, and can determine, for example, the conditions of culture medium or temperature and time. Depending on the vector used, an induction period (corresponding to an increase in the production of enzymes E and / or D) can be observed. The use of a weak (e.g., arabinose in the case of vector pBad) or strong (e.g., isopropyl β-D-1-thiogalactoside (IPTG) in the case of vectors pET22b, pRSF, etc.) inducer can be considered. The production of enzymes E and / or D by the host cells can be confirmed using techniques such as SDS-PAGE electrophoresis or Western blotting.

[0146] An expression vector is a DNA molecule whose size has been reduced so that a desired base sequence can be inserted. There are many known expression vectors to choose from, such as plasmids, cosmids, and phages.

[0147] The vector is selected particularly as a function of the cellular host to be used. The expression vector may be, for example, one described in International Publication No. WO83 / 004261.

[0148] <Incorporation of the coding sequence for enzymes E and / or D into the genome of a host cell in the absence of an expression vector> The nucleotide sequence encoding the enzymes E and / or D can be integrated into the genome of the host cell by any known method, for example, by homologous recombination or a system such as CRISPR-Cas9. The production of the enzymes E and / or D by the host cell can be confirmed using a method such as SDS-PAGE electrophoresis or Western blotting.

[0149] Isolation and / or purification of enzymes E and / or D for use in isolated and / or purified form Transformation and culture / incubation of the transformed host cells may be followed by a step of isolation and optionally purification of the enzymes E and / or D. Thus, the process according to the invention is carried out with the enzymes E and / or D in solution (preferably in aqueous solution) in composition M, and not in the presence of host cells.

[0150] The isolation and / or purification of said produced enzymes E and / or D can be carried out by any means known to the person skilled in the art, for example by techniques selected from electrophoresis, molecular sieving, ultracentrifugation, differential precipitation, for example with ammonium sulfate, ultrafiltration, membrane or gel filtration, ion exchange, separation by hydrophobic interaction or affinity chromatography, such as IMAC.

[0151] <Host cell lysis pattern and preparation of crude extract from disrupted cells> Cell lysates can be obtained by a variety of known techniques, such as sonication, pressure (French press), through the use of chemical agents (e.g. Triton), etc. The resulting lysate represents a crude extract of the disrupted cells.

[0152] Composition and Use The present invention also relates to a composition comprising: (1) Sulfide, (2) optionally an oxidizing agent; (3) an organic compound having at least one thiol group; (4) an enzyme E that catalyzes the oxidation of the sulfide to a sulfoxide or to a sulfone; (5) an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) a cofactor common to the two enzymes E and D, and (7) Mercaptans.

[0153] The present invention also relates to the use of mercaptans for regenerating or recycling a cofactor (enzymatic cofactor, in particular as defined above), preferably a cofactor used in the enzymatic oxidation of sulfides. By "regeneration" or "recycle" is understood in particular the passage of the cofactor from an oxidized form to a reduced form or vice versa.

[0154] The present invention also relates to the use of a mercaptan for reducing disulfide bridges formed between two equivalents of an organic compound having at least one thiol group, said mercaptan being preferably converted to the corresponding disulfide. Preferably, the mercaptan is methyl mercaptan and the organic compound is glutathione. This use of the mercaptan is envisaged as a route for regenerating or recycling cofactors, in particular those used in the enzymatic oxidation of sulfides.

[0155] The present invention also relates to a process for the enzymatic oxidation of sulfide, which includes a step of regenerating or recycling the cofactor with a mercaptan.

[0156] The present invention also relates to a process using a mercaptan for reducing a disulfide bridge formed between two equivalents of an organic compound having at least one thiol group, said mercaptan being preferably converted to the corresponding disulfide.

[0157] More specifically, the sulfide, the oxidant, the organic compound, the enzyme E, the enzyme D, the cofactor, the mercaptan, and the enzymatic and chemical reactions are as defined above, particularly in the context of the co-production process according to the present invention.

[0158] <Drawing Description> Figure 1 shows the concentrations (in mM) of diethyl sulfide (DES), diethyl sulfoxide (DESO) and bis(2-hydroxyethyl) disulfide (disulfide) present in the reaction as a function of time (expressed in hours) when the oxidation reaction was catalyzed by the enzyme CHMO. An element of the cascade allowing the recycling of the cofactor NADP was also added: cells overexpressing glutathione reductase (GR) from Escherichia coli were added at the same concentration as CHMO-expressing cells, oxidized glutathione (GSSG) was introduced in catalytic amounts, and 2-mercaptoethanol was introduced at twice the concentration relative to DES. Figure 2 shows the amount of diethyl sulfoxide (DESO) and diethyl sulfone (DESO) present in the reaction solution when the oxidation reaction was catalyzed by the enzyme CHMO. 2 ), and bis(2-hydroxyethyl)disulfide (disulfide) concentrations (in mM) as a function of time (expressed in hours). Elements of the cascade allowing recycling of the cofactor NADP were also added: cells overexpressing glutathione reductase (GR) from E. coli were added at the same concentration as in CHMO-expressing cells, oxidized glutathione (GSSG) was introduced in catalytic amounts, and 2-mercaptoethanol was introduced at twice the concentration relative to DESO. Figure 3 shows the concentration (mM) of diethyl sulfoxide (DESO) present in the reaction as a function of time (expressed in hours) when the reaction is catalyzed by the enzyme CHMO. In this example, 2-mercaptoethanol was replaced by methyl mercaptan (MeSH) as the proton donor. An element of the cascade allowing the recycling of the cofactor NADP was also added: cells overexpressing glutathione reductase (GR) from E. coli were added at the same concentration as the cells expressing CHMO, oxidized glutathione (GSSG) was introduced in catalytic amounts, and MeSH was gradually added to the reaction at a flow rate of about 4 mmol / L / h. FIG. 4 is a diagram illustrating the enzyme cascade according to the present invention.

[0159] The mercaptan and sulfide are introduced into a reaction solution containing: CHMO (corresponding to enzyme E), NADPH (a common cofactor), Glutathione reductase (corresponding to enzyme D), · Glutathione disulfide or glutathione (corresponding to a dimer or organic compound containing at least one thiol group).

[0160] In particular, by continuously introducing air it is possible to oxidize the sulfides to sulfoxides or sulfones, depending on how the sulfides are introduced, thus forming an enzyme cascade.

[0161] The common cofactor, NADPH, is oxidized to NADP+ with the concomitant production of sulfoxide or sulfone. NADP+ is reduced back to NADPH,H+ in the presence of two glutathione molecules, and two glutathione molecules (2GSH) become glutathione disulfide (GSSG). To complete the cascade, glutathione disulfide can oxidize, through chemical equilibrium, two mercaptans to the corresponding disulfides, thereby regenerating two glutathione molecules.

[0162] The following examples are offered by way of illustration and not by way of limitation of the present invention.

[0163] <Example> Example 1: Selective synthesis of diethyl sulfide from diethyl sulfide using 2-mercaptoethanol (I) Preparation of biocatalyst: [Cyclohexanone monooxygenase (CHMO) production] An E. coli BL21(DE3) (Merck Millipore) strain expressing the chnB gene inserted into the plasmid pET22b (Promega, Qiagen) was previously constructed, which allows heterologous expression of cyclohexanone monooxygenase (CHMO) derived from Acinetobacter sp.

[0164] It is understood that the above strain contains both CHMO and its cofactors NADP and FAD.

[0165] This strain has been pre-cultured and cultivated by techniques well known to those skilled in the art.

[0166] After inducing an induction phase by adding isopropyl β-D-1-thiogalactoside (IPTG) at a final concentration of 0.85 mmol / L, an aliquot of the culture was centrifuged (10 min, 5000 g, 4° C.) to obtain the desired amount of cells.

[0167] [Glutathione reductase (GR) production] An E. coli BL21(DE3) (Merck Millipore) strain expressing the gor gene inserted in the plasmid pET26b+ (Promega, Qiagen) was designed according to techniques well known to those skilled in the art, allowing the heterologous expression of glutathione reductase (GR) from E. coli.

[0168] It is understood that the above strain contains both GR and NADP, a cofactor for GR.

[0169] This strain has been pre-cultured and cultivated using techniques known to those skilled in the art.

[0170] After inducing an induction phase by addition of isopropyl β-D-1-thiogalactoside (IPTG), an aliquot of the culture is centrifuged (10 min, 5000 g, 4° C.) to obtain the desired amount of cells.

[0171] (II) Bioconversion: In this example, fresh cell pellets were resuspended in 32 mL of 0.1 mol / L phosphate buffer, pH 8. This resulted in a cell concentration of 62 ODU / mL, or 20 g for cells overexpressing CHMO and GR. CDW / L (where CDW stands for cell dry weight).

[0172] In a 250 mL flask, initial concentrations of 3 mmol / L diethyl sulfide (DES), 6 mmol / L 2-mercaptoethanol, and 0.25 mmol / L oxidized glutathione (GSSG) are present at t=0 with a final volume of 32 mL.

[0173] At regular intervals, 200 μL of the reaction mixture was withdrawn and diluted with 1000 μL of acetonitrile solution. After centrifugation (5 min, 12500 g), the supernatant was injected into a GC, and the diethyl sulfoxide (DESO) and bis(2-hydroxyethyl) disulfide (disulfide) produced during the reaction were quantitatively measured using the previously obtained calibration standard range. Under the analytical conditions used, the minimum measurable concentration was 50 μM.

[0174] A linear increase in the amount of DESO was measured over time, but the sulfone (DESO 2 ) is not detectable. The initial oxidation rate of sulfides is 1 mmol of DES oxidized per liter of medium per hour (Figure 1). The same amount of bis(2-hydroxyethyl)disulfide is produced simultaneously with the formation of DESO, confirming that the GSSG / GR coupled system recycling the cofactor is functioning properly. Note that the concentration of bis(2-hydroxyethyl)disulfide as a function of time was determined after subtracting the background noise associated with the slow spontaneous oxidation of 2-mercaptoethanol (measured under the same conditions in the presence of all components of the cascade except for cells overexpressing CHMO).

[0175] DESO is DESO 2 The CHMO catalysis is chemoselective since the conversion of sulfone to sulfone occurs only when DES is completely consumed. Conversely, sulfone is not produced in the presence of DES in the reaction medium.

[0176] The selectivity obtained is about 100%. If DES is still present in the reaction mixture, the sulfone is not detected by the analyzer used. At the end of the reaction (t=4 h), about 100% DESO is obtained, and the analyzer used does not detect DESO. 2 is not detected.

[0177] Example 2: Selective synthesis of diethyl sulfone from diethyl sulfoxide using 2-mercaptoethanol as hydrogen donor (I) Preparation of biocatalyst: In this example, the same strains as described in Example 1 were used, namely, E. coli overexpressing CHMO and GR.

[0178] Centrifuge (10 min, 5000 g, 4°C) and suspend the pellet in 32 mL of 0.1 mol / L phosphate buffer (pH 8). CDW / L) and used in the reaction test.

[0179] (II) Biotransformation: To a 250 mL flask, 3 mmol / L diethylsulfoxide (DESO), 6 mmol / L 2-mercaptoethanol, and 0.25 mmol / L oxidized glutathione (GSSG) are added simultaneously at t=0 to a final volume of 32 mL.

[0180] At regular intervals, 200 μL of the reaction mixture was withdrawn and diluted with 1000 μL of acetonitrile solution. After centrifugation (5 min, 12500 g), the supernatant was injected into a GC to measure diethylsulfone (DESO 2 ) and bis(2-hydroxyethyl) disulfide (disulfide) are quantitatively measured using previously obtained calibration standard ranges.

[0181] DESO over time 2 A linear increase in the amount of DESO is then measured. The initial oxidation rate of sulfoxide is then 0.75 mmol of DESO per liter of medium per hour (Figure 2). In parallel, the DESO 2Simultaneously with the production of , an equal amount of bis(2-hydroxyethyl) disulfide is also produced. As a result, the added 2-mercaptoethanol actually makes it possible to recycle the cofactor NADP in a cascade-dependent pathway. Note that background noise was subtracted in the same manner as in Example 1.

[0182] Example 3: Selective synthesis of diethyl sulfide from diethyl sulfide using methyl mercaptan (MeSH) as a hydrogen donor (I) Preparation of biocatalyst: In this example, the same strains were used as described in Examples 1 and 2, i.e., strains overexpressing E. coli CHMO and GR. In this biocatalytic reaction, 2-mercaptoethanol was replaced by MeSH.

[0183] (II) Biotransformation: In this example, a fresh cell pellet is resuspended in 200 mL of 0.1 mol / L phosphate buffer at pH 8. The cell concentration thus obtained is 62 ODU / mL, or 20 g for cells overexpressing CHMO and GR. CDW / L (where CDW stands for cell dry weight).

[0184] In a 1 L reactor, 10 mmol / L diethyl sulfide (DES), 0.25 mmol / L oxidized glutathione (GSSG), and the enzyme solution prepared in advance are simultaneously charged at t=0. Furthermore, methyl mercaptan is gradually added to the reaction solution by sulfuric acid acidification of sodium methyl mercaptan (flow rate is adjusted to approximately 4 mmol / L / h). After that, pure diethyl sulfide is gradually added at a flow rate of 40 μL / h to keep the DES concentration constant, and oxygen required for the reaction is periodically added by a bladder. The reaction is started with stirring at a controlled temperature of 30 °C.

[0185] At regular intervals, 200 μL of the reaction mixture was withdrawn and diluted with 1000 μL of acetonitrile solution. After centrifugation (5 min, 12500 g), the supernatant was injected into a GC and the diethyl sulfoxide (DESO) and diethyl sulfone (DESO) potentially formed during the reaction were analyzed using a previously obtained calibration standard range. 2 ) is quantitatively measured. Under the analytical conditions used here, the minimum measurable concentration is 50 μM.

[0186] A linear increase in the amount of DESO was measured over time, but the sulfone (DESO 2 ) is not detected. The initial oxidation rate of sulfide is then 0.2 mmol of DES oxidized per liter of medium per hour (Figure 3). Concomitant with the production of DESO is the production of dimethyl disulfide (DMDS), confirming that the cofactor-recycling GSSG / GR coupled system is functioning properly.

[0187] In the reaction catalyzed by CHMO, DES is present in the reaction mixture throughout the reaction, so DESO is 2 No reaction occurs that converts it to, so it can be said to be a chemoselective reaction. The present invention includes the following aspects. <1> A process for co-producing a disulfide and a sulfoxide or sulfone, comprising the steps of: (a) (1) Sulfide, (2) optionally an oxidizing agent; (3) an organic compound having at least one thiol group; (4) an enzyme E that catalyzes the oxidation of the sulfide to a sulfoxide or to a sulfone; (5) an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) a cofactor common to the two enzymes E and D; providing a composition M comprising: (b) carrying out the (preferably simultaneous) enzymatic reactions of oxidation of sulfides and formation of disulfide bridges, Sulfoxides or sulfones, and ·Dimer to obtain; (c) reducing the dimer obtained in step (b) by reaction with a mercaptan, the corresponding disulfides, and The organic compound having at least one thiol group to obtain; (d) optionally, the disulfide obtained in step (c), and / or The sulfoxide or sulfone obtained in step (b) To recover Here, the mercaptan may be added in any one of steps (a), (b) and (c), and preferably the mercaptan is added in step (a). <2> The mercaptan has the general formula R 3 -SH, Here, R 3 is a hydrocarbon radical, which is optionally substituted, saturated, linear, branched or cyclic; <1> The co-generation process described in. <3> R 3 is selected from the group consisting of methyl, ethyl, octyl, and dodecyl, preferably R 3 is methyl, <2> The co-generation process described in. <4> the mercaptan is selected from the group consisting of mercaptoethanol, methyl mercaptan, ethyl mercaptan, n-propyl mercaptan, isopropyl mercaptan (or 2-propanethiol), n-butyl mercaptan, sec-butyl mercaptan, tert-butyl mercaptan, n-octyl mercaptan, tert-octyl mercaptan, n-dodecyl mercaptan, tert-dodecyl mercaptan, benzyl mercaptan, thioglycolic acid, 3-mercaptopropionic acid, cysteine, and homocysteine; <2> The co-generation process described in. <5> The organic compound is selected from the group consisting of an amino acid having a thiol group, a peptide having a thiol group, mycothiol, and dihydrolipoic acid, and preferably the organic compound is an amino acid having a thiol group or a peptide having a thiol group. <1> ~ <4> 13. The co-generation process according to any one of claims 1 to 12. <6> The organic compound is selected from the group consisting of cysteine, homocysteine, glutathione, thioredoxin, mycothiol, and dihydrolipoic acid, and is preferably glutathione; <1> ~ <5> 13. The co-generation process according to any one of claims 1 to 12. <7> The enzyme E is an oxidoreductase (preferably an oxidoreductase selected from the group consisting of monooxygenases and dioxygenases), more preferably a monooxygenase; <1> ~ <6> 13. The co-generation process according to any one of claims 1 to 12. <8> The enzyme D is a reductase or dehydrogenase (preferably selected from the group consisting of glutathione reductase, thioredoxin reductase, cysteine ​​reductase, homocysteine ​​reductase, mycothiol disulfide reductase, and dihydrolipoyl dihydrolipoyl), and preferably the enzyme D is glutathione reductase; <1> ~ <7> 13. The co-generation process according to any one of claims 1 to 12. <9> The sulfide has the general formula: R 1 -SR 2 Here, R 1 and R 2 may be the same or different, and each independently represents (C 1 -C 20 ) alkyl, (C 2 -C 20 ) alkenyl, (C 2 -C 20 ) alkynyl, (C3 -C 10 ) cycloalkyl, and (C 6 -C 10 ) aryl; or R 1 and R 2 together with the sulfur atom to which they are attached form a heterocycloalkane or heteroarene, The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkane, and heteroarene groups may be optionally substituted with one or more substituents; The alkyl, alkenyl, alkynyl, cycloalkyl, and aryl groups may contain one or more heteroatoms. <1> ~ <8> 13. The co-generation process according to any one of claims 1 to 12. <10> The sulfide radicals R1 and R2 are the same; <9> The co-generation process described in. <11> The sulfide is selected from the group consisting of dimethyl sulfide, diethyl sulfide, dipropyl sulfide, dibutyl sulfide, dioctyl sulfide, didodecyl sulfide, and tetrahydrothiophene, preferably dimethyl sulfide; <1> ~ <10> 13. The co-generation process according to any one of claims 1 to 12. <12> The oxidant is selected from the group consisting of air, oxygen-depleted air, oxygen-enriched air, and pure oxygen. <1> ~ <11> 13. The co-generation process according to any one of claims 1 to 12. <13> The cofactor is selected from a nicotine cofactor and a flavin cofactor; <1> ~ <12> 13. The co-generation process according to any one of claims 1 to 12. <14> The sulfide is dimethyl sulfide, the organic compound having at least one thiol group is glutathione; Enzyme E is a Baeyer-Villiger monooxygenase (BVMO), preferably a cyclohexanone monooxygenase (CHMO); Enzyme D is glutathione reductase, The cofactor common to the two enzymes E and D is NADP. <1> ~ <13> 13. The co-generation process according to any one of claims 1 to 12. <15> (1) Sulfide, (2) optionally an oxidizing agent; (3) an organic compound having at least one thiol group; (4) an enzyme E that catalyzes the oxidation of the sulfide to a sulfoxide or to a sulfone; (5) an enzyme D that catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) a cofactor common to the two enzymes E and D; and (7) Mercaptan A composition comprising: <16> Use of a mercaptan to regenerate or reuse a cofactor used in the enzymatic oxidation of a sulfide to a sulfoxide or to a sulfone. <17> To reduce disulfide bridges formed between two equivalents of an organic compound having at least one thiol group, <16> The use of a mercaptan according to The mercaptan is preferably converted to the corresponding disulfide. <18> The mercaptan is methyl mercaptan and the organic compound is glutathione; <17> The use described in.

Claims

1. A process for co-producing a disulfide and a sulfoxide or sulfone, comprising the steps of: (a) (1) Sulfide, (2) an oxidizing agent, (3) an organic compound having at least one thiol group; (4) Enzyme E, an oxidoreductase, which catalyzes the oxidation of the sulfide to a sulfoxide or to a sulfone; (5) an enzyme D which is a reductase or dehydrogenase, which catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) a cofactor common to the two enzymes E and D; providing a composition M comprising: (b) carrying out an enzymatic reaction of oxidation of sulfides and formation of disulfide bridges, sulfoxides or sulfones, and ・Dimer to obtain; (c) reducing the dimer obtained in step (b) by reaction with a mercaptan, the disulfide corresponding to said mercaptan, and The organic compound having at least one thiol group to obtain; (d) optionally, the disulfide obtained in step (c), and / or The sulfoxide or sulfone obtained in step (b) To recover Here, the mercaptan may be added in any one of steps (a), (b), and (c).

2. The mercaptan has the general formula R ３ -SH, Here, R ３ 2. The co-production process of claim 1 , wherein is an optionally substituted, saturated, linear, branched, or cyclic hydrocarbon radical.

3. R ３ 3. The coproduction process of claim 2, wherein is selected from the group consisting of methyl, ethyl, octyl, and dodecyl.

4. 3. The coproduction process of claim 2, wherein the mercaptan is selected from the group consisting of mercaptoethanol, methyl mercaptan, ethyl mercaptan, n-propyl mercaptan, isopropyl mercaptan (or 2-propanethiol), n-butyl mercaptan, sec-butyl mercaptan, tert-butyl mercaptan, n-octyl mercaptan, tert-octyl mercaptan, n-dodecyl mercaptan, tert-dodecyl mercaptan, benzyl mercaptan, thioglycolic acid, 3-mercaptopropionic acid, cysteine, and homocysteine.

5. The co-production process according to any one of claims 1 to 4, wherein the organic compound is selected from the group consisting of an amino acid having a thiol group, a peptide having a thiol group, mycothiol, and dihydrolipoic acid.

6. The co-production process of any one of claims 1 to 5, wherein the organic compound is selected from the group consisting of cysteine, homocysteine, glutathione, thioredoxin, mycothiol, and dihydrolipoic acid.

7. 7. The co-production process of any one of claims 1 to 6, wherein the enzyme E is an oxidoreductase selected from the group consisting of monooxygenases and dioxygenases.

8. The coproduction process of any one of claims 1 to 7, wherein the enzyme D is a reductase or dehydrogenase selected from the group consisting of glutathione reductase, thioredoxin reductase, cysteine ​​reductase, homocysteine ​​reductase, mycothiol disulfide reductase, and dihydrolipoyl dehydrogenase.

9. The sulfide is of the general formula: R １ -S-R ２ Here, R １ and R ２ may be the same or different, and each independently represents １ -C ２０ ) alkyl, (C ２ -C ２０ ) alkenyl, (C ２ -C ２０ ) alkynyl, (C ３ -C １０ ) cycloalkyl, and (C ６ -C １０ ) aryl; or R １ and R ２ together with the sulfur atom to which they are attached form a heterocycloalkane or heteroarene, The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkane, and heteroarene groups may be optionally substituted with one or more substituents; The alkyl, alkenyl, alkynyl, cycloalkyl, and aryl groups may contain one or more heteroatoms. A co-production process according to any one of claims 1 to 8.

10. The radical R of the sulfide １ and R ２ 10. The co-production process of claim 9, wherein

11. 11. The co-production process of any one of claims 1 to 10, wherein the sulfide is selected from the group consisting of dimethyl sulfide, diethyl sulfide, dipropyl sulfide, dibutyl sulfide, dioctyl sulfide, didodecyl sulfide, and tetrahydrothiophene.

12. The co-production process of any one of claims 1 to 11, wherein the oxidant is selected from the group consisting of air, oxygen-depleted air, oxygen-enriched air, and pure oxygen.

13. 13. The co-production process of any one of claims 1 to 12, wherein the cofactor is selected from a nicotine cofactor and a flavin cofactor.

14. The sulfide is dimethyl sulfide, the organic compound having at least one thiol group is glutathione; Enzyme E is Baeyer-Villiger monooxygenase (BVMO), Enzyme D is glutathione reductase, The cofactor common to the two enzymes E and D is NADP. A co-production process according to any one of claims 1 to 13.

15. (1) Sulfide, (2) an oxidizing agent, (3) an organic compound having at least one thiol group; (4) Enzyme E, an oxidoreductase, which catalyzes the oxidation of the sulfide to a sulfoxide or to a sulfone; (5) an enzyme D which is a reductase or dehydrogenase, which catalyzes the formation of a disulfide bridge between two equivalents of the above organic compound having at least one thiol group to form a dimer; (6) a cofactor common to the two enzymes E and D; and (7) Mercaptan A composition comprising:

16. The use of a mercaptan to regenerate or reuse a cofactor used in the enzymatic oxidation of sulfides to sulfoxides or to sulfones in the coproduction process of claim 1.

17. 17. Use of the mercaptan according to claim 16 for reducing disulfide bridges formed between two equivalents of an organic compound having at least one thiol group.

18. 18. The use according to claim 17, wherein the mercaptan is methyl mercaptan and the organic compound is glutathione.

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