WO2020085436A1 - Système de production de trihydroxybenzène - Google Patents

Système de production de trihydroxybenzène Download PDF

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WO2020085436A1
WO2020085436A1 PCT/JP2019/041724 JP2019041724W WO2020085436A1 WO 2020085436 A1 WO2020085436 A1 WO 2020085436A1 JP 2019041724 W JP2019041724 W JP 2019041724W WO 2020085436 A1 WO2020085436 A1 WO 2020085436A1
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thb
doi
culture
heating
bacterial
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PCT/JP2019/041724
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English (en)
Japanese (ja)
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修 吉川
充幸 中島
千夏 皆川
光史 和田
達雄 宮崎
修久 井坂
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株式会社Ihi
三井化学株式会社
学校法人 新潟科学技術学園
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Publication of WO2020085436A1 publication Critical patent/WO2020085436A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/06—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by conversion of non-aromatic six-membered rings or of such rings formed in situ into aromatic six-membered rings, e.g. by dehydrogenation
    • C07C37/07—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by conversion of non-aromatic six-membered rings or of such rings formed in situ into aromatic six-membered rings, e.g. by dehydrogenation with simultaneous reduction of C=O group in that ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/72—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with no unsaturation outside the aromatic ring
    • C07C39/10—Polyhydroxy benzenes; Alkylated derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • the present disclosure relates to a system for producing trihydroxybenzene (THB).
  • DOI Deoxyscylinosose
  • a bioprocess using a bacterium which expresses a specific enzyme (WO 2006/109479 (Patent Document 1), and thus WO 2010/053052 ( From Patent Document 2), International Publication No. 2015/005451 (Patent Document 3)), from the viewpoint of escape from dependence on fossil resources and global environment conservation, it is a particularly attractive chemical substance.
  • a plant material mainly composed of cellulose, such as wood chips is decomposed into a monosaccharide such as glucose by a known technique, and the monosaccharide is converted into a DOI by the bioprocess described above. It is possible to realize chemical product manufacturing that effectively uses fossil resources.
  • THB trihydroxybenzene
  • THB is a compound produced by a dehydration reaction from DOI (a total of two molecules of water are eliminated from one molecule of DOI).
  • THB can be further derivatized into useful compounds such as quercitol, carbaglucose, catechol, adipic acid derivatives, hydroquinone, trimethoxybenzene, trimethoxybenzoic acid phenyl ester and sesamol.
  • THB-derived useful compounds can be used in applications such as antidiabetic agents, nylon, photographic agents, polymerization inhibitors, Li secondary battery additives, cellulose film additives, antidepressants, etc. (Biotechnology, No. 1). 93, 533-535, 2015 (Non-patent document 1)).
  • the present disclosure provides a system for manufacturing THB with high efficiency.
  • the present inventor has discovered that THB is produced at a high yield by directly heating a culture solution of a bacterium that synthesizes DOI.
  • the present disclosure is based on this finding.
  • a culture tank for holding a liquid medium containing bacteria that synthesize deoxyscylinosose (DOI) and culturing the bacteria to produce a bacterial culture solution;
  • a heating device connected to the culture tank to heat the bacterial culture at a high temperature of 80 ° C. or higher to generate a product solution containing trihydroxybenzene (THB); System for producing trihydroxybenzene (THB).
  • TLB trihydroxybenzene
  • the culture tank is connected to the heating device via a liquid supply pipe, and the heating device heats the bacterial culture solution sent from the culture tank inside the heating device, [1] The system described in.
  • the culture tank is one of a stirring device for stirring the medium, a shaking device for shaking the medium, an aeration device for aerating the medium, and a heating device for heating the medium.
  • the system according to any one of [1] to [7], which has the above.
  • the embodiments of the present disclosure significantly improve the efficiency of producing THB. That is, the number of steps of the process from the synthesis of DOI by the bacterium to the production of THB can be significantly simplified and a part or all of the process can be continuously performed.
  • FIG. 1 shows an outline of a THB manufacturing system according to one embodiment.
  • FIG. 2 shows an outline of a THB manufacturing system according to another embodiment.
  • FIG. 3 shows the appearance (a) and the DOI content (b) of the DOI fermentation liquor (stock solution) and the heated one.
  • FIG. 4 shows a 13 C-NMR spectrum of a THB product solution (bottom) obtained from a DOI fermentation broth and a THB sample (top) after solvent extraction.
  • the present disclosure includes the step of heating a bacterial culture containing deoxyscylinosose (DOI) at an elevated temperature of 80 ° C. or higher to obtain a product solution containing trihydroxybenzene (THB).
  • DOI deoxyscylinosose
  • TAB trihydroxybenzene
  • DOI deoxyscylinosose
  • TAB trihydroxybenzene
  • the THB produced in this embodiment contains 1,2,4-trihydroxybenzene and 1,2,3 -Trihydroxybenzene (pyrogallol) may also be included.
  • the product solution may also include catechol.
  • the concentration of DOI in the heated bacterial culture is preferably 1 g / L or more, more preferably 10 g / L or more, More preferably, it is 50 g / L or more.
  • the upper limit of the DOI concentration is not particularly limited, but is usually 100 g / L or less, for example, 90 g / L or less, or 80 g / L or less.
  • Bacterial culture is a conditioned liquid medium containing DOI obtained after culturing bacteria that synthesize DOI in liquid medium under DOI synthesis conditions.
  • Liquid media suitable for culturing bacteria that synthesize DOI are known to those of ordinary skill in the art or can be prepared by those of ordinary skill in the art, and typically 10 in stationary phase. It can support culturing at a cell density of 7 cells / mL or more, preferably 10 8 cells / mL or more.
  • a liquid medium for culturing bacteria usually contains a carbon source (in particular, sugar), a nitrogen source (such as amino acid), vitamins, inorganic salts and the like.
  • the liquid medium capable of supporting the culture at a relatively high cell density and DOI synthesis as described above.
  • sugars other than glucose can also be metabolized to glucose, it is preferable that the liquid medium contains glucose itself because it provides a substrate for DOI synthesis directly and is efficient.
  • YE medium which contains BD Bacto Tryptone (pancreatic digest of casein) 50 per 6.5 L of water. ⁇ 200g (104g in 2xYE medium), 60 ⁇ 260g BD Bacto Yeast Extract (water soluble extract of yeast) (130g in 2xYE medium), 15-60g NaCl (32.5g in 2xYE medium) ), Glucose 200 to 800 g (377 x 390 g in 2 x YE medium) and mannitol 150 to 650 g (335 g in 2 x YE medium), optionally containing antibiotics such as ampicillin, phytic acid, antifoaming agent, etc.
  • antibiotics such as ampicillin, phytic acid, antifoaming agent, etc.
  • Additives may be included in small amounts (eg, 5 g or less total).
  • Phytic acid is an additive component capable of improving DOI production by bacteria, and for example, 1 to 20 g of a 50% (w / w) aqueous phytic acid solution can be added per the above amount of YE medium.
  • Another specific example of the liquid medium (CSL medium) is 150 to 700 g (standard 325 g) of CSL (corn steep liquor), 1000 to 5000 g of glucose (standard 2010 g), and fructose of 1000 to 52.6 kg of water.
  • the product contains 2 to 10 g (4.55 g as standard).
  • the CSL medium may further include 40 to 200 g (91 g by standard) of 50% (w / w) phytic acid per 52.6 kg of water.
  • the CSL medium may include an antifoaming agent (eg 5 to 30 g of Adecanol antifoaming agent per the above amount of CSL medium).
  • an antifoaming agent eg 5 to 30 g of Adecanol antifoaming agent per the above amount of CSL medium.
  • part or all of the medium components may be supplemented at any time. For example, it is preferable to supplement the sugar component during the culture.
  • the heated bacterial culture may or may not contain bacterial cells. That is, in one embodiment, the heated bacterial culture is a liquid medium after culturing the bacteria, which contains the bacteria or after the bacteria have been removed.
  • solution means a liquid in which at least one substance different from the solvent is dissolved or suspended.
  • An insoluble substance or a solid substance may be suspended in the solution.
  • the insoluble substance and the solid substance as used herein may include biological cells.
  • the bacterial culture solution to be heated may contain bacterial cells of DOI-synthesizing bacteria.
  • the liquid medium containing the bacteria and the DOI that is, the bacterial culture solution
  • the method of the present embodiment can be carried out by heating.
  • Such an embodiment allows THB to be produced and used bacteria inactivated / sterilized at the same time, which not only greatly simplifies the overall procedure, but also in terms of biosafety and hygiene. Will also be advantageous.
  • the method further comprises the step of culturing the DOI-synthesizing bacteria in the liquid medium under DOI synthesis conditions, before the step of heating the bacterial culture.
  • the liquid medium after culturing provides the bacterial broth to be heated.
  • the bacterial broth to be heated may be one that has been cultured for preferably 12 hours or more, more preferably 16 hours or more, for example, 24 hours or more, or 32 hours or more.
  • a cell density of preferably 10 7 cells / mL or more, more preferably 10 8 cells / mL or more can be achieved within 24 hours from a cell density of 10 4 cells / mL or less.
  • a bacterium that synthesizes DOI preferably 1 g / L or more, more preferably 10 g / L or more, still more preferably 50 g / L or more DOI accumulates in the bacterial culture.
  • the culture temperature is usually 25 to 38 ° C, preferably 30 to 37 ° C. Culture conditions for synthesizing DOI in bacteria can be appropriately adjusted by those skilled in the art based on ordinary knowledge.
  • the bacterium that synthesizes DOI is known to those skilled in the art, or can be identified or obtained by those skilled in the art based on known information. Specific examples of such bacterial species include E. coli, Bacillus circulans, Bacillus amyloliquefaciens, Bacillus subtilis, Corynebacterium glutamicum, and Geobacillus stearothermophilus. Not limited to. E. coli is particularly preferred.
  • the bacterium preferably expresses 2-deoxyscylinosose synthase. Most preferably, the bacterium that synthesizes DOI is Escherichia coli that expresses 2-deoxyscylinosose synthase.
  • a preferred example of 2-deoxyscylinosose synthase is the product of the btrC gene derived from Bacillus circulans (Patent Document 1), but as long as the DOI synthetic activity is maintained, a homologous gene product derived from another bacterium. It will be understood by those skilled in the art that a modified natural sequence can be substituted. Such a gene may be endogenous, but is preferably exogenously introduced by a genetic engineering technique. Means for increasing and / or optimizing gene expression, such as plasmid vector selection, promoter selection, codon optimization, etc., are known to those of skill in the art.
  • the promoter may be a constitutively expressed promoter or an expression-inducible promoter.
  • genes may be modified, added, or deleted in the host bacterium into which 2-deoxyscylinosose synthase is introduced.
  • pgi, zwf, and pgm which are genes that metabolize glucose-6-phosphate as a substrate for DOI synthesis into another compound, and / or a protein in stationary phase
  • the yield of DOI can be increased by using, as a host, Escherichia coli in which rmf, which is a gene involved in synthesis suppression, is destroyed (Patent Document 1). In that case, it may be preferable to add a carbon source other than glucose, such as mannitol, to the medium.
  • the liquid medium (bacterial culture medium) after the bacteria have synthesized DOI contains 2-deoxyscylinosose (DOI) and a small amount (for example, a total of one or more related compounds selected from the following). , 20% or less, 15% or less, 10% or less, or 5% or less by weight of 2-deoxycylinosose (DOI)): 1-epi-DOI, ⁇ , ⁇ -unsaturated ketone, (+ ) Vibo-quercitol, and scyllo-quercitol.
  • DOI 2-deoxyscylinosose
  • the bacterial culture may be heated while the bacteria are still contained, but a step of removing bacterial cells from the bacterial culture may be added before heating. By this step, it is possible to avoid the possibility that a large amount of insoluble substance released from the heated microbial cell may reduce the purity of the product solution or inhibit the reaction.
  • the technique used in the step of removing bacterial cells from the bacterial culture can be appropriately selected from those known by those skilled in the art, and examples thereof include filter filtration and centrifugation. Filter filtration is preferable because it can be performed by a relatively simple mechanism.
  • a filter with a cutoff of 1 ⁇ m or less for example, a filter with a pore size of 0.2 ⁇ m can be used.
  • a plurality of filtration filters having different pore sizes may be combined.
  • THB purity in the product solution after heating can be improved by removing components unnecessary for THB synthesis from the bacterial broth by appropriate filtration. Unnecessary components for THB synthesis can also be removed by salting out.
  • the temperature of the“ high temperature ” that heats the bacterial culture is 80 ° C or higher. If the temperature is lower than 80 ° C., the DOI conversion reaction itself may occur, but it is considered that sufficient conversion efficiency cannot be obtained.
  • the high temperature is preferably 90 ° C or higher, more preferably 100 ° C or higher, more preferably 130 ° C or higher, further preferably 140 ° C or higher, particularly preferably 160 ° C or higher, most preferably 170 ° C or higher.
  • the upper limit is not particularly limited, but it is usually 300 ° C or lower, or 200 ° C or lower.
  • the elevated temperature is typically 130-180 ° C.
  • High temperatures may be combined with high pressures above atmospheric pressure. Such high pressure is, for example, 1 MPa or less, 0.8 MPa or less, 0.5 MPa or less, or 0.2 MPa or less.
  • the heating time of the bacterial culture at elevated temperature may vary depending on the temperature used and the desired THB yield, but is usually at least 30 seconds, preferably at least 1 minute, more preferably at least 2 minutes, more preferably at least 5 minutes. , More preferably at least 10 minutes, even more preferably at least 30 minutes.
  • the heating may be performed for 1 hour or more, 2 hours or more, or 4 hours or more.
  • the heating may be continuous over the above time, or the cumulative time of reaching a high temperature by intermittent heating may be the above time.
  • the upper limit of the heating time is not particularly limited, but is, for example, 24 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, or 10 minutes or less.
  • Heating the bacterial culture at an elevated temperature can be accomplished using techniques known to those skilled in the art, such as injecting hot air (steam) or hot liquid into the bacterial culture, solidifying the bacterial culture. It can be achieved by contacting with a heat source, heating the inner wall of the vessel or tube containing the bacterial culture, energizing the bacterial culture and / or applying microwaves to the bacterial culture.
  • the atmosphere during heating of the bacterial culture is preferably air, an inert gas such as nitrogen and argon, or a mixture thereof.
  • the atmosphere during the heating of the bacterial culture is preferably a substantially hydrogen (H 2 ) -free atmosphere.
  • the substantially hydrogen-free atmosphere has, for example, a hydrogen volume concentration of less than 1 ppm.
  • the heating of the bacterial culture is preferably carried out in the substantial absence of a metal catalyst or reduction catalyst such as palladium, rhodium, ruthenium, platinum, iridium, nickel, cobalt and copper.
  • Process solution means a solution in which THB is dissolved or suspended, which is obtained after heating the bacterial culture.
  • Filter filtration or centrifugation may be performed to remove impurities from the product solution.
  • a bacterial culture solution containing bacterial cells is heated, a large amount of insoluble matter derived from the modified bacterial cells may be contained in the product solution, and thus these treatments may be particularly significant.
  • the method of this embodiment may further include a step of separating THB from the product solution.
  • a known method can be appropriately used for the THB separation.
  • THB is extracted from the product solution by solvent extraction.
  • the solvent extraction may be repeated once or more.
  • As the extraction solvent ethyl acetate or methyl acetate is preferable, and ethyl acetate is particularly preferable, but not limited thereto.
  • the volume of the extraction solvent used for extraction is preferably equal to or larger than the volume of the product solution.
  • Solvent extraction can be carried out by mixing the product solution with an organic solvent and then separating into an organic layer and an aqueous layer and recovering an organic layer containing THB, as will be understood by those skilled in the art.
  • Water-soluble salts may be added to the product solution for solvent extraction. For example, adding less than saturated concentration of sodium chloride to the product solution followed by solvent extraction may improve yield. Also, the yield may be improved by adding an acid, such as hydrochloric acid, to the product solution to lower the pH before solvent extraction.
  • the pH is preferably less than 2, more preferably less than 1.5, even more preferably less than 1.
  • the insoluble matter can be removed by, for example, filter filtration.
  • the THB sample after separation has a THB content of preferably 50% or more, more preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, in particular, by dry weight after removing insoluble matter and solvent. It is preferably 90% or more.
  • the THB sample after separation has a catechol content of preferably 5% or less, more preferably 3% or less, still more preferably 2% or less, and particularly preferably 1.5% by dry weight after removing the insoluble matter and the solvent. % Or less.
  • steps from (a) the synthesis of DOI by culturing the bacteria to the production of THB by heating at high temperature and / or (b) the production of THB to the isolation of THB are carried out continuously. obtain. These steps may be performed batch by batch.
  • the present disclosure provides a culture tank that holds a liquid medium containing DOI-synthesizing bacteria and cultures the bacteria to produce a bacterial culture, and the bacterial culture connected to the culture tank. Is heated at a high temperature of 80 ° C. or higher to produce a product solution containing trihydroxybenzene (THB), and a heating device is provided to provide a system for producing trihydroxybenzene (THB).
  • a heating device is provided to provide a system for producing trihydroxybenzene (THB).
  • the liquid medium is capable of supporting culture at a cell density of 10 7 cells / mL or more, preferably 10 8 cells / mL or more, typically in the stationary phase as described above.
  • the bacteria are also as described above.
  • the culture tank may be equipped with auxiliary equipment for providing conditions suitable for culturing bacteria.
  • the culture tank is one of a stirring device for stirring the culture medium, a shaking device for shaking the culture medium, an aeration device for aerating the culture medium, and a heating device for heating the culture medium. It may have more than one.
  • a liquid medium supply line may be connected to the culture tank to supply a part or all of the components of the fresh liquid medium into the culture tank.
  • the liquid culture medium supply line has a switching valve so that the culture tank can be switched and connected between the liquid culture medium supply line and a liquid feeding pipe described later.
  • the culture tank can be connected to a heating device via a liquid feeding tube.
  • the heating device in this case heats the liquid sent from the culture tank inside the heating device.
  • the heating device may have a structure in which a continuous heating passage is provided inside and the bacterial culture solution passing through the heating passage is heated.
  • the heating passage can heat the liquid culture medium, for example, by injecting hot air or a hot liquid into the passing bacterial culture, or by heating the inner wall of the passage or a solid heat source arranged in the passage, The heating method is not limited to these.
  • Hot air is, for example, heated air, steam, or nitrogen.
  • the hot liquid is, for example, heated water or fresh liquid medium.
  • the heating temperature, holding time, and flow rate in the heating passage can be adjusted arbitrarily.
  • the heating passage may have a plurality of heating zones that can be set at different temperatures.
  • the heating device may be provided with an outlet for collecting the product solution that is heated to produce THB.
  • the culture tank may be connected to the heating device via a bacterial cell removing device that removes bacterial cells of the bacterial solution.
  • the microbial cell removal device may include a filtration filter or a centrifuge.
  • a bacterial cell removal device including a filtration filter for removing bacterial cells is preferable.
  • a plurality of filtration filters having different pore sizes may be provided in the bacterial cell removing device, and unnecessary components other than bacterial cells may be removed at this stage.
  • the bacterial cell removing device may be provided in the middle of the liquid feeding pipe.
  • the liquid transfer tube includes one or more of a bacterial cell removal device, a pump for transferring the liquid, a safety valve for preventing excessive internal pressure (for example, 10 bar or more), and a flow rate control device for controlling the flow rate.
  • a bacterial cell removal device for transferring the liquid
  • a safety valve for preventing excessive internal pressure (for example, 10 bar or more)
  • a flow rate control device for controlling the flow rate.
  • the heating device may be an integrated heating device that heats the bacterial culture solution inside the culture tank itself.
  • the heating in this case can be achieved, for example, by injecting hot air or a hot liquid into the inside of the culture tank, or by heating the inner wall of the culture tank or a solid heat source arranged in the culture tank, but possible heating is possible.
  • the method is not limited to these.
  • Hot air is, for example, heated air, steam, or nitrogen.
  • the hot liquid is, for example, heated water or fresh liquid medium.
  • the heating temperature and the heating time by the heating device are as described in the embodiment of the THB manufacturing method.
  • a purification device for removing solid substances and insoluble substances from the product solution may be connected to the heating device (including the culture tank provided with the heating device in the case of an integrated heating device).
  • the purification device can have, for example, a filtration filter or a centrifuge. If the heated bacterial culture contains bacterial cells, the product solution may contain a large amount of insoluble matter derived from denatured bacterial cells. You can
  • the purification device may be provided with a plurality of filtration filters having different pore sizes.
  • the heating device may be connected to a cooling device for reducing the temperature of the liquid. The liquid after heating at a high temperature is cooled to, for example, room temperature, and the cooling device can accelerate this cooling.
  • the heating device in the case of an integrated heating device, including the culture tank provided with it
  • the separation device is preferably connected downstream of the purification device described above.
  • Separation devices may include known solvent extraction devices such as centrifugal extractors, mixer settlers, microreactors, flow reactors, pulse columns and the like.
  • the solvent extraction device may have an organic layer outlet for discharging an organic layer containing THB.
  • the solvent extraction device may further have a water layer outlet for discharging the water layer.
  • the heating device may be connected to the separation device via a liquid feeding pipe.
  • This liquid supply pipe may also have an auxiliary equipment such as a pump, a safety valve, a cooling device, a purifying device, and a flow rate adjusting device as described above in any number and arrangement order.
  • the entire system of this embodiment may be integrated as one device.
  • the present disclosure provides a method of manufacturing THB using the THB manufacturing system of this embodiment.
  • FIG. 1 An example of the system according to this embodiment is shown in FIG.
  • the bacteria 101 that synthesize DOI and the liquid medium 102 are held in the culture tank 103, and the culture is performed in the culture tank 103. That is, the liquid medium 102 becomes a bacterial culture liquid 102 '.
  • the culture tank 103 can be connected to either the liquid medium supply line 105 or the liquid feeding pipe 106 by a switching valve 104.
  • the liquid feed pipe 106 is a bacterial cell removal device 107 having a filtration filter, a pump 108 for transferring the liquid (ie, the filtered bacterial culture liquid 102 '), and an excessive internal pressure (for example, 10 bar or more) for preventing it.
  • a safety valve 109 and a flow rate adjusting device 110 for adjusting the flow rate are provided and are connected to the heating device 111.
  • the bacterial culture solution passes through the heating passage 112 and is heated at that time.
  • three heating zones 113A, 113B, 113C are provided in the heating device 111, each having one or more heating passages so that the liquid culture medium can be heated at different temperatures.
  • the bacterial culture solution that has been heated at a high temperature becomes a product solution containing THB, and exits the heating device 111 from the outlet 114.
  • the outlet 114 is connected to a separation device 117 including a centrifugal extractor via a cooling device 115 for cooling the product solution and a purification device 116 having a filtration filter for removing solid substances and insoluble substances from the product solution. ing.
  • the separation device 117 performs extraction by adding an organic solvent (eg, ethyl acetate) to the product solution and discharges the organic layer 118 and the aqueous layer 119. THB is separated in the organic layer 118.
  • an organic solvent eg, ethyl acetate
  • FIG. Another example of the system according to this embodiment is shown in FIG. Also in the system 200 of this example, the bacteria 201 that synthesize DOI and the liquid medium 202 are held in the culture tank 203, and the culture is performed in the culture tank 203. That is, the liquid medium 202 becomes a bacterial culture fluid 202 '.
  • the culture tank 203 is equipped with an integrated heating device 211.
  • the integrated heating device 211 heats the liquid culture medium 202 after culture, that is, the bacterial culture liquid 202 ′ to a high temperature by injecting hot air 220 into the culture tank 203. Therefore, the bacterial culture solution 202 ′ is converted into a THB-containing product solution in the culture tank 203.
  • the culture tank 203 can be connected to either the liquid medium supply line 205 or the liquid feeding pipe 206 by a switching valve 204.
  • the liquid supply pipe 206 has a pump 208 for transferring the product solution, a safety valve 209 for preventing an excessive internal pressure, a cooling device 215 for cooling the product solution, and a filtration filter for removing solid substances and insoluble substances from the product solution. It has a purifying device 216 for removal and a flow rate adjusting device 210 for adjusting the flow rate, and is connected to a separating device 217 including a centrifugal extractor.
  • the separator 217 adds an organic solvent (for example, ethyl acetate) to the product solution to perform extraction, and discharges the organic layer 218 and the aqueous layer 219. THB is separated in the organic layer 218.
  • FIG. 3 shows DOI fermentation liquor (stock solution) at pH 6 or pH 3 and what was heated at 95 ° C. for 30 minutes, at 105 ° C. for 30 minutes, at 132 ° C. for 20 minutes, and at 132 ° C.
  • a purified DOI aqueous solution DOI concentration 70 g / L
  • a DOI fermentation broth 0.2 ⁇ m MF membrane
  • This DOI fermentation broth was obtained by culturing Escherichia coli expressing the btrC gene in the CSL medium for 32 to 40 hours as described above. The analysis results are shown in Table 1 below.
  • Table 1 the same test conditions for the purified DOI aqueous solution sample and the DOI fermentation liquid sample are shown in the same row.
  • the DOI residual ratio represents how much of the DOI contained in the sample before heating remained after the heating, and the 1,2,4-THB conversion rate was the% of DOI contained in the sample before heating. Represents the conversion to 1,2,4-THB (the conversion rate of mono-dehydrate is the same).
  • production of 1,2,3-THB was also confirmed (data not shown).
  • the description “160 ° C., 30 seconds, repeated 5 times” means that heating at 160 ° C. for 30 seconds was repeated 5 times, that is, the sample was heated at 160 ° C. for a cumulative time of 2.5 minutes. means.
  • the conversion of DOI to THB was achieved with significantly higher efficiency in the DOI fermentation broth compared to the purified DOI aqueous solution. That is, the THB conversion rate in the purified DOI aqueous solution sample 2 was 2.33%, while the THB conversion rate in the DOI fermentation liquid sample 5 under the same heating conditions was 27.19%. Similarly, the THB conversion rate of the purified DOI aqueous solution sample 3 was 5.23%, while the THB conversion rate of the DOI fermentation liquid sample 6 was 43.40%, and the THB conversion rate of the purified DOI aqueous solution sample 4 was The THB conversion rate of DOI fermentation liquor sample 7 was 54.66%, whereas it was 9.55%.
  • THB yield was improved more than when the DOI fermented liquor not filtered was heated, and salting out treatment was combined with the filtration. In some cases THB yields were further improved (data not shown).
  • THB aqueous solution, a THB product-containing solution obtained by heating an aqueous DOI solution, or a product solution obtained by heating a DOI fermentation broth was subjected to solvent extraction of THB using ethyl acetate as an extraction solvent. Tested. It was confirmed that most of THB can be recovered by the first solvent extraction. The recovery of THB is improved when hydrochloric acid is added to the THB product solution to lower the pH to less than 2 before solvent extraction or when less than saturated sodium chloride is added to the THB product solution. Was observed. The yield of THB finally extracted varies depending on the conditions and the degree of the THB production reaction prior to the extraction.
  • DOI synthesis by culture of DOI synthetic bacteria heating of a bacterial culture solution (DOI fermentation solution), and separation of THB by solvent extraction are continuously performed will be described below.
  • 150 mL of DOI fermentation broth (DOI concentration: 60 g / L) obtained by culturing DOI synthetic bacteria was reacted for 2 hours at 170 ° C. to convert DOI to THB.
  • the obtained THB-containing reaction solution was filtered through a membrane filter (0.45 ⁇ m) to prepare a THB product solution ( 13 C-NMR spectrum is shown in the lower part of FIG. 4).
  • THB product solution (1 mL / min) and ethyl acetate (1 mL / min, 1.5 mL / min, 2 mL / min, and 3 mL / min, four conditions) were used.
  • the solution was sent and the eluate was taken out (elution time 1 minute).
  • the ethyl acetate layer of the obtained eluate was separated and concentrated under reduced pressure to obtain THB yield (all experiments were carried out in triplicate).
  • Table 2 The results are summarized in Table 2 below.
  • the THB yield was 74.7% on average when the ratio of the THB product solution to ethyl acetate was 1: 1.
  • the yield tended to increase as the ratio of ethyl acetate increased, and it was 86.9% on average when the ratio of the THB product solution and ethyl acetate was 1: 3.
  • the 13 C-NMR spectrum of the obtained THB extracted sample is shown in FIG.

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Abstract

L'invention concerne un système de production hautement efficace de THB. Le système de production de trihydroxybenzène (THB) comprend : un réservoir de culture qui maintient une culture liquide, cultive les bactéries et génère un fluide de culture bactérien, ladite culture liquide comprenant des bactéries qui synthétisent du désoxy-scyllo-inosose (DOI); et un dispositif de chauffage qui est relié au réservoir de culture, chauffe le fluide de culture bactérienne à l'aide d'une température élevée d'au moins 80°C, et génère une solution de produit comprenant du trihydroxybenzène (THB).
PCT/JP2019/041724 2018-10-25 2019-10-24 Système de production de trihydroxybenzène WO2020085436A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112000A1 (fr) * 2005-03-30 2006-10-26 The Niigata Institute Of Science And Technology Procede permettant de synthetiser un 2-deoxy-scyllo-inosose au moyen d'une souche e. coli modifiee, procede de purification correspondant et 2-deoxy-scyllo-inosose ainsi obtenu
WO2009125581A1 (fr) * 2008-04-11 2009-10-15 三井化学株式会社 Procédé de fabrication de catéchol
WO2010125807A1 (fr) * 2009-04-28 2010-11-04 三井化学株式会社 Procédé de fabrication d'un phénol polyvalent
WO2015005451A1 (fr) * 2013-07-12 2015-01-15 三井化学株式会社 Procédé de production de 2-désoxy-scyllo-inosose

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112000A1 (fr) * 2005-03-30 2006-10-26 The Niigata Institute Of Science And Technology Procede permettant de synthetiser un 2-deoxy-scyllo-inosose au moyen d'une souche e. coli modifiee, procede de purification correspondant et 2-deoxy-scyllo-inosose ainsi obtenu
WO2009125581A1 (fr) * 2008-04-11 2009-10-15 三井化学株式会社 Procédé de fabrication de catéchol
WO2010125807A1 (fr) * 2009-04-28 2010-11-04 三井化学株式会社 Procédé de fabrication d'un phénol polyvalent
WO2015005451A1 (fr) * 2013-07-12 2015-01-15 三井化学株式会社 Procédé de production de 2-désoxy-scyllo-inosose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HANSEN, C.A. ET AL.: "Deoxygenation of Polyhydroxybenzenes: An Alternative Strategy for the Benzene-Free Synthesis of Aromatic Chemicals", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, 2002, pages 5926 - 5927, XP002397677, DOI: 10.1021/ja0176346 *
KAKINUMA, K. ET AL.: "An expeditious chemo-enzyma tic route from glucose to catechol by the use of 2-deoxy-scyllo-inosose synthase", TETRAHEDRON LETTERS, vol. 41, 2000, pages 1935 - 1938 *

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