WO2021193846A1 - Method for producing pha using agricultural waste - Google Patents

Method for producing pha using agricultural waste Download PDF

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Publication number
WO2021193846A1
WO2021193846A1 PCT/JP2021/012642 JP2021012642W WO2021193846A1 WO 2021193846 A1 WO2021193846 A1 WO 2021193846A1 JP 2021012642 W JP2021012642 W JP 2021012642W WO 2021193846 A1 WO2021193846 A1 WO 2021193846A1
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Prior art keywords
acid
present
carbon source
production method
pha
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PCT/JP2021/012642
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French (fr)
Japanese (ja)
Inventor
光太郎 猪野
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住友林業株式会社
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Priority to JP2022510677A priority Critical patent/JPWO2021193846A1/ja
Publication of WO2021193846A1 publication Critical patent/WO2021193846A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/16—Compositions of unspecified macromolecular compounds the macromolecular compounds being biodegradable
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a more efficient method for producing polyhydroxyalkanoates (PHA).
  • Biomass plastics and biodegradable plastics can be mentioned as solutions to such problems.
  • Biomass plastic is a plastic whose raw material is not fossil fuel but biomass such as squeezed waste such as corn and sugar cane and wood, and biodegradable plastic is decomposed into water and carbon dioxide by the power of microorganisms in the environment after use. It is a plastic.
  • PHA Polyhydroxyalkanoates
  • the present invention has an object of providing a more efficient method for producing PHA.
  • the present invention relates to at least the following inventions: [1] Method for producing polyhydroxyalkanoic acid, which comprises the following steps: (A) A step of culturing a microorganism using a medium containing agricultural waste that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture. [2] The production method according to [1], wherein the agricultural waste is rice bran. [3] The production method according to [1] or [2], wherein the microorganism is a highly halophilic bacterium.
  • sugar as a carbon source other than the carbon source derived from agricultural waste, and the sugar is (a) glucose and / or its derivative only (b) glucose and its derivative and xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose. , One or more from sucrose and its derivatives, or (c) one or more from xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives.
  • the production method according to [6] wherein a polyhydroxyalkanoic acid having a weight average molecular weight of 1 million or more is obtained.
  • a method for producing PHA at low cost by using an inexpensive carbon source such as agricultural waste such as rice bran without performing biological or chemical pretreatment There have been reports on the production of PHA using rice bran, whey, wheat bran, etc., and among them, reports on the production of PHA using highly halophilic bacteria. However, all the rice bran disclosed in this report has been biologically pretreated and used, and PHA has not been produced from rice bran that has only been physically crushed as appropriate. It wasn't done.
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2015-100283 describes a PHA production method using sap, but the production method requires pretreatment.
  • Non-Patent Document 1 J Ind Mivrobiol Biotechnol (2006) 33: 701 describes that rice bran decomposed by amylase is used for culturing to produce PHBV, but pretreatment is an indispensable step. be.
  • Non-Patent Document 2 (Brazilian Journal of Microbiology (2012): 1094-1102) discloses that rice bran or wheat bran hydrolyzed with amylase is used as a carbon source to produce PHA. Pretreatment (pretreatment with amylase) is also performed in this technique.
  • Non-Patent Document 3 (Bioresource Technology 181 (2015) 283-290) describes that when pretreated rice bran is given to recombinant Escherichia coli as a carbon source, PHA or a copolymer of PHA and lactic acid is produced. However, the pretreatment of rice bran requires heating at 121 ° C. for 1 h and hydrolysis with amylase.
  • Non-Patent Document 4 Macromolecular Bioscience 2007, 7, 2178
  • the obtained PHBV has a molecular weight of 696,000 and a 3HV fraction of 8 to 10%.
  • the whey that has been hydrolyzed with amylase is used as the whey.
  • a method for producing polyhydroxyalkanoic acid using biologically and chemically untreated agricultural waste such as rice bran has not been known.
  • the production method of the present invention which is a method for producing polyhydroxyalkanoic acid using biologically and chemically untreated agricultural waste such as rice bran, is not only unthinkable even by those skilled in the art from the prior art. Although the pretreatment step which has been conventionally performed is omitted, the PHA production efficiency is improved, which is an excellent effect.
  • the principle that the present invention exerts such an effect is that it originally exists in agricultural waste by not biologically and chemically attaching agricultural waste such as rice bran. Substances that inhibit the growth of microorganisms, which may be due to the more effective use of substances that have an advantageous effect on the growth of microorganisms, and / or the biological and chemical attachment of the agricultural waste. It may be due to the prevention of the occurrence of.
  • the molecular weight of PHA in the present specification is a numerical value measured by gel filtration chromatography using a polystyrene standard unless otherwise specified.
  • the symbol "-" represents a numerical range (including the two numerical values) specified by a range between two numerical values connected by the symbol.
  • the description "200,000 to 3 million” means a range composed of all numerical values existing between 200,000 and 3 million, including 200,000 and 3 million.
  • the notation "200,000 or more and 3 million or less" may be used.
  • the production method of the present invention is as follows: Method for producing polyhydroxyalkanoic acid, which comprises the following steps: (A) A step of culturing a microorganism using a medium containing agricultural waste that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture.
  • the chemical pretreatment includes chemical decomposition such as hydrolysis with hydrochloric acid, sulfuric acid or the like.
  • the biological pretreatment includes decomposition by enzymes such as amylase and decomposition in vivo using metabolism of microorganisms. In addition to these steps, physical decomposition (crushing, grinding, etc.) may be combined as appropriate.
  • the agricultural waste in the production method of the present invention is not limited as long as it has the desired effect in the present invention, but rice bran, wheat bran, wheat bran, milk syrup, sugar syrup, food residue in general, sugar cane pomace, and corn.
  • Examples include squeezed dregs, discarded konjac potatoes, sake pomace, and beer pomace.
  • Rice bran is a product in which the light brown layer covering the surface of brown rice becomes powdery during rice milling and is separated from the milled rice.
  • Rice bran contains dietary fiber, oils, sugars, proteins, and inorganic salts.
  • the production method of the present invention is due to the fact that the growth of microorganisms is guaranteed by these nutritional components contained in agricultural waste.
  • the type of rice bran in the present invention is not limited, and rice bran produced when brown rice is milled may be used. In addition, defatted rice bran may be used.
  • the degree of rice polishing in the step of milling brown rice when obtaining rice bran in the present invention is not limited as long as the obtained rice bran exerts the desired effect of the present invention.
  • the degree of rice polishing is exemplified by 1 minute to 8 minutes. That is, the rice bran used in the production method of the present invention may be rice bran obtained by grinding brown rice for one minute to eight minutes.
  • the rice bran obtained when producing wash-free rice is preferable.
  • Non-washed rice is white rice from which the skin bran that remains a little in the normal rice milling process has been removed in advance. Manufactured by a manufacturing method such as the method.
  • the rice bran in the present invention may be rice bran produced by any of these methods.
  • the type and variety of rice used as a raw material for rice bran in the present invention is not limited as long as it exhibits the desired effect of the present invention.
  • Such types and varieties include Koshihikari, Sasanishiki, Hitomebore, Hinohikari, Akitakomachi, Nanatsuboshi, Haenuki, Milky Queen, Premonition of Love, Tsuyahime, Yukiwakamaru, and Kinuhikari.
  • Sake rice such as Miyama Nishiki, Dewa Sasanishiki, and Omachi; glutinous rice; and rice from land rice are exemplified.
  • the origin of these rice is also not limited.
  • the rice bran in the present invention may be used after being crushed in advance by a crusher such as a cutter mill.
  • the particle size and the degree of pulverization of rice bran in the present invention are not limited as long as they exhibit the desired effects of the present invention.
  • the particle size is, for example, 5 to 1000 ⁇ m.
  • Rice bran having a particle size of 5 to 50 ⁇ m is preferable.
  • sugars other than sugars derived from agricultural waste may be added to the medium.
  • Such sugars do not include sugars contained in other nutrient sources such as yeast extract.
  • the sugar refers to a sugar that is supplied separately from other nutrient sources such as yeast extract.
  • the sugar used in the production method of the present invention also includes derivatives of each of the above sugars.
  • the sugar derivative the above-mentioned sugar is assumed to be etherified and esterified.
  • the etherified derivative is a methoxylated derivative
  • the esterified derivative is an acetylated derivative.
  • each is exemplified.
  • PHA can be produced by controlling the molecular weight of PHA to a weight average molecular weight of 1 million to 3 million, but PHA is further added by adding a furfural compound as a carbon source. As a result, a PHA having a lower molecular weight can be obtained, and a PHA having a wider molecular weight can be produced.
  • the molecular weight of the produced PHA is not limited, for example, the molecular weight of the PHA is controlled to a weight average molecular weight of 200,000 to 3 million. Can be manufactured.
  • the method of the present invention in which a furfural compound is used will be further described later.
  • a method of producing PHA by controlling the molecular weight of PHA to a weight average molecular weight of 1 million to 3 million is preferable.
  • the carbon source in the production method of the present invention includes a carbon source derived from the agricultural waste contained in the agricultural waste.
  • the carbon source in the production method of the present invention may include a carbon source other than the carbon source derived from agricultural waste in addition to the carbon source derived from agricultural waste.
  • a carbon source (A) When only glucose and / or its derivative is used, the molecular weight of PHA produced tends to be large.
  • the carbon source (b) one or more of glucose and its derivatives and xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives.
  • the molecular weight of the produced PHA tends to be smaller than that in the case of (a) above. It is produced by using one or more of (c) xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives as the carbon source and not using glucose and / or its derivatives. The molecular weight of PHA tends to be even smaller than in the case of (b) above.
  • the present invention also provides a method of controlling the molecular weight of PHA by selecting such a sugar as a carbon source.
  • the method for controlling the molecular weight of PHA according to the present invention is that the carbon source in the culture contains a sugar and the sugar is (a) glucose and / or a derivative thereof only (b) glucose and a derivative thereof and xylose, cellobiose, glucuronic acid, One or more from arabinose, mannose, galactose, sucrose and their derivatives, or (c) one or more from xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives And then A method of controlling the weight average molecular weight of the produced polyhydroxyalkanoates between 1 million and 3 million. As a control method of the present invention, a method of controlling the molecular weight of PHA to a weight average molecular weight of 1 million to 2.5 million is preferable.
  • the amount of sugar used in the production method of the present invention is not limited, and is exemplified by about 5 g / L to 910 g / L.
  • the amount of sugar is preferably from about 5 g / L to about 500 g / L, more preferably from about 10 g / L to 50 g / L.
  • the sugar used may be added in a plurality of times.
  • the number and timing of input are not limited, and the number of times may be 2, 3, 4, 5, or 6 or more. Further, it is also possible to continuously supply sugar, and in that case, a method of controlling so as to keep the sugar concentration constant may be used.
  • the molecular weight of PHA is controlled to a broader molecular weight, for example, a weight average molecular weight of 200,000 to 3,000,000. PHA can be produced.
  • Furfural compounds are a type of aldehyde produced from sugar as a raw material, and 5-hydroxymethylfurfural (HMF) from 6 monosaccharides and furfural from 5 monosaccharides are produced by heating the sugar under acidic conditions. Is known to produce.
  • furfural is a common name for 2-furfural aldehyde.
  • the "furfural compound” in the present invention means furfural, 5-hydroxymethylfurfural, and derivatives thereof.
  • the elongation of the polymer chain is stopped by promoting the movement of the growth end of the polymer chain having a relatively low molecular weight by the furfural compound, and the next polymer different from the polymer is used. Since the extension of the chain is started, it can be considered that the molecular weight of the obtained PHA is smaller than that in the case where the furfural compound is not used.
  • furfural compounds used in the production method or control method of the present invention in which furfural compounds are used are not limited, and furfural and 5-hydroxymethylfurfural, and derivatives thereof are exemplified.
  • examples of such derivatives include 5-hydroxymethylfurancarboxylic acid, 5-hydroxyfurfuryl alcohol, furan-2,5-bismethanol and those obtained by etherifying or esterifying them.
  • Methoxylation is exemplified as etherification
  • acetylation and methyl esterification are exemplified as esterification.
  • the sugar is one or more from glucose and its derivatives, as well as xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives, and the furfural compounds are furfural and / or 5-hydroxymethylfurfural.
  • the production method of the present invention or the control method of the present invention, which is a derivative thereof, is preferable.
  • the amount of the furfural compound used in the production method of the present invention is not limited, and the initial concentration of the culture is exemplified by about 0.01 to about 3.0 g / L.
  • the initial concentration of the furfural compound is preferably 0.01 g / L to 1 g / L.
  • the ratio of the amount of furfural compound used to the amount of sugar is not limited, and is exemplified by about 0.0025 to about 0.6.
  • the production method of the present invention or the control method of the present invention in which the ratio of the amount of the furfural compound used to the amount of sugar is 0.01 to 0.2 is preferable.
  • microorganisms used in the production method of the present invention is not limited, highly halophilic bacteria are suitable as the microorganism. This is because not only the production capacity of PHA is high, but also the produced PHA can be easily recovered.
  • the conditions other than the above carbon source and microorganisms are not limited, and conditions usually used in the present technology and known conditions may be used.
  • the medium used for culturing in the production method of the present invention may be solid, liquid or gel, but a liquid medium is preferable from the viewpoint of production speed.
  • the total salt concentration is, for example, 100 g / L to 300 g / L. It is preferable that the total salt concentration is 150 g / L to 250 g / L.
  • Examples of the medium used include those containing inorganic salts such as NH 4 Cl, KH 2 PO 4 , FeCl 3 , NaCl, MgCl 2 , DDL 4 , CaCl 2 , KCl, LVDS 3 and NaBr.
  • inorganic salts such as NH 4 Cl, KH 2 PO 4 , FeCl 3 , NaCl, MgCl 2 , DDL 4 , CaCl 2 , KCl, LVDS 3 and NaBr.
  • the types and amounts of these inorganic salts are not limited, and for example, for NH 4 Cl, KH 2 PO 4 , FeCl 3 , KCl, LVDS 3 , NaCl and NaCl, the following amounts (g / L) are exemplified.
  • a medium obtained by substituting and modifying the sugar content in a known medium with the sugar content contained in agricultural waste is preferable.
  • Such known media are not limited, but a list of media published on the website of BSM medium and NBRC (National Institute of Technology and Evaluation Biotechnology Center) (https://www.nite.go.jp/nbrc) The medium (medium of No. 1380, etc.) in /cultures/cultures/culture-list.html) is exemplified.
  • the medium composition of No. 1380 is shown by quoting from the above medium list:
  • the nutrient source contained in the medium used for culturing the microorganism in the production method of the present invention at least one of the above sugars is used as the carbon source.
  • a carbon source different from the above-mentioned sugar at the time of culturing a carbon source usually used for culturing microorganisms such as amino acids, alcohols, fats and oils, and / or fatty acids may be used.
  • As amino acids glycine, alanine, phenylalanine, lysine, leucine
  • As alcohols methanol, ethanol, 1-propanol, 2-propanol, glycerol
  • Oils and fats include palm oil, palm kernel oil, corn oil, palm oil, olive oil, soybean oil, and rapeseed oil
  • As fatty acids fatty acid salts such as acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and crotonic acid and its sodium salt and ammonium salt. When fatty acids are used, the amount of the fatty acids is preferably small.
  • the medium used for culturing microorganisms in the production method of the present invention may contain a nitrogen source, inorganic salts and the like.
  • the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as well as peptone, meat extract, yeast extract, and the like.
  • the inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like.
  • the temperature of culturing when culturing a microorganism in the production method of the present invention is not limited as long as the microorganism can grow, and examples thereof include about 10 ° C. to about 65 ° C., preferably 20 ° C. to 50 ° C. , 35 ° C to 45 ° C are more preferable.
  • the timing of adding the carbon source, the inorganic salt, and the organic nutrient source is not limited, but generally used methods such as batch culture, fed-batch culture, and continuous culture can be used depending on the purpose. It can be appropriately selected and used.
  • Examples of the culture time of microorganisms in the production method of the present invention include a long period of 24 hours to 168 hours for batch culture, 72 hours to 14 days for fed-batch culture, and more than 14 days for continuous culture. Not limited to.
  • the pH of the medium is not limited in the production method of the present invention, it is preferable that the initial pH of the medium is about 6.5 to about 7.5. It is more preferable that the initial pH of the medium is about 6.5 to about 7.5 and the pH of the medium being cultured is maintained at about 6.5 to about 7.5. It is more preferable to set these pH to about 7.0 to about 7.4.
  • control method of the present invention other culture conditions used in the production method of the present invention can also be used.
  • the type of PHA product in the production method of the present invention is not limited, but is PHBV, PHB (polyhydroxybutyrate), poly (hydroxybutyrate / hydroxyhexanoate, and poly (3 hydroxybutyrate / 4-hydroxy). Butyrate) is exemplified. Of these, PHBV and / or PHB are suitable products.
  • the PHA produced by the production method of the present invention can be recovered from the cells by a known method by centrifugation, filtration or precipitation using an organic solvent, a surfactant, a low-concentration sodium chloride aqueous solution or water.
  • the production amount and 3HV fraction of PHA produced by the microorganism in the production method of the present invention may be measured by using a known method such as the GC-MS method.
  • the 3HV fraction may also be determined using a known method such as the GC-MS method.
  • the concentration of glucose or its derivative, other sugars, and furfural compounds For the measurement of the concentration of glucose or its derivative, other sugars, and furfural compounds, a measurement using a commercially available concentration measurement kit or a known method such as an HPLC method may be used.
  • the production amount of the microbial cells is known such as an absorbance method or a dry cell weight measurement method. It may be measured by the method.
  • the molecular weight of PHA obtained by the production method of the present invention may also be measured by a known method, for example, by gel filtration chromatography using a polystyrene standard. As described above, for the molecular weight of PHA in the present specification, the numerical value measured by gel filtration chromatography using a polystyrene standard is used unless otherwise specified.
  • the weight average molecular weight of PHA produced by the method of the present invention is about 1 million to about 3 million when the furfural compound is not used, and about 200,000 to about 3 million when the furfural compound is used. be.
  • the molecular weight of PHA obtained in each range can be controlled or adjusted in the production method of the present invention.
  • PHBV a copolymer in which 3-hydroxybutanoic acid (3HB) and 3-hydroxyvalerylic acid (3HV) are randomly copolymerized (hereinafter, may be referred to as "PHBV") is also provided.
  • the PHBV of the present invention includes the structural units shown below as any combination (m and n represent integers in the following equation):
  • the structure, monomer composition and molecular weight of PHBV of the present invention are not limited.
  • the PHBV of the present invention is linear, and the PHBV of the present invention, which is at least substantially linear, is preferred.
  • the PHBV of the present invention is a copolymer in which 3HB and 3HV are randomly copolymerized as described above, and a copolymer produced by alternating copolymerization of 3HB and 3HV or block copolymerization is also included.
  • the molar ratio of 3-hydroxybutanoic acid and 3-hydroxyvalerylic acid constituting PHBV is not limited.
  • the 3HV fraction (the ratio of the number of moles of 3-hydroxyvalerylic acid to the total number of moles of 3-hydroxybutanoic acid and 3-hydroxyvalerylic acid constituting the copolymer) is about 5.0%.
  • the PHBV of the present invention which is about 40.0%, is preferable, and the PHBV of the present invention, which is about 5.0% to about 28.0%, is more preferable, and the PHBV of the present invention is about 7.0% to 18.0%.
  • the PHBV of the present invention is even more preferable, and the PHBV of the present invention, which is about 9.0% to about 15.0%, is even more preferable.
  • the method for producing PHBV of the present invention is not limited as long as it is a method including the above steps (a) and (b), and is a method capable of producing PHBV having a weight average molecular weight of about 3 million. It's okay.
  • a method of culturing a microorganism to obtain PHBV of the present invention as a culture product is exemplified.
  • a method of using a highly halophilic bacterium as a microorganism in the culture and glucose and / or a derivative thereof as a carbon source is used.
  • the method using glucose is particularly preferred. This is because according to these methods, PHBV having a weight average molecular weight of about 3 million can be efficiently produced, and the produced PHBV can be easily recovered.
  • the saccharides as carbon sources are (a) glucose and / or its derivative only, or (b) glucose and its derivative and xylose, cellobiose, etc.
  • the PHBV of the present invention is produced by using a yeast extract without adding a carbon source other than the carbon source such as amino acids contained in the yeast extract.
  • a carbon source other than the carbon source such as amino acids contained in yeast extract, the PHBV of the present invention can be produced at a lower cost.
  • the guard column used was TSKgel guardcolumn Super HZ-H, and the column used was two TSK gel Super HZM-H connected in series.
  • Chloroform (0.6 mL / min) was used as the mobile phase, and the column temperature was 40 ° C.
  • the sample concentration was about 0.5 mg / mL, and the sample injection volume was 10 ⁇ L.
  • a polystyrene standard was used to prepare the calibration curve.
  • the 3-hydroxyvalerylic acid (3HV) fraction and PHBV production of the copolymer were measured using gas chromatography-mass spectrometry (GC-MS method) as follows (device name: Agilent 6890 /). 5973 GCMS System).
  • the values of PHA production, molecular weight, and 3HV fraction specified by these methods are accurately compared with the values of PHA production, molecular weight, and 3HV fraction described in publicly known documents. Is something that can be done.
  • the weight of dried cells can be measured by a known method such as a freeze-drying method.
  • a method of thermally decomposing all organic substances other than the inorganic salts at a high temperature may be used.
  • Example ⁇ (Material and method) For the Haloferax mediterranei NBRC14739 strain, the sugar (carbon source) in the BMS medium shown in Table 2 was used as the material using rice bran (“rice bran 1”, “rice bran 2”, “rice bran 3”, Alternatively, a medium group containing a medium composed by substituting with sugar derived from "rice bran 4") was prepared. Using these media, in a 300 mL Erlenmeyer flask, the total volume of the culture solution was 50 mL, and the culture was carried out for 72 hours at 37 ° C. The ingredients of rice bran were as follows. The amount of the material added using rice bran was 10.2 g / L, which corresponds to the sugar concentration of 5 g / L in terms of glucose in the BMS medium. The composition of each test plot was as shown in the table below:
  • composition of the BMS medium is as shown below:
  • PHBV was produced in plot No. 1 using rice bran that had not been chemically and biologically pretreated as a carbon source.
  • the production volume of PHBV in the section No. 1 exceeded the production volume in the sections No. 2 and 3 corresponding to the prior art. Therefore, it was clarified that PHBV can be efficiently produced by using untreated rice bran as a carbon source.
  • the medium of NBRC No. 1380 has a high PHA production amount, a high 3HV fraction, and a high molecular weight, and can produce an unprecedented type of PHA.
  • PHBV having various molecular weights can be produced and the molecular weight can be controlled by changing the concentration of furfural and the concentration of HMF. It is considered that the molecular weight of PHBV produced by a furfural compound such as furfural or HMF is controlled even when a sugar other than glucose is used.
  • the present invention there is provided a method for producing PHA at low cost by using an inexpensive carbon source such as agricultural waste such as rice bran without performing biological or chemical pretreatment. Therefore, the present invention greatly contributes to the development of the PHA manufacturing industry and related industries.

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Abstract

A method for producing a polyhydroxyalkanoate that includes the following steps: (a) a step for culturing a micro-organism with a culture medium containing agricultural waste that has not been chemically or biologically pre-treated; and (b) a step for obtaining a polyhydroxyalkanoate as product of the culture.

Description

農産廃棄物を用いたＰＨＡの製造方法PHA manufacturing method using agricultural waste
　本発明は、ポリヒドロキシアルカン酸（ＰＨＡ）のより効率的な製造方法に関する。 The present invention relates to a more efficient method for producing polyhydroxyalkanoates (PHA).
　２０世紀後半、石油からプラスチックを作る技術が急速に発展し、プラスチックの普及によって人々の生活が一変した。プラスチックに関するノーベル賞の受賞も多数ある（カロザースのナイロン、チーグラーとナッタのポリエチレン、ポリプロピレンなど）。 In the latter half of the 20th century, the technology for making plastic from petroleum developed rapidly, and the spread of plastic changed people's lives. He has also received numerous Nobel Prizes for plastics (Carothers nylon, Ziegler-Natta polyethylene, polypropylene, etc.).
　現在では、プラスチックは日本だけでも年間で１０７５万ｔ生産されている（２０１６年）。一方で、プラスチックのほとんどは石油から作られており、その廃棄時の二酸化炭素の排出による温暖化への影響や石油資源の安定供給性などが問題となっている。さらに、通常のプラスチックは一度環境中に放出されると自然界で分解されないため、プラスチックがそのまま海に漂ってしまうことによる、マイクロプラスチックの問題が深刻化している。
　このような問題の解決策としてバイオマスプラスチック、生分解プラスチックが挙げられる。バイオマスプラスチックは、原料が化石燃料ではなくトウモロコシやサトウキビなどの搾りかすや木質などのバイオマスであるプラスチックであり、生分解プラスチックは、使用後に環境中で微生物の力で水と二酸化炭素にまで分解されるプラスチックである。 At present, 10.75 million tons of plastic are produced annually in Japan alone (2016). On the other hand, most plastics are made from petroleum, and there are problems such as the impact of carbon dioxide emissions at the time of disposal on global warming and the stable supply of petroleum resources. Furthermore, since ordinary plastics are not decomposed in nature once they are released into the environment, the problem of microplastics is exacerbated by the fact that the plastics float in the sea as they are.
Biomass plastics and biodegradable plastics can be mentioned as solutions to such problems. Biomass plastic is a plastic whose raw material is not fossil fuel but biomass such as squeezed waste such as corn and sugar cane and wood, and biodegradable plastic is decomposed into water and carbon dioxide by the power of microorganisms in the environment after use. It is a plastic.
　ポリヒドロキシアルカン酸（ＰＨＡ）はある種の微生物に脂質や糖質などのバイオマスを炭素源（栄養）として与えるとエネルギー貯蔵物質として蓄えるプラスチックであり、使用後は環境中で生分解する環境負荷の小さいバイオプラスチックである。
　ＰＨＡとして最初に発見されたのはポリ［３－ヒドロキシブタン酸］（「Ｐ（３ＨＢ）」と表記されることがある）である。非特許文献３には、モノマー成分として３－ヒドロキシブタン酸以外に第２成分として３－ヒドロキシバレリル酸（「３ＨＶ」と表記されることがある）についての開示がある。 Polyhydroxyalkanoates (PHA) is a plastic that stores biomass such as lipids and sugars as a carbon source (nutrient) to certain microorganisms as an energy storage substance, and is an environmental load that biodegrades in the environment after use. It is a small bioplastic.
The first to be discovered as PHA is poly [3-hydroxybutanoic acid] (sometimes referred to as "P (3HB)"). Non-Patent Document 3 discloses 3-hydroxyvaleryl acid (sometimes referred to as "3HV") as a second component in addition to 3-hydroxybutanoic acid as a monomer component.
特開２０１５－１００２８３号公報JP-A-2015-100233
　本発明はＰＨＡのより効率的な製造方法を提供することを課題とした。 The present invention has an object of providing a more efficient method for producing PHA.
　本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、従来用いられることがなかった資材を用いることによりＰＨＡを製造する効率を高めることができる可能性があることを見出し、さらに研究を進めた結果本発明を完成するに至った。 As a result of intensive research to solve the above problems, the present inventors have found that there is a possibility that the efficiency of producing PHA can be improved by using a material that has not been used in the past. As a result of advancing research, the present invention has been completed.
　すなわち本発明は、少なくとも以下の発明に関する：
　［１］
　　以下の工程を含む、ポリヒドロキシアルカン酸の製造方法：
　（ａ）化学的及び生物的な前処理がなされていない農産廃棄物を含む培地を用いて微生物を培養する工程；及び
　（ｂ）前記培養における培養産物としてポリヒドロキシアルカン酸を得る工程。
　［２］
　農産廃棄物が米ぬかである、［１］に記載の製造方法。
　［３］
　微生物が高度好塩菌である［１］又は［２］に記載の製造方法。
　［４］
　ポリヒドロキシアルカン酸が、３－ヒドロキシブタン酸と３－ヒドロキシバレリル酸とがランダムに共重合した共重合体である［１］～［３］のいずれかに記載の製造方法。
　［５］
　共重合体を構成する３－ヒドロキシブタン酸のモル数と３－ヒドロキシバレリル酸のモル数の総和に対する３－ヒドロキシバレリル酸のモル数の割合（３ＨＶ分率）が５．０％～４０．０％である［４］に記載の方法。
　［６］
　炭素源として農産廃棄物由来の炭素源以外の炭素源を含む、［１］～［５］のいずれかに記載の製造方法。
　［７］
　農産廃棄物由来の炭素源以外の炭素源として糖を含有し、該糖を
　（ａ）グルコース及び／又はその誘導体のみ
　（ｂ）グルコース及びその誘導体ならびにキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上、又は
　（ｃ）キシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上
にして、
　重量平均分子量が１００万以上であるポリヒドロキシアルカン酸を得る、［６］に記載の製造方法。
　［８］
　（ｂ）又は（ｃ）におけるキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上がキシロース及び／又はセロビオースである［７］に記載の製造方法。
　［９］
　以下の工程を含む、ポリヒドロキシアルカン酸の製造方法における、米ぬかの使用：
　（ａ）化学的及び生物的な前処理がなされていない米ぬかを含む培地を用いて微生物を培養する工程；及び
　（ｂ）前記培養における培養産物としてポリヒドロキシアルカン酸を得る工程。 That is, the present invention relates to at least the following inventions:
[1]
Method for producing polyhydroxyalkanoic acid, which comprises the following steps:
(A) A step of culturing a microorganism using a medium containing agricultural waste that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture.
[2]
The production method according to [1], wherein the agricultural waste is rice bran.
[3]
The production method according to [1] or [2], wherein the microorganism is a highly halophilic bacterium.
[4]
The production method according to any one of [1] to [3], wherein the polyhydroxyalkanoic acid is a copolymer in which 3-hydroxybutanoic acid and 3-hydroxyvalerylic acid are randomly copolymerized.
[5]
The ratio of the number of moles of 3-hydroxyvaleryl acid (3HV fraction) to the total number of moles of 3-hydroxybutanoic acid and 3-hydroxyvaleryl acid constituting the copolymer is 5.0% to 40. The method according to [4], which is 0.0%.
[6]
The production method according to any one of [1] to [5], which comprises a carbon source other than the carbon source derived from agricultural waste as the carbon source.
[7]
It contains sugar as a carbon source other than the carbon source derived from agricultural waste, and the sugar is (a) glucose and / or its derivative only (b) glucose and its derivative and xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose. , One or more from sucrose and its derivatives, or (c) one or more from xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives.
The production method according to [6], wherein a polyhydroxyalkanoic acid having a weight average molecular weight of 1 million or more is obtained.
[8]
The production method according to [7], wherein one or more of xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and derivatives thereof in (b) or (c) are xylose and / or cellobiose. ..
[9]
Use of rice bran in a method for producing polyhydroxyalkanoates, which comprises the following steps:
(A) A step of culturing a microorganism using a medium containing rice bran that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture.
　本発明によれば、米ぬかなどの農産廃棄物といった安価な炭素源を用いて、生物的、化学的な前処理を行わず、ＰＨＡを低コストで製造する方法が提供される。
　米ぬか、乳清、小麦ふすまなどを用いたＰＨＡの製造に関する報告や、その中でも特に高度好塩菌を用いてＰＨＡを生産した報告は過去になされている。しかしながら、かかる報告に開示されている米ぬかはすべて生物学的に前処理して用いられており、適宜物理的な破砕のみが行われた米ぬかによりＰＨＡを製造することはこれまでなされていないし、知られていなかった。 According to the present invention, there is provided a method for producing PHA at low cost by using an inexpensive carbon source such as agricultural waste such as rice bran without performing biological or chemical pretreatment.
There have been reports on the production of PHA using rice bran, whey, wheat bran, etc., and among them, reports on the production of PHA using highly halophilic bacteria. However, all the rice bran disclosed in this report has been biologically pretreated and used, and PHA has not been produced from rice bran that has only been physically crushed as appropriate. It wasn't done.
　従来、米ぬかをポリヒドロキシアルカン酸の生産に利用する際は、アミラーゼなど分解酵素による生物的前処理や酸、アルカリなどを利用する化学的前処理が必要であった。例えば特許文献１（特開２０１５－１００２８３号公報）には樹液を用いたＰＨＡ製造方法に関する記載があるが、同製造方法においては前処理が必要である。
　非特許文献１（J Ind Mivrobiol Biotechnol(2006)33:701）においては、アミラーゼにより分解した米ぬかを用いて培養を行いＰＨＢＶの生産を行ことについて記載されているが、前処理が不可欠の工程である。
　非特許文献２（Brazilian Journal of Microbiology (2012): 1094-1102）においてはアミラーゼで加水分解した米ぬか、小麦ふすま(麦ぬか) を炭素源に用いてＰＨＡを生産することについての開示があるところ、同技術においても前処理（アミラーゼによる前処理）が行われる。
　非特許文献３（Bioresource Technology 181 (2015) 283-290）においては、組み換え大腸菌に前処理した米ぬかを炭素源として与えると、ＰＨＡやＰＨＡと乳酸の共重合体を生産することについて記載されているが、米ぬかの前処理に１２１℃、１ｈの加熱が必要なうえにアミラーゼによる加水分解が必要とされている。
　非特許文献４（Macromolecular Bioscience 2007, 7, 218）においては、乳清を炭素源として微生物によりＰＨＢＶを生産した場合、得られたＰＨＢＶの分子量が６９．６万、３ＨＶ分率が８～１０％であったこと等が開示されているが、前記乳清としてアミラーゼを用いて加水分解したものが用いられている。
　このように従来技術においては生物的・化学的に未処理の米ぬかなどの農産廃棄物を用いたポリヒドロキシアルカン酸の製造方法は知られていなかった。生物的・化学的に未処理の米ぬかなどの農産廃棄物を用いたポリヒドロキシアルカン酸の製造方法である本発明の製造方法は、従来技術からは当業者といえども想到しえないばかりでなく、従来行われていた前処理の工程を省略しているにもかかわらずＰＨＡの製造効率を高めるといった、優れた効果を奏するものである。
　理論に束縛されるものではないが、本発明がこのような効果を奏する原理として、米ぬかのような農産廃棄物を生物的・化学的に付さないことにより、農産廃棄物にもともと存在する、微生物の生育に有利に作用する物質がより有効に利用されることによるものである可能性、及び／又は前記農産廃棄物を生物的・化学的に付すことにより生じる、微生物の生育を阻害する物質の発生を防ぐことによるものである可能性、がある。 Conventionally, when rice bran is used for the production of polyhydroxyalkanoic acid, biological pretreatment with a degrading enzyme such as amylase or chemical pretreatment using an acid or alkali has been required. For example, Patent Document 1 (Japanese Unexamined Patent Publication No. 2015-100283) describes a PHA production method using sap, but the production method requires pretreatment.
Non-Patent Document 1 (J Ind Mivrobiol Biotechnol (2006) 33: 701) describes that rice bran decomposed by amylase is used for culturing to produce PHBV, but pretreatment is an indispensable step. be.
Non-Patent Document 2 (Brazilian Journal of Microbiology (2012): 1094-1102) discloses that rice bran or wheat bran hydrolyzed with amylase is used as a carbon source to produce PHA. Pretreatment (pretreatment with amylase) is also performed in this technique.
Non-Patent Document 3 (Bioresource Technology 181 (2015) 283-290) describes that when pretreated rice bran is given to recombinant Escherichia coli as a carbon source, PHA or a copolymer of PHA and lactic acid is produced. However, the pretreatment of rice bran requires heating at 121 ° C. for 1 h and hydrolysis with amylase.
In Non-Patent Document 4 (Macromolecular Bioscience 2007, 7, 218), when PHBV is produced by a microorganism using whey as a carbon source, the obtained PHBV has a molecular weight of 696,000 and a 3HV fraction of 8 to 10%. However, the whey that has been hydrolyzed with amylase is used as the whey.
As described above, in the prior art, a method for producing polyhydroxyalkanoic acid using biologically and chemically untreated agricultural waste such as rice bran has not been known. The production method of the present invention, which is a method for producing polyhydroxyalkanoic acid using biologically and chemically untreated agricultural waste such as rice bran, is not only unthinkable even by those skilled in the art from the prior art. Although the pretreatment step which has been conventionally performed is omitted, the PHA production efficiency is improved, which is an excellent effect.
Although not bound by theory, the principle that the present invention exerts such an effect is that it originally exists in agricultural waste by not biologically and chemically attaching agricultural waste such as rice bran. Substances that inhibit the growth of microorganisms, which may be due to the more effective use of substances that have an advantageous effect on the growth of microorganisms, and / or the biological and chemical attachment of the agricultural waste. It may be due to the prevention of the occurrence of.
　以下に本発明について、より詳細に説明する。なお本明細書におけるＰＨＡの分子量は、他の記載がない限りポリスチレン標準を用いたゲルろ過クロマトグラフィーにより測定された数値である。 The present invention will be described in more detail below. The molecular weight of PHA in the present specification is a numerical value measured by gel filtration chromatography using a polystyrene standard unless otherwise specified.
　本明細書において「～」の記号は、該記号により結ばれた２つの数値の間の範囲により特定される数値範囲（前記２つの数値を包含する）を表す。例えば「２０万～３００万」の記載は２０万及び３００万を含む、２０万から３００万の間に存在するすべての数値により構成される範囲を意味する。同じ意味を表すために、「２０万以上３００万以下」の表記が用いられることがある。
　本明細書においてポリマーの分子量を数値により表す場合、本技術分野における当業者により理解される数値の幅を含むことにより特定される数値が意図される。 In the present specification, the symbol "-" represents a numerical range (including the two numerical values) specified by a range between two numerical values connected by the symbol. For example, the description "200,000 to 3 million" means a range composed of all numerical values existing between 200,000 and 3 million, including 200,000 and 3 million. In order to express the same meaning, the notation "200,000 or more and 3 million or less" may be used.
When the molecular weight of a polymer is expressed numerically in the present specification, a numerical value specified by including a range of numerical values understood by those skilled in the art is intended.
　本発明の製造方法は、下記のものである：
以下の工程を含む、ポリヒドロキシアルカン酸の製造方法：
　（ａ）化学的及び生物的な前処理がなされていない農産廃棄物を含む培地を用いて微生物を培養する工程；及び
　（ｂ）前記培養における培養産物としてポリヒドロキシアルカン酸を得る工程。
　上記化学的前処理には、塩酸又は硫酸等による加水分解のような化学的な分解が包含される。上記生物的前処理には、アミラーゼ等の酵素による分解や微生物の代謝を利用した生体内での分解が包含される。
　これらの工程に加え、適宜物理的な分解（粉砕、摩砕など）を組み合わせてよい。 The production method of the present invention is as follows:
Method for producing polyhydroxyalkanoic acid, which comprises the following steps:
(A) A step of culturing a microorganism using a medium containing agricultural waste that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture.
The chemical pretreatment includes chemical decomposition such as hydrolysis with hydrochloric acid, sulfuric acid or the like. The biological pretreatment includes decomposition by enzymes such as amylase and decomposition in vivo using metabolism of microorganisms.
In addition to these steps, physical decomposition (crushing, grinding, etc.) may be combined as appropriate.
　本発明の製造方法における農産廃棄物は本願発明における所期の効果を奏するものであれば限定されないところ、米ぬか、麦ぬか、小麦ふすま、乳清、糖蜜、食品残渣全般、サトウキビの搾りかす、とうもろこしの搾りかす、廃棄こんにゃく芋、酒かす、ビールかすが例示される。 The agricultural waste in the production method of the present invention is not limited as long as it has the desired effect in the present invention, but rice bran, wheat bran, wheat bran, milk syrup, sugar syrup, food residue in general, sugar cane pomace, and corn. Examples include squeezed dregs, discarded konjac potatoes, sake pomace, and beer pomace.
　本発明の製造方法について、農産廃棄物として米ぬかを用いる場合についてより詳細に説明する。
　米ぬかは玄米の表面を覆う薄茶色の層が精米時に粉状になり、精米された米から分離してなる生成物である。
　米ぬかは食物繊維、油分、糖質、タンパク質、及び無機塩類を含有する。理論に束縛されるものではないが、本発明の製造方法は、農産廃棄物に含有されるこれらの栄養成分により微生物の生育が担保されるためであると推察することができる。 The production method of the present invention will be described in more detail when rice bran is used as agricultural waste.
Rice bran is a product in which the light brown layer covering the surface of brown rice becomes powdery during rice milling and is separated from the milled rice.
Rice bran contains dietary fiber, oils, sugars, proteins, and inorganic salts. Although not bound by theory, it can be inferred that the production method of the present invention is due to the fact that the growth of microorganisms is guaranteed by these nutritional components contained in agricultural waste.
　本発明における米ぬかの種類は限定されないところ、玄米を精米する際に生じる米ぬかを用いてよい。また、脱脂米ぬかを用いてよい。
　本発明における米ぬかを得る際の玄米を精米する工程における精米度合いは、得られる米ぬかが本発明の所望の効果を奏するものであれば限定されない。かかる精米度合いとして一分～八分が例示される。すなわち本発明の製造方法に用いられる米ぬかは、玄米を一分搗き～八分搗きして得られる米ぬかであってよい。
　本発明における米ぬかのうち、無洗米を製造する際に得られる米ぬかは好ましい。
　無洗米とは、通常の精米化の工程では少し残ってしまう肌ぬか）をあらかじめ取り去った白米であり、ＢＧ精米製法、水洗い乾燥法・湿式法、ＮＴＷＰ（Neo Tasty White Process） 加工法、及び乾式法といった製法により製造される。本発明における米ぬかは、これらの方法のいずれにより生成される米ぬかであってよい。 The type of rice bran in the present invention is not limited, and rice bran produced when brown rice is milled may be used. In addition, defatted rice bran may be used.
The degree of rice polishing in the step of milling brown rice when obtaining rice bran in the present invention is not limited as long as the obtained rice bran exerts the desired effect of the present invention. The degree of rice polishing is exemplified by 1 minute to 8 minutes. That is, the rice bran used in the production method of the present invention may be rice bran obtained by grinding brown rice for one minute to eight minutes.
Among the rice bran in the present invention, the rice bran obtained when producing wash-free rice is preferable.
Non-washed rice is white rice from which the skin bran that remains a little in the normal rice milling process has been removed in advance. Manufactured by a manufacturing method such as the method. The rice bran in the present invention may be rice bran produced by any of these methods.
　本発明における米ぬかの原料となる米の種類及び品種は、本発明の所望の効果を奏するものであれば限定されない。かかる種類・品種として、コシヒカリ、ササニシキ、ひとめぼれ、ヒノヒカリ、あきたこまち、ななつぼし、はえぬき、ミルキークウィーン、恋の予感、つや姫、雪若丸及びキヌヒカリといった水稲うるち米；山田錦、五百万石、美山錦、出羽燦々、及び雄町といった酒米；もち米；ならびに陸稲からの米、が例示される。これらの米の産地も限定されない。
　本発明における米ぬかは、カッターミル等の粉砕機によりあらかじめ粉砕して用いてよい。また、本発明における米ぬかの粒子径や粉砕度は、本発明の所望の効果を奏するものであれば限定されない。粒子径としては、例えば5～1000μmがあげられる。粒子径が5～50μmである米ぬかは好ましい。 The type and variety of rice used as a raw material for rice bran in the present invention is not limited as long as it exhibits the desired effect of the present invention. Such types and varieties include Koshihikari, Sasanishiki, Hitomebore, Hinohikari, Akitakomachi, Nanatsuboshi, Haenuki, Milky Queen, Premonition of Love, Tsuyahime, Yukiwakamaru, and Kinuhikari. Sake rice such as Miyama Nishiki, Dewa Sasanishiki, and Omachi; glutinous rice; and rice from land rice are exemplified. The origin of these rice is also not limited.
The rice bran in the present invention may be used after being crushed in advance by a crusher such as a cutter mill. Further, the particle size and the degree of pulverization of rice bran in the present invention are not limited as long as they exhibit the desired effects of the present invention. The particle size is, for example, 5 to 1000 μm. Rice bran having a particle size of 5 to 50 μm is preferable.
　本発明の製造方法においては、農産廃棄物由来の糖以外の糖を培地に加えてよい。かかる糖には、例えば酵母エキスのような他の栄養源に含有される糖は包含されない。上記糖は、酵母エキスのような他の栄養源とは別に供給される糖を指す。
　本発明の製造方法において用いられる糖には、上記各糖の誘導体も包含される。糖の誘導体としては、上記糖がエーテル化、エステル化されたものが想定され、とくにエーテル化されたものとしてはメトキシ化されてなる誘導体が、エステル化されたものとしてはアセチル化されてなる誘導体が、それぞれ例示される。 In the production method of the present invention, sugars other than sugars derived from agricultural waste may be added to the medium. Such sugars do not include sugars contained in other nutrient sources such as yeast extract. The sugar refers to a sugar that is supplied separately from other nutrient sources such as yeast extract.
The sugar used in the production method of the present invention also includes derivatives of each of the above sugars. As the sugar derivative, the above-mentioned sugar is assumed to be etherified and esterified. In particular, the etherified derivative is a methoxylated derivative, and the esterified derivative is an acetylated derivative. However, each is exemplified.
　本発明の製造方法においては、例えば１００万～３００万の重量平均分子量に、ＰＨＡの分子量を制御してＰＨＡを製造することができるところ、炭素源としてのフルフラール系化合物をさらに添加することによりＰＨＡとしてより低分子量のものが得られ、さらに広範囲な分子量を有するＰＨＡを製造することができる。炭素源としてのフルフラール系化合物をさらに添加する本発明の製造方法においては、製造されるＰＨＡの分子量は限定されないところ、例えば２０万～３００万の重量平均分子量に、ＰＨＡの分子量を制御してＰＨＡを製造することができる。フルフラール系化合物が用いられる本発明の方法については、さらに後述する。
　本発明の製造方法として、ＰＨＡの分子量を１００万～３００万の重量平均分子量に制御してＰＨＡを製造する方法は好ましい。 In the production method of the present invention, for example, PHA can be produced by controlling the molecular weight of PHA to a weight average molecular weight of 1 million to 3 million, but PHA is further added by adding a furfural compound as a carbon source. As a result, a PHA having a lower molecular weight can be obtained, and a PHA having a wider molecular weight can be produced. In the production method of the present invention in which a furfural compound as a carbon source is further added, the molecular weight of the produced PHA is not limited, for example, the molecular weight of the PHA is controlled to a weight average molecular weight of 200,000 to 3 million. Can be manufactured. The method of the present invention in which a furfural compound is used will be further described later.
As the production method of the present invention, a method of producing PHA by controlling the molecular weight of PHA to a weight average molecular weight of 1 million to 3 million is preferable.
＜炭素源＞
　本発明の製造方法における炭素源は、前記農産廃棄物に含まれていた、該農産廃棄物由来の炭素源を含む。
　本発明の製造方法における炭素源として、農産廃棄物由来の炭素源に加えて、農産廃棄物由来の炭素源以外の炭素源を含んでよい。
　かかる炭素源として、
　（ａ）グルコース及び／又はその誘導体のみ
を用いると、生成されるＰＨＡの分子量は大きくなる傾向にあり、
　上記炭素源として
　（ｂ）グルコース及びその誘導体ならびにキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上、
を用いると、生成されるＰＨＡの分子量は上記（ａ）の場合より小さくなる傾向にあり、
　上記炭素源として
　（ｃ）キシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上を用いグルコース及び／又はその誘導体を用いないことにより、生成されるＰＨＡの分子量は上記（ｂ）の場合よりさらに小さくなる傾向にある。
　このような炭素源としての糖を選択することにより、ＰＨＡの分子量を制御する方法も本発明により提供される。本発明によるＰＨＡの分子量を制御する方法は、培養における炭素源が糖を含有し、該糖を
　（ａ）グルコース及び／又はその誘導体のみ
　（ｂ）グルコース及びその誘導体ならびにキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上、又は
　（ｃ）キシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上
にして、
　製造されるポリヒドロキシアルカン酸の重量平均分子量を１００万から３００万の間で制御する、方法である。
　本発明の制御する方法として、ＰＨＡの分子量を１００万～２５０万の重量平均分子量に制御する方法は好ましい。 <Carbon source>
The carbon source in the production method of the present invention includes a carbon source derived from the agricultural waste contained in the agricultural waste.
The carbon source in the production method of the present invention may include a carbon source other than the carbon source derived from agricultural waste in addition to the carbon source derived from agricultural waste.
As such a carbon source
(A) When only glucose and / or its derivative is used, the molecular weight of PHA produced tends to be large.
As the carbon source, (b) one or more of glucose and its derivatives and xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives.
When is used, the molecular weight of the produced PHA tends to be smaller than that in the case of (a) above.
It is produced by using one or more of (c) xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives as the carbon source and not using glucose and / or its derivatives. The molecular weight of PHA tends to be even smaller than in the case of (b) above.
The present invention also provides a method of controlling the molecular weight of PHA by selecting such a sugar as a carbon source. The method for controlling the molecular weight of PHA according to the present invention is that the carbon source in the culture contains a sugar and the sugar is (a) glucose and / or a derivative thereof only (b) glucose and a derivative thereof and xylose, cellobiose, glucuronic acid, One or more from arabinose, mannose, galactose, sucrose and their derivatives, or (c) one or more from xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives And then
A method of controlling the weight average molecular weight of the produced polyhydroxyalkanoates between 1 million and 3 million.
As a control method of the present invention, a method of controlling the molecular weight of PHA to a weight average molecular weight of 1 million to 2.5 million is preferable.
　本発明の製造方法に用いられる糖の量（培養の初期濃度）は限定されないところ、約5g/L～910g/Lが例示される。糖の量として約5g/L～約500g/Lは好ましく、約10g/L～50g/Lはより好ましい。
　本発明の製造方法において、用いられる糖は複数回に分けて投入されてよい。投入される回数及びタイミングは限定されず、回数としては２回、３回、４回、５回、又は６回以上であってよい。
　また、糖の供給を連続的に行うことも可能で、その場合には糖の濃度を一定に保つようなコントロールをする方法を用いてよい。 The amount of sugar used in the production method of the present invention (initial concentration of culture) is not limited, and is exemplified by about 5 g / L to 910 g / L. The amount of sugar is preferably from about 5 g / L to about 500 g / L, more preferably from about 10 g / L to 50 g / L.
In the production method of the present invention, the sugar used may be added in a plurality of times. The number and timing of input are not limited, and the number of times may be 2, 3, 4, 5, or 6 or more.
Further, it is also possible to continuously supply sugar, and in that case, a method of controlling so as to keep the sugar concentration constant may be used.
　本発明の製造方法においては、上述のとおり、炭素源としてのフルフラール系化合物をさらに添加することにより、さらに広範囲な分子量、例えば２０万～３００万の重量平均分子量に、ＰＨＡの分子量を制御してＰＨＡを製造することができる。 In the production method of the present invention, as described above, by further adding a furfural compound as a carbon source, the molecular weight of PHA is controlled to a broader molecular weight, for example, a weight average molecular weight of 200,000 to 3,000,000. PHA can be produced.
　フルフラール系化合物は糖質を原料に生産されるアルデヒドの一種であり、糖質を酸性下で加熱することなどにより６単糖からは５-ヒドロキシメチルフルフラール（HMF）が、５単糖からはフルフラールが生成することが知られている。フルフラール系化合物のうち、「フルフラール」は２－フリルアルデヒドの通称である。
　本発明における「フルフラール系化合物」とは、フルフラール及び５－ヒドロキシメチルフルフラール、ならびにそれらの誘導体を意味する。
　理論に束縛されるものではないが、フルフラール系化合物によりさらに広範囲な分子量にＰＨＡの分子量を制御してＰＨＡを製造することができる理由は、フルフラール系化合物が、重合により生成されたポリマー鎖の移動反応を起こしやすくすることによるものである可能性がある。重合によりポリマーが生成するときに、フルフラール系化合物により比較的低い分子量のポリマー鎖の成長末端が移動を促進されることにより該ポリマー鎖の伸長は停止され、該ポリマーとは別異の次のポリマー鎖の伸長が開始されることとなるため、得られるＰＨＡの分子量はフルフラール系化合物を用いない場合より小さくなると考えることができる。 Furfural compounds are a type of aldehyde produced from sugar as a raw material, and 5-hydroxymethylfurfural (HMF) from 6 monosaccharides and furfural from 5 monosaccharides are produced by heating the sugar under acidic conditions. Is known to produce. Among the furfural compounds, "furfural" is a common name for 2-furfural aldehyde.
The "furfural compound" in the present invention means furfural, 5-hydroxymethylfurfural, and derivatives thereof.
Although not bound by theory, the reason why furfural compounds can produce PHA by controlling the molecular weight of PHA over a wider range of molecular weights is that furfural compounds transfer polymer chains produced by polymerization. It may be due to making the reaction more likely. When a polymer is produced by polymerization, the elongation of the polymer chain is stopped by promoting the movement of the growth end of the polymer chain having a relatively low molecular weight by the furfural compound, and the next polymer different from the polymer is used. Since the extension of the chain is started, it can be considered that the molecular weight of the obtained PHA is smaller than that in the case where the furfural compound is not used.
　フルフラール系化合物が用いられる本発明の製造方法又は制御方法において用いられるフルフラール系化合物の種類は限定されず、フルフラール及び５－ヒドロキシメチルフルフラール、ならびにそれらの誘導体が例示される。かかる誘導体として、５－ヒドロキシメチルフランカルボン酸、５－ヒドロキシフルフリルアルコール、フラン－２，５－ビスメタノールとそれらをエーテル化又はエステル化されてなるものが挙げられる。エーテル化としてはメトキシ化が、エステル化としてはアセチル化及びメチルエステル化が、それぞれ例示される。
　糖がグルコース及びその誘導体、ならびにキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上であり、フルフラール系化合物がフルフラール及び/又は５－ヒドロキシメチルフルフラール又はそれらの誘導体である本発明の製造方法又は本発明の制御方法は、好ましい。 The types of furfural compounds used in the production method or control method of the present invention in which furfural compounds are used are not limited, and furfural and 5-hydroxymethylfurfural, and derivatives thereof are exemplified. Examples of such derivatives include 5-hydroxymethylfurancarboxylic acid, 5-hydroxyfurfuryl alcohol, furan-2,5-bismethanol and those obtained by etherifying or esterifying them. Methoxylation is exemplified as etherification, and acetylation and methyl esterification are exemplified as esterification.
The sugar is one or more from glucose and its derivatives, as well as xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives, and the furfural compounds are furfural and / or 5-hydroxymethylfurfural. Alternatively, the production method of the present invention or the control method of the present invention, which is a derivative thereof, is preferable.
　本発明の製造方法において用いられるフルフラール系化合物の量は限定されず、培養の初期濃度として約０．０１～約３．０g/Lが例示される。フルフラール系化合物の初期濃度として、０．０１ｇ/Ｌ～１ｇ/Ｌは好ましい。
　用いられるフルフラール系化合物の量の糖の量に対する割合は限定されず、約０．００２５～約０．６が例示される。用いられるフルフラール系化合物の量の糖の量に対する割合が、０．０１～０．２である本発明の製造方法又は本発明の制御方法は好ましい。 The amount of the furfural compound used in the production method of the present invention is not limited, and the initial concentration of the culture is exemplified by about 0.01 to about 3.0 g / L. The initial concentration of the furfural compound is preferably 0.01 g / L to 1 g / L.
The ratio of the amount of furfural compound used to the amount of sugar is not limited, and is exemplified by about 0.0025 to about 0.6. The production method of the present invention or the control method of the present invention in which the ratio of the amount of the furfural compound used to the amount of sugar is 0.01 to 0.2 is preferable.
＜微生物＞
　本発明の製造方法において用いられる微生物は限定されないところ、該微生物として高度好塩菌は好適である。ＰＨＡの生産能が高いばかりでなく、生産されたＰＨＡの回収が容易だからである。
　高度好塩菌のうちHaloferax属、Halalkalicoccus属、Haloarchaeobius属、Haloarcula属、Halobacterium属、Halobaculum属、Halococcus属、Halogranum属、Halomarina属、Halorubrum属、Haloterrigena属、Natrialba属、Natronobacterium属は好ましく、Haloferax mediterraneiはとくに好ましい。 <Microorganisms>
Although the microorganism used in the production method of the present invention is not limited, highly halophilic bacteria are suitable as the microorganism. This is because not only the production capacity of PHA is high, but also the produced PHA can be easily recovered.
Advanced Haloferax genus of halophilic, Halalkalicoccus genus, Haloarchaeobius genus, Haloarcula genus, Halobacterium sp, Halobaculum genus, Halococcus genus, Halogranum genus, Halomarina genus, Halorubrum genus, Haloterrigena genus, Natrialba genus, Natronobacterium genus preferably, Haloferax mediterranei is Especially preferable.
＜その他の培養条件＞
　本発明のＰＨＡの製造方法における微生物の培養に関し、上記炭素源及び微生物以外の条件については限定されず、本技術分野において通常用いられる条件や公知の条件を用いてよい。
　また本発明の製造方法における培養に用いられる培地としては、固体であっても液体であってもゲル状であっても構わないが、生産スピードの観点から液体培地であることは好ましい。
　また本発明の製造方法における培養において、総塩濃度としては例えば100g/L～300g/Lが挙げられる。総塩濃度が150g/L～250g/Lであることは好ましい。
　用いられる培地としては、例えばNH4Cl、KH2PO4、FeCl3、NaCl、MgCl2、MgSO4、CaCl2、KCl、NaHCO3及びNaBr等の無機塩類を含有するものが挙げられる。これらの無機塩類の種類と量は限定されず、例えばNH4Cl、KH2PO4、FeCl3、KCl、NaHCO3、NaBr及びNaClについては、それぞれ以下の量(g/L)が例示される：
　NH4Cl：１．５～２．５、KH2PO4：０．００５～０．３５、FeCl3 ：０．００１～０．０１、KCl：３～７、NaHCO3 ：０．１～１、NaBr：０．３～０．７、 NaCl： １００～３００。
　本発明のＰＨＡの製造方法において用いられる培地として、公知の培地における糖分を農産廃棄物に含有される糖分により代替して改変したものは好ましい。かかる公知の培地は限定されないところ、ＢＳＭ培地及びＮＢＲＣ（独立行政法人製品評価技術基盤機構バイオテクノロジーセンター）がウェブサイトにおいて公開している培地一覧（https://www.nite.go.jp/nbrc/cultures/cultures/culture-list.html）における培地（No.1380の培地等）が例示される。No.1380の培地組成を上記培地一覧から引用して示す：
Figure JPOXMLDOC01-appb-I000001
　 <Other culture conditions>
Regarding the culture of microorganisms in the method for producing PHA of the present invention, the conditions other than the above carbon source and microorganisms are not limited, and conditions usually used in the present technology and known conditions may be used.
The medium used for culturing in the production method of the present invention may be solid, liquid or gel, but a liquid medium is preferable from the viewpoint of production speed.
Further, in the culture in the production method of the present invention, the total salt concentration is, for example, 100 g / L to 300 g / L. It is preferable that the total salt concentration is 150 g / L to 250 g / L.
Examples of the medium used include those containing inorganic salts such as NH 4 Cl, KH 2 PO 4 , FeCl 3 , NaCl, MgCl 2 , DDL 4 , CaCl 2 , KCl, LVDS 3 and NaBr. The types and amounts of these inorganic salts are not limited, and for example, for NH 4 Cl, KH 2 PO 4 , FeCl 3 , KCl, LVDS 3 , NaCl and NaCl, the following amounts (g / L) are exemplified. :
NH 4 Cl: 1.5 to 2.5, KH 2 PO 4 : 0.005 to 0.35, FeCl 3 : 0.001 to 0.01, KCl: 3 to 7, LVDS 3 : 0.1 to 1 , NaBr: 0.3-0.7, NaCl: 100-300.
As the medium used in the method for producing PHA of the present invention, a medium obtained by substituting and modifying the sugar content in a known medium with the sugar content contained in agricultural waste is preferable. Such known media are not limited, but a list of media published on the website of BSM medium and NBRC (National Institute of Technology and Evaluation Biotechnology Center) (https://www.nite.go.jp/nbrc) The medium (medium of No. 1380, etc.) in /cultures/cultures/culture-list.html) is exemplified. The medium composition of No. 1380 is shown by quoting from the above medium list:
Figure JPOXMLDOC01-appb-I000001
　本発明の製造方法における微生物の培養に用いられる培地に含有される栄養源についてさらに説明するに、炭素源としては、上記いずれかの糖が少なくとも用いられることは上述のとおりであるところ、微生物を培養する際の上記糖とは別の炭素源として、アミノ酸類、アルコール類、油脂類、及び／又は脂肪酸類等の通常微生物培養に用いられる炭素源を用いてよい。具体的には以下のものが例示される：
　アミノ酸類として、グリシン、アラニン、フェニルアラニン、リシン、ロイシン；
　アルコール類として、メタノール、エタノール、１－プロパノール、２－プロパノール、グリセロール；
　油脂類として、パーム油、パーム核油、コーン油、やし油、オリーブ油、大豆油、及び菜種油；
　脂肪酸類として、酢酸、プロピオン酸、酪酸、吉草酸、カプロン酸、及びクロトン酸とそのナトリウム塩やアンモニウム塩などの脂肪酸塩。脂肪酸類が用いられる場合、該脂肪酸類の量は少量であることが好ましい。 To further explain the nutrient source contained in the medium used for culturing the microorganism in the production method of the present invention, as described above, at least one of the above sugars is used as the carbon source. As a carbon source different from the above-mentioned sugar at the time of culturing, a carbon source usually used for culturing microorganisms such as amino acids, alcohols, fats and oils, and / or fatty acids may be used. Specifically, the following are exemplified:
As amino acids, glycine, alanine, phenylalanine, lysine, leucine;
As alcohols, methanol, ethanol, 1-propanol, 2-propanol, glycerol;
Oils and fats include palm oil, palm kernel oil, corn oil, palm oil, olive oil, soybean oil, and rapeseed oil;
As fatty acids, fatty acid salts such as acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and crotonic acid and its sodium salt and ammonium salt. When fatty acids are used, the amount of the fatty acids is preferably small.
　本発明の製造方法における微生物の培養に用いられる培地には、窒素源や無機塩類等を含んでよい。
　窒素源としては、例えばアンモニア、塩化アンモニウム、硫酸アンモニウム、リン酸アンモニウム等のアンモニウム塩のほか、ペプトン、肉エキス、酵母エキス等が挙げられる。
　無機塩類としては、例えばリン酸２水素カリウム、リン酸水素２ナトリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム等が挙げられる。 The medium used for culturing microorganisms in the production method of the present invention may contain a nitrogen source, inorganic salts and the like.
Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as well as peptone, meat extract, yeast extract, and the like.
Examples of the inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like.
　本発明の製造方法において微生物を培養する際の培養の温度は、該微生物が生育可能な温度であれば限定されないところ、例えば約１０℃～約６５℃が挙げられ、２０℃～５０℃は好ましく、３５℃～４５℃はより好ましい。
　本発明の製造方法において、炭素源、無機塩類、有機栄養源を添加するタイミングに関しては限定されないところ、一般的に用いられている回分培養、流加培養、連続培養等の方法を目的に応じて適宜選択して用いることができる。
本発明の製造方法における微生物の培養時間としては、回分培養では２４時間～１６８時間、流加培養では７２時間～１４日間、連続培養では１４日間を超える長期間が例示されるが、これらの時間に限定されない。 The temperature of culturing when culturing a microorganism in the production method of the present invention is not limited as long as the microorganism can grow, and examples thereof include about 10 ° C. to about 65 ° C., preferably 20 ° C. to 50 ° C. , 35 ° C to 45 ° C are more preferable.
In the production method of the present invention, the timing of adding the carbon source, the inorganic salt, and the organic nutrient source is not limited, but generally used methods such as batch culture, fed-batch culture, and continuous culture can be used depending on the purpose. It can be appropriately selected and used.
Examples of the culture time of microorganisms in the production method of the present invention include a long period of 24 hours to 168 hours for batch culture, 72 hours to 14 days for fed-batch culture, and more than 14 days for continuous culture. Not limited to.
　本発明の製造方法において、培地における溶存酸素量を大きくすることは好ましく、培地における溶存酸素量を飽和量に保つことはより好ましい。
　本発明の製造方法において培地のｐＨは限定されないところ、培地の初期のｐＨを約６．５～約７．５とすることは好ましい。培地の初期のｐＨを約６．５～約７．５とし、培養中の培地のｐＨを約６．５～約７．５に維持することはより好ましい。これらのｐＨを約７．０～約７．４にすることはさらに好ましい。 In the production method of the present invention, it is preferable to increase the amount of dissolved oxygen in the medium, and it is more preferable to keep the amount of dissolved oxygen in the medium at a saturated amount.
Although the pH of the medium is not limited in the production method of the present invention, it is preferable that the initial pH of the medium is about 6.5 to about 7.5. It is more preferable that the initial pH of the medium is about 6.5 to about 7.5 and the pH of the medium being cultured is maintained at about 6.5 to about 7.5. It is more preferable to set these pH to about 7.0 to about 7.4.
　本発明の制御方法においても、本発明の製造方法に用いられるその他の培養条件を用いることができる。 In the control method of the present invention, other culture conditions used in the production method of the present invention can also be used.
＜本発明の製造方法により製造されるＰＨＡ＞
　本発明の製造方法における生成物であるＰＨＡの種類は限定されないところ、ＰＨＢＶ、ＰＨＢ（ポリヒドロキシブチレート）、ポリ(ヒドロキシブチレート／ヒドロキシヘキサノエート、及びポリ（３ヒドロキシブチレート／４－ヒドロキシブチレート）が例示される。これらのうち、ＰＨＢＶ及び／又はＰＨＢが好適な生成物である。 <PHA produced by the production method of the present invention>
The type of PHA product in the production method of the present invention is not limited, but is PHBV, PHB (polyhydroxybutyrate), poly (hydroxybutyrate / hydroxyhexanoate, and poly (3 hydroxybutyrate / 4-hydroxy). Butyrate) is exemplified. Of these, PHBV and / or PHB are suitable products.
　本発明の製造方法により製造されたＰＨＡは、有機溶媒や界面活性剤、低濃度塩化ナトリウム水溶液又は水を用いる遠心分離や濾過・沈殿による公知の方法により菌体から回収することができる。
　本発明の製造方法において微生物が産生するＰＨＡの生産量と３HV分率は、ＧＣ－ＭＳ法等の公知の方法を用いて測定してよい。本発明の製造方法により得られるＰＨＡがＰＨＢＶである場合、３ＨＶ分率もＧＣ－ＭＳ法等の公知の方法を用いて決定してよい。
　グルコース又はその誘導体やその他の糖質、フルフラール系化合物の濃度の測定には、市販の濃度測定キットによる測定や、ＨＰＬＣ法などの公知の方法を用いてよい。
　本発明の製造方法において微生物が培養された際の微生物菌体の生産量を特定することが必要な場合には、微生物菌体の生産量は吸光度法や乾燥菌体重量測定法などの公知の方法で測定してよい。 The PHA produced by the production method of the present invention can be recovered from the cells by a known method by centrifugation, filtration or precipitation using an organic solvent, a surfactant, a low-concentration sodium chloride aqueous solution or water.
The production amount and 3HV fraction of PHA produced by the microorganism in the production method of the present invention may be measured by using a known method such as the GC-MS method. When the PHA obtained by the production method of the present invention is PHBV, the 3HV fraction may also be determined using a known method such as the GC-MS method.
For the measurement of the concentration of glucose or its derivative, other sugars, and furfural compounds, a measurement using a commercially available concentration measurement kit or a known method such as an HPLC method may be used.
When it is necessary to specify the production amount of the microbial cells when the microorganisms are cultured in the production method of the present invention, the production amount of the microbial cells is known such as an absorbance method or a dry cell weight measurement method. It may be measured by the method.
　また本発明の製造方法により得られるＰＨＡの分子量の測定も公知の方法により行ってよく、例えばポリスチレン標準を用いたゲルろ過クロマトグラフィーにより行うことができる。本明細書におけるＰＨＡの分子量には、他の記載がない限りポリスチレン標準を用いたゲルろ過クロマトグラフィーにより測定された数値を用いたことは前述のとおりである。
　本発明の方法で製造されるＰＨＡの重量平均分子量は、フルフラール系化合物を用いない場合には約１００万から約３００万であり、フルフラール系化合物を用いる場合には約２０万から約３００万である。それぞれの範囲において得られるＰＨＡの分子量を、本発明の製造方法においては制御又は調節することができる。 The molecular weight of PHA obtained by the production method of the present invention may also be measured by a known method, for example, by gel filtration chromatography using a polystyrene standard. As described above, for the molecular weight of PHA in the present specification, the numerical value measured by gel filtration chromatography using a polystyrene standard is used unless otherwise specified.
The weight average molecular weight of PHA produced by the method of the present invention is about 1 million to about 3 million when the furfural compound is not used, and about 200,000 to about 3 million when the furfural compound is used. be. The molecular weight of PHA obtained in each range can be controlled or adjusted in the production method of the present invention.
２．ＰＨＢＶ
　本発明の製造方法により、３－ヒドロキシブタン酸（３ＨＢ）と３－ヒドロキシバレリル酸（３ＨＶ）とがランダムに共重合した共重合体（以下「ＰＨＢＶ」ということがある）も提供される。
　本発明のＰＨＢＶは、以下で示される構造単位を、任意の組み合わせとして含む（下式においてｍ及びｎは整数を表す）：
Figure JPOXMLDOC01-appb-I000002

 
　本発明のＰＨＢＶの構造、モノマーの組成及び分子量は限定されない。本発明のＰＨＢＶは線状であり、少なくとも実質的に直鎖状である本発明のＰＨＢＶは好ましい。
　本発明のＰＨＢＶは上記のとおり３ＨＢと３ＨＶとがランダムに共重合した共重合体であるところ、３ＨＢと３ＨＶとの交互共重合又はブロック共重合により生成される共重合体も包含される。 2. PHBV
According to the production method of the present invention, a copolymer in which 3-hydroxybutanoic acid (3HB) and 3-hydroxyvalerylic acid (3HV) are randomly copolymerized (hereinafter, may be referred to as "PHBV") is also provided.
The PHBV of the present invention includes the structural units shown below as any combination (m and n represent integers in the following equation):
Figure JPOXMLDOC01-appb-I000002


The structure, monomer composition and molecular weight of PHBV of the present invention are not limited. The PHBV of the present invention is linear, and the PHBV of the present invention, which is at least substantially linear, is preferred.
The PHBV of the present invention is a copolymer in which 3HB and 3HV are randomly copolymerized as described above, and a copolymer produced by alternating copolymerization of 3HB and 3HV or block copolymerization is also included.
　本発明のＰＨＢＶにおいて、ＰＨＢＶを構成する３－ヒドロキシブタン酸と３－ヒドロキシバレリル酸とのモル比は限定されない。３ＨＶ分率（共重合体を構成する３－ヒドロキシブタン酸のモル数と３－ヒドロキシバレリル酸のモル数の総和に対する３－ヒドロキシバレリル酸のモル数の割合）が、約５．０％～約４０．０％である本発明のＰＨＢＶは好ましく、約５．０％～約２８．０％である本発明のＰＨＢＶはより好ましく、約７．０％～１８．０％である本発明のＰＨＢＶは一層より好ましく、約９．０％～約１５．０％である本発明のＰＨＢＶは一層さらにより好ましい。 In the PHBV of the present invention, the molar ratio of 3-hydroxybutanoic acid and 3-hydroxyvalerylic acid constituting PHBV is not limited. The 3HV fraction (the ratio of the number of moles of 3-hydroxyvalerylic acid to the total number of moles of 3-hydroxybutanoic acid and 3-hydroxyvalerylic acid constituting the copolymer) is about 5.0%. The PHBV of the present invention, which is about 40.0%, is preferable, and the PHBV of the present invention, which is about 5.0% to about 28.0%, is more preferable, and the PHBV of the present invention is about 7.0% to 18.0%. The PHBV of the present invention is even more preferable, and the PHBV of the present invention, which is about 9.0% to about 15.0%, is even more preferable.
　本発明のＰＨＢＶを製造するための方法は、上記工程（ａ）及び（ｂ）を含む方法であれば限定されず、重量平均分子量が約３００万に達するＰＨＢＶを製造することができる方法であってよい。
　かかる方法として、微生物を培養し、培養産物として本発明のＰＨＢＶを得る方法が例示されるところ、前記培養における微生物として高度好塩菌を用い、炭素源としてグルコース及び／又はその誘導体を用いる方法は好ましく、グルコースを用いる方法はとくに好ましい。これらの方法によれば、重量平均分子量が約３００万に達するＰＨＢＶを効率的に製造することができ、また製造されたＰＨＢＶを簡便に回収することができるからである。 The method for producing PHBV of the present invention is not limited as long as it is a method including the above steps (a) and (b), and is a method capable of producing PHBV having a weight average molecular weight of about 3 million. It's okay.
As such a method, a method of culturing a microorganism to obtain PHBV of the present invention as a culture product is exemplified. A method of using a highly halophilic bacterium as a microorganism in the culture and glucose and / or a derivative thereof as a carbon source is used. Preferably, the method using glucose is particularly preferred. This is because according to these methods, PHBV having a weight average molecular weight of about 3 million can be efficiently produced, and the produced PHBV can be easily recovered.
　本発明の上記ＰＨＡの製造方法を用いて本発明のＰＨＢＶを製造する場合、炭素源としての糖類は
　（ａ）グルコース及び／又はその誘導体のみ、又は
　（ｂ）グルコース及びその誘導体ならびにキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上、
であってよい。
　本発明のＰＨＢＶは、酵母エキスを用い、酵母エキスに含有されるアミノ酸などの炭素源以外の炭素源を添加しなくても製造される。酵母エキスに含有されるアミノ酸などの炭素源以外の炭素源を用いることにより、より小さいコストで本発明のＰＨＢＶを製造することができる。 When the PHBV of the present invention is produced using the above-mentioned method for producing PHA of the present invention, the saccharides as carbon sources are (a) glucose and / or its derivative only, or (b) glucose and its derivative and xylose, cellobiose, etc. One or more from glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives,
May be.
The PHBV of the present invention is produced by using a yeast extract without adding a carbon source other than the carbon source such as amino acids contained in the yeast extract. By using a carbon source other than the carbon source such as amino acids contained in yeast extract, the PHBV of the present invention can be produced at a lower cost.
（本発明の例）
　本発明を実施例及び参考例によりさらに具体的に説明する。本発明は、いかなる意味においてもかかる実施例に限定されるものではない。
　すべての実施例又は参考例において他に記載がない限りNBRCより提供の菌株 Haloferax mediterranei NBRC 14739を用いて実験を行った。
　すべての実施例又は参考例において他に記載がない限り、上記菌を各例の項において示される方法・条件にて培養後、培養により得られたＰＨＡ（ＰＨＢＶ）の分子量をポリスチレン標準を用いたGPC(ゲルろ過クロマトグラフィー)により測定し、分子量として示した。
　GPC測定には、東ソー株式会社製EcoSEC　HLC-8320GPCを使用した。ガードカラムはTSKgel guardcolumn SuperHZ-Hを、カラムはTSKgel Super HZM-H２本を直列につないで用いた。移動相にはクロロホルム（０．６mL/min）を用い、カラム温度は４０℃とした。サンプル濃度は約0.5 mg/mLとし、サンプル注入量は10μLとした。検量線の作成には、ポリスチレン標準を用いた。 
　共重合体の3-ヒドロキシバレリル酸(３ＨＶ)分率とPHBV生産量の測定はガスクロマトグラフィー-質量分析(GC-MS法)を用いて以下のように行った（機器名：Agilent 6890 / 5973 GCMS System ）。すなわち、乾燥菌体の約2mg～25mgに2mlの硫酸－メタノール混液(15:85)と2mlのクロロホルムを添加して密栓し、100℃で140分間加熱することでポリエステル分解物のメチルエステルを得て、これに水１mLを加えて攪拌後、クロロホルム層をGC-MS法により分析して行った。
　これらのＰＨＡの生産量、分子量、３HV分率の測定方法は、ＰＨＡの生産量、分子量、３HV分率を特定するための手法として本技術分野において通常用いられるものである。また、これらの方法により特定されたＰＨＡの生産量、分子量及び３HV分率の数値は公知文献に記載されているＰＨＡの生産量、分子量及び３HV分率の数値と、その大小をそれぞれ正確に比較することができるものである。
　乾燥菌体重量は、凍結乾燥法などの公知の方法により測定することができる。乾燥菌体に含まれる無機塩を定量したいときは、高温で無機塩以外の有機物をすべて熱分解する方法を用いてもよい。 (Example of the present invention)
The present invention will be described in more detail with reference to Examples and Reference Examples. The present invention is not limited to such examples in any sense.
Experiments were performed using the strain Haloferax mediterranei NBRC 14739 provided by NBRC unless otherwise stated in all Examples or References.
Unless otherwise stated in all Examples or Reference Examples, after culturing the above bacteria under the methods and conditions shown in each example, the molecular weight of PHA (PHBV) obtained by culturing was measured using a polystyrene standard. It was measured by GPC (gel permeation chromatography) and shown as a molecular weight.
An EcoSEC HLC-8320 GPC manufactured by Tosoh Corporation was used for GPC measurement. The guard column used was TSKgel guardcolumn Super HZ-H, and the column used was two TSK gel Super HZM-H connected in series. Chloroform (0.6 mL / min) was used as the mobile phase, and the column temperature was 40 ° C. The sample concentration was about 0.5 mg / mL, and the sample injection volume was 10 μL. A polystyrene standard was used to prepare the calibration curve.
The 3-hydroxyvalerylic acid (3HV) fraction and PHBV production of the copolymer were measured using gas chromatography-mass spectrometry (GC-MS method) as follows (device name: Agilent 6890 /). 5973 GCMS System). That is, 2 ml of sulfuric acid-methanol mixed solution (15:85) and 2 ml of chloroform were added to about 2 mg to 25 mg of dried cells, the mixture was sealed, and heated at 100 ° C. for 140 minutes to obtain the methyl ester of the polyester decomposition product. Then, 1 mL of water was added thereto, and the mixture was stirred, and then the chloroform layer was analyzed by the GC-MS method.
These methods for measuring the production amount, molecular weight, and 3HV fraction of PHA are usually used in the present technical field as a method for specifying the production amount, molecular weight, and 3HV fraction of PHA. In addition, the values of PHA production, molecular weight, and 3HV fraction specified by these methods are accurately compared with the values of PHA production, molecular weight, and 3HV fraction described in publicly known documents. Is something that can be done.
The weight of dried cells can be measured by a known method such as a freeze-drying method. When it is desired to quantify the inorganic salts contained in the dried cells, a method of thermally decomposing all organic substances other than the inorganic salts at a high temperature may be used.
実施例
●（材料と方法）　Haloferax mediterranei ＮＢＲＣ１４７３９株について、表２に示す、ＢＭＳ培地における糖（炭素源）を、米ぬかを用いた材料（「米ぬか１」、「米ぬか２」、「米ぬか３」、又は「米ぬか４」）由来の糖により代替するなどして構成した培地を含む培地群を作製した。これらの培地を用い、300 mL三角フラスコにおいて、培養液総量を50 mLにして培養を７２時間、３７℃において行った。米ぬかの成分は、下記の通りであった。
Figure JPOXMLDOC01-appb-T000003
 
　米ぬかを用いた材料の添加量は１０．２g/Lとして、ＢＭＳ培地におけるグルコース換算による糖濃度である５g/Lに相当する量とした。各試験区の構成は下表に示すとおりであった：
Figure JPOXMLDOC01-appb-T000004
 Example ● (Material and method) For the Haloferax mediterranei NBRC14739 strain, the sugar (carbon source) in the BMS medium shown in Table 2 was used as the material using rice bran (“rice bran 1”, “rice bran 2”, “rice bran 3”, Alternatively, a medium group containing a medium composed by substituting with sugar derived from "rice bran 4") was prepared. Using these media, in a 300 mL Erlenmeyer flask, the total volume of the culture solution was 50 mL, and the culture was carried out for 72 hours at 37 ° C. The ingredients of rice bran were as follows.
Figure JPOXMLDOC01-appb-T000003

The amount of the material added using rice bran was 10.2 g / L, which corresponds to the sugar concentration of 5 g / L in terms of glucose in the BMS medium. The composition of each test plot was as shown in the table below:
Figure JPOXMLDOC01-appb-T000004
　ＢＭＳ培地の組成は以下に示すとおりである：
Figure JPOXMLDOC01-appb-T000005
  The composition of the BMS medium is as shown below:
Figure JPOXMLDOC01-appb-T000005
●結果
　下表に示すとおりであった：
 
Figure JPOXMLDOC01-appb-T000006
 
　化学的及び生物的な前処理を行っていない米ぬかを炭素源として用いた区No.１において、ＰＨＢＶが生産された。区No.１におけるＰＨＢＶの生産量は、従来技術に相当する区No.２及び３におけるにおける生産量を上回った。
　したがって、前処理を行っていない米ぬかを炭素源として用いることにより、ＰＨＢＶを効率的に生産できることが明らかになった。 ● Results As shown in the table below:

Figure JPOXMLDOC01-appb-T000006

PHBV was produced in plot No. 1 using rice bran that had not been chemically and biologically pretreated as a carbon source. The production volume of PHBV in the section No. 1 exceeded the production volume in the sections No. 2 and 3 corresponding to the prior art.
Therefore, it was clarified that PHBV can be efficiently produced by using untreated rice bran as a carbon source.
Ｊ．参考例１５＜培地の評価＞
●（材料と方法）　Haloferax mediterraneiについて、表１０に示す高度好塩菌用のＮＢＲＣ指定培地+5g/Lのグルコースを用いて、300 mL三角フラスコに、培養液総量を50 mLにして培養を行った。指定培地中にグルコースが含まれている場合には、もともと含まれているグルコース以外にさらに5 g/Lのグルコースを追加した。
　各指定培地の成分は、ＮＢＲＣがウェブサイトにおいて公開している培地一覧（https://www.nite.go.jp/nbrc/cultures/cultures/culture-list.html）を引用して以下に示す。
 
Figure JPOXMLDOC01-appb-I000007

Figure JPOXMLDOC01-appb-I000008

 
Figure JPOXMLDOC01-appb-I000009

Figure JPOXMLDOC01-appb-I000010

Figure JPOXMLDOC01-appb-I000011

Figure JPOXMLDOC01-appb-I000012
Figure JPOXMLDOC01-appb-I000013

Figure JPOXMLDOC01-appb-I000014

 
●(結果)下表に示すとおりであった。
　培地を変更することにより、重量平均分子量の範囲が１３０万～５４０万であるＰＨＡを生産することが可能であった。
　またＮＢＲＣNo.1380の培地では、PHA生産量が高いうえに、3HV分率、分子量が高くこれまでにないタイプのPHAを生産可能であることが明らかになった。
Figure JPOXMLDOC01-appb-T000015
  J. Reference Example 15 <Evaluation of medium>
● (Materials and methods) Haloferax mediterranei was cultured in a 300 mL Erlenmeyer flask with a total volume of culture solution of 50 mL using the NBRC designated medium for highly halophilic bacteria + 5 g / L glucose shown in Table 10. rice field. When glucose was contained in the designated medium, 5 g / L of glucose was added in addition to the glucose originally contained.
The components of each designated medium are shown below by quoting the medium list (https://www.nite.go.jp/nbrc/cultures/cultures/culture-list.html) published by NBRC on its website. ..

Figure JPOXMLDOC01-appb-I000007

Figure JPOXMLDOC01-appb-I000008


Figure JPOXMLDOC01-appb-I000009

Figure JPOXMLDOC01-appb-I000010

Figure JPOXMLDOC01-appb-I000011

Figure JPOXMLDOC01-appb-I000012
Figure JPOXMLDOC01-appb-I000013

Figure JPOXMLDOC01-appb-I000014


● (Result) It was as shown in the table below.
By changing the medium, it was possible to produce PHA with a weight average molecular weight range of 1.3 million to 5.4 million.
In addition, it was clarified that the medium of NBRC No. 1380 has a high PHA production amount, a high 3HV fraction, and a high molecular weight, and can produce an unprecedented type of PHA.
Figure JPOXMLDOC01-appb-T000015
Ｋ．参考例１６～２５　＜フルフラール系化合物の評価＞
　フルフラール系化合物をさらに用いた以外は実施例１～１１と同様の方法で、初期グルコース濃度を10 g/L、フルフラール濃度、HMF（５-ヒドロキシメチルフルフラール）濃度をそれぞれ0.06g/Lから1.6g/Lまで変化させ、Haloferax mediterranei NBRC14739のフラスコ培養を行った。より具体的には以下のとおりである。
●（材料と方法）300 mL三角フラスコに、培養液総量を50 mLにして以下の各成分を加えて72 h、37 ℃、200 rpmで振とう培養を行った。初期pHは 7.2に合わせた。
　培地中の成分(g/L)：NH4Cl 2、KH2PO4  0.0375、FeCl3 0.005、NaCl 194、
　　　　　　　　　　MgCl2 16、MgSO4 24、CaCl2 1、KCl 5、NaHCO3 0.2、
　　　　　　　　　　NaBr 0.5、酵母エキス 5
　上記成分からなる培地に、炭素源として、下表に示す糖質とフルフラール系化合物を所定量入れて、72 hの培養を行った。
●（結果）各条件で生産されたＰＨＢＶの３ＨＶ分率と重量平均分子量、多分散度、乾燥菌体重量、ＰＨＢＶ生産量を下表に示す。
Figure JPOXMLDOC01-appb-T000016
 
　フルフラールの濃度及びHMF濃度を変化させることで、様々な分子量のＰＨＢＶを製造し、分子量を制御できること明らかになった。フルフラール又はHMFといったフルフラール系化合物による製造されるＰＨＢＶの分子量の制御は、グルコース以外の糖が用いられた場合においてもなされると考えられる。 K. Reference Examples 16 to 25 <Evaluation of Furfural Compounds>
The initial glucose concentration was 10 g / L, the furfural concentration, and the HMF (5-hydroxymethylfurfural) concentration were 0.06 g / L to 1.6 g, respectively, in the same manner as in Examples 1 to 11 except that the furfural compound was further used. The concentration was changed to / L, and a flask of Haloferax mediterranei NBRC14739 was cultured. More specifically, it is as follows.
● (Materials and methods) In a 300 mL Erlenmeyer flask, the total volume of the culture solution was 50 mL, the following components were added, and the culture was shaken at 72 h, 37 ° C, and 200 rpm. The initial pH was adjusted to 7.2.
Ingredients in medium (g / L): NH 4 Cl 2, KH 2 PO 4 0.0375, FeCl 3 0.005, NaCl 194,
MgCl 2 16, DDL 4 24, CaCl 2 1, KCl 5, LVDS 3 0.2,
NaBr 0.5, yeast extract 5
A predetermined amount of the sugar and furfural compound shown in the table below were added as a carbon source to a medium composed of the above components, and the culture was carried out for 72 h.
● (Results) The table below shows the 3HV fraction, weight average molecular weight, polydispersity, dry cell weight, and PHBV production of PHBV produced under each condition.
Figure JPOXMLDOC01-appb-T000016

It was clarified that PHBV having various molecular weights can be produced and the molecular weight can be controlled by changing the concentration of furfural and the concentration of HMF. It is considered that the molecular weight of PHBV produced by a furfural compound such as furfural or HMF is controlled even when a sugar other than glucose is used.
　本発明によれば米ぬかなどの農産廃棄物といった安価な炭素源を用いて、生物的、化学的な前処理を行わず、ＰＨＡを低コストで製造する方法が提供される。したがって本発明はＰＨＡ製造業及びその関連産業の発展に寄与するところ大である。 According to the present invention, there is provided a method for producing PHA at low cost by using an inexpensive carbon source such as agricultural waste such as rice bran without performing biological or chemical pretreatment. Therefore, the present invention greatly contributes to the development of the PHA manufacturing industry and related industries.

Claims (9)

  1. 　以下の工程を含む、ポリヒドロキシアルカン酸の製造方法：
    　（ａ）化学的及び生物的な前処理がなされていない農産廃棄物を含む培地を用いて微生物を培養する工程；及び
    　（ｂ）前記培養における培養産物としてポリヒドロキシアルカン酸を得る工程。     Method for producing polyhydroxyalkanoic acid, which comprises the following steps:
    (A) A step of culturing a microorganism using a medium containing agricultural waste that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture.
  2. 　農産廃棄物が米ぬかである、請求項１に記載の製造方法。 The manufacturing method according to claim 1, wherein the agricultural waste is rice bran.
  3. 　微生物が高度好塩菌である請求項１又は２に記載の製造方法。 The production method according to claim 1 or 2, wherein the microorganism is a highly halophilic bacterium.
  4. 　ポリヒドロキシアルカン酸が、３－ヒドロキシブタン酸と３－ヒドロキシバレリル酸とがランダムに共重合した共重合体である請求項１～３のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 3, wherein the polyhydroxyalkanoic acid is a copolymer in which 3-hydroxybutanoic acid and 3-hydroxyvalerylic acid are randomly copolymerized.
  5. 　共重合体を構成する３－ヒドロキシブタン酸のモル数と３－ヒドロキシバレリル酸のモル数の総和に対する３－ヒドロキシバレリル酸のモル数の割合（３ＨＶ分率）が５．０％～４０．０％である請求項４に記載の方法。 The ratio of the number of moles of 3-hydroxyvaleryl acid (3HV fraction) to the total number of moles of 3-hydroxybutanoic acid and 3-hydroxyvaleryl acid constituting the copolymer is 5.0% to 40. The method according to claim 4, which is 0.0%.
  6. 　炭素源として農産廃棄物由来の炭素源以外の炭素源を含む、請求項１～５のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 5, wherein the carbon source includes a carbon source other than the carbon source derived from agricultural waste.
  7. 　農産廃棄物由来の炭素源以外の炭素源として糖を含有し、該糖を
    　（ａ）グルコース及び／又はその誘導体のみ
    　（ｂ）グルコース及びその誘導体ならびにキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上、又は
    　（ｃ）キシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上
    にして、
    　重量平均分子量が１００万以上であるポリヒドロキシアルカン酸を得る、請求項６に記載の製造方法。     It contains sugar as a carbon source other than the carbon source derived from agricultural waste, and the sugar is (a) glucose and / or its derivative only (b) glucose and its derivative and xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose. , One or more from sucrose and its derivatives, or (c) one or more from xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and their derivatives.
    The production method according to claim 6, wherein a polyhydroxyalkanoic acid having a weight average molecular weight of 1 million or more is obtained.
  8. 　（ｂ）又は（ｃ）におけるキシロース、セロビオース、グルクロン酸、アラビノース、マンノース、ガラクトース、スクロース及びそれらの誘導体からの１種もしくは２種以上がキシロース及び／又はセロビオースである請求項７に記載の製造方法。 The production method according to claim 7, wherein one or more of xylose, cellobiose, glucuronic acid, arabinose, mannose, galactose, sucrose and derivatives thereof in (b) or (c) are xylose and / or cellobiose. ..
  9. 　以下の工程を含む、ポリヒドロキシアルカン酸の製造方法における、米ぬかの使用：
    　（ａ）化学的及び生物的な前処理がなされていない米ぬかを含む培地を用いて微生物を培養する工程；及び
    　（ｂ）前記培養における培養産物としてポリヒドロキシアルカン酸を得る工程。     Use of rice bran in a method for producing polyhydroxyalkanoates, which comprises the following steps:
    (A) A step of culturing a microorganism using a medium containing rice bran that has not been chemically and biologically pretreated; and (b) a step of obtaining polyhydroxyalkanoate as a culture product in the culture.
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