Method for producing polyhydroxyalkanoic acid and composition for producing same

Abstract

To provide a method and a composition for producing polyhydroxyalkanoic acid.SOLUTION: A method for producing polyhydroxyalkanoic acid includes causing Ideonella sakaiensis to act on a sample containing a carbon source.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for producing polyhydroxyalkanoic acid, a composition for producing polyhydroxyalkanoic acid, and the like. [Background technology]

[0002] Most plastics are manufactured using petroleum as a raw material. Furthermore, most used plastics are disposed of by landfill or incineration, or released into the environment, and the low rate of recycling is a problem.

[0003] In recent years, therefore, there has been active development of biodegradable plastics such as polyhydroxyalkanoates (PHAs) that can be decomposed by microorganisms.

[0004] On the other hand, Ideonella sakaiensis, which decomposes polyethylene terephthalate (PET), a type of plastic, has been discovered (Non-Patent Document 1), and its applications are expected. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2008-199957 A [Non-patent literature]

[0006] [Non-Patent Document 1] Yoshida et al., Science 351 (6278), 1196-1199, 2016 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present disclosure is to provide a method for producing polyhydroxyalkanoic acid. [Means for solving the problem]

[0008] The present inventors discovered that Ideonella sakaiensis can degrade PET and produce polyhydroxyalkanoic acid, and have made further improvements.

[0009] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. A method for producing polyhydroxyalkanoic acid, comprising allowing Ideonella sakaiensis to act on a sample containing a carbon source. Section 2. Item 2. The method according to Item 1, wherein the Ideonella sakaiensis is Ideonella sakaiensis NBRC 110686. Section 3. Item 3. The method according to item 1 or 2, wherein the carbon source is at least one selected from the group consisting of polyethylene terephthalic acid and sugars. Section 4. Item 4. The method according to any one of Items 1 to 3, wherein the polyhydroxyalkanoic acid has 3-hydroxybutanoic acid as a constituent unit. Section 5. A composition for producing polyhydroxyalkanoic acid, comprising Ideonella sakaiensis. Section 6. Item 6. The composition for producing a polyhydroxyalkanoic acid according to Item 5, wherein the Ideonella sakaiensis is Ideonella sakaiensis NBRC 110686. Section 7. Item 7. The composition for producing a polyhydroxyalkanoic acid according to item 5 or 6, further comprising a carbon source. Section 8. 8. The composition for producing a polyhydroxyalkanoic acid according to any one of Items 5 to 7, wherein the carbon source is at least one selected from the group consisting of polyethylene terephthalic acid and sugars. Section 9. Item 9. A composition for producing a polyhydroxyalkanoic acid according to any one of Items 5 to 8, wherein the polyhydroxyalkanoic acid has 3-hydroxybutanoic acid as a constituent unit. Effect of the Invention

[0010] A method for producing polyhydroxyalkanoic acid using Ideonella sakaiensis is provided. Also provided is a composition for producing polyhydroxyalkanoic acid, comprising Ideonella sakaiensis. [Brief description of the drawings]

[0011] [Figure 1] An electron microscope photograph of Ideonella sakaiensis is shown. [Diagram 2] The results of analysis of polyhydroxyalkanoic acid decomposition products in the cells of Ideonella sakaiensis when it was allowed to act on a medium containing sterilized A-PET granules as a carbon source are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Each embodiment included in the present disclosure will be described in further detail below.

[0013] The present disclosure encompasses a composition for producing polyhydroxyalkanoic acid, comprising Ideonella sakaiensis. In this specification, the composition may be referred to as the "composition for producing polyhydroxyalkanoic acid of the present disclosure."

[0014] An example of Ideonella sakaiensis used in the present disclosure is Ideonella sakaiensis NBRC 110686 (see Non-Patent Document 1 above). Ideonella sakaiensis NBRC 110686 is also called Ideonella sakaiensis 201-F6 or Ideonella sakaiensis TISTR 2288.

[0015] The Ideonella sakaiensis used in the present disclosure preferably has a base sequence encoding PETase (EC3.1.1.101). The Ideonella sakaiensis used in the present disclosure can degrade PET into monohydroxyethyl terephthalic acid by PETase. The amino acid sequence of PETase from Ideonella sakaiensis NBRC 110686 is GenBank accession number GAP38373.1.

[0016] The Ideonella sakaiensis used in the present disclosure preferably has a base sequence encoding MHETase (EC3.1.1.102). The Ideonella sakaiensis used in the present disclosure can decompose monohydroxyethyl terephthalate into ethylene glycol and terephthalic acid by MHETase. The amino acid sequence of PETase from Ideonella sakaiensis NBRC 110686 is GenBank accession number GAP38911.1.

[0017] Polyhydroxyalkanoic acid is a polyester produced by microorganisms and the like. The polyhydroxyalkanoic acid obtained by the present disclosure may be a homopolymer consisting of one type of monomer, or may be a copolymer consisting of at least two or more types of monomers. Examples of the monomers that constitute the polyhydroxyalkanoic acid include 3-hydroxyalkanoic acid such as 3-hydroxybutanoic acid, 3-hydroxypropionic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, and 3-hydroxyoctanoic acid, 4-hydroxyalkanoic acid such as 4-hydroxybutanoic acid, and 2-hydroxyalkanoic acid such as lactic acid and 2-hydroxybutanoic acid. The polyhydroxyalkanoic acid obtained by the present disclosure preferably has 3-hydroxybutanoic acid as a constituent unit. Examples of the polyhydroxyalkanoic acid obtained by the present disclosure include poly(3-hydroxybutanoic acid), which is a homopolymer of a 3-hydroxybutanoic acid monomer.

[0018] The content of Ideonella sakaiensis in the composition for producing a polyhydroxyalkanoic acid can be appropriately set and can be, for example, 0.00001 to 100% by mass. More specifically, it may be 0.00001 to 90% by mass.

[0019] The composition for producing a polyhydroxyalkanoic acid may further contain a carbon source. The carbon source used in the present disclosure may be, for example, a carbon source generally used in the culture of a microorganism. More specifically, examples of the carbon source include saccharides such as monosaccharides, oligosaccharides, and polysaccharides; organic acids; and the like. Examples of monosaccharides include aldoses such as allose, talose, gulose, glucose, altrose, mannose, galactose, idose, ribose, lyxose, xylose, arabinose, erythrose, threose, and glyceraldehyde; ketoses such as psicose, fructose, sorbose, tagatose, ribulose, xylulose, erythrulose, and dihydroxyacetone; and N-acetylglucosamine. Of these, N-acetylglucosamine is preferred. Examples of oligosaccharides include disaccharides such as sucrose, lactulose, lactose, maltose, trehalose, and cellobiose; and trisaccharides such as raffinose, melezitose, and maltotriose. Of these, maltose is preferred. Examples of polysaccharides include starch, cellulose, glycogen, dextran, glucomannan, and xylan. The sugar may be a mixture of sugars such as molasses, honey, high fructose glucose liquid sugar, starch hydrolysate, starch syrup, etc. The organic acid may be, for example, gluconic acid, adipic acid, malic acid, citric acid, etc. In addition, for example, PET can be used as the carbon source used in the present disclosure. PET is a polycondensate of terephthalic acid, which is a dicarboxylic acid component, and ethylene glycol, which is a diol component. PET may be crystalline PET (also called C-PET) or non-crystalline PET (also called amorphous PET or A-PET). Among them, non-crystalline PET can be preferably used. In addition, for example, isophthalic acid copolymer PET can be used as the carbon source used in the present disclosure. In addition, for example, terephthalic acid or ethylene glycol, which are components of PET, can be used as the carbon source used in the present disclosure. In addition, hydrolysates of PET such as monohydroxyethyl terephthalic acid (MHET) and bishydroxyethyl terephthalic acid (BHET) can be used as the carbon source used in the present disclosure. The form of the PET is not particularly limited, and examples thereof include granules, fibers, films, blocks, bottles, grains, flakes, pellets, mixtures of these, and the like. These carbon sources can be used alone or in combination of two or more. In the composition for producing polyhydroxyalkanoic acid, for example, the carbon source may be only PET. In other words, the composition for producing polyhydroxyalkanoic acid may not contain saccharides such as monosaccharides, oligosaccharides, polysaccharides, etc., organic acids, etc., other than PET.

[0020] The content of the carbon source in the composition for producing a polyhydroxyalkanoic acid can be appropriately set and can be, for example, 1 to 99.9999 mass %.

[0021] The content ratio of Ideonella sakaiensis and the carbon source in the composition for producing polyhydroxyalkanoic acid can be appropriately set and can be, for example, 0.0001:99.9999 to 90:10.

[0022] The composition for producing a polyhydroxyalkanoic acid may further contain other components in addition to the components described above.

[0023] Examples of other components include solvents, dispersion media (e.g., glycerol, DMSO, skim milk, etc.), preservatives, excipients, activators, antiseptics, pH adjusters, stabilizers, and medium components (e.g., agar; nitrogen sources such as ammonium salts, nitrates, yeast extract, and peptone; inorganic salts such as sodium bicarbonate and calcium carbonate; vitamin sources; trace elements; antibiotics, etc.). These can be used alone or in combination of two or more. The composition for producing polyhydroxyalkanoic acid of the present disclosure may be prepared just before use by mixing Ideonella sakaiensis with a carbon source or other components.

[0024] The composition for producing a polyhydroxyalkanoic acid according to the present disclosure may be in a solid form (eg, powder, granules, gel, paste, etc.) or in a liquid form (eg, solution, suspension, etc.).

[0025] The composition for producing polyhydroxyalkanoic acid according to the present disclosure can be prepared by a conventional method by combining Ideonella sakaiensis with a carbon source or other components as necessary.

[0026] The composition for producing a polyhydroxyalkanoic acid according to the present disclosure can be suitably used as a composition for producing a polyhydroxyalkanoic acid.

[0027] The present disclosure also encompasses a method for producing a polyhydroxyalkanoic acid. In this specification, the method may be referred to as the "method for producing a polyhydroxyalkanoic acid of the present disclosure."

[0028] The method for producing polyhydroxyalkanoic acid of the present disclosure is characterized by comprising allowing Ideonella sakaiensis to act on a sample containing a carbon source. More specifically, the method for producing polyhydroxyalkanoic acid of the present disclosure is characterized by comprising culturing Ideonella sakaiensis using a sample containing a carbon source. In addition, the method for producing polyhydroxyalkanoic acid of the present disclosure may be one that uses the composition for producing polyhydroxyalkanoic acid of the present disclosure.

[0029] The sample containing the carbon source is not particularly limited as long as it contains the carbon source used in the present disclosure. The above description can be used for the carbon source used in the present disclosure. In addition, in the method for producing polyhydroxyalkanoic acid of the present disclosure, for example, the carbon source may be only PET. In other words, in the method for producing polyhydroxyalkanoic acid of the present disclosure, the carbon source may not include saccharides such as monosaccharides, oligosaccharides, polysaccharides, etc., organic acids, etc., other than PET. When the method for producing polyhydroxyalkanoic acid according to the present disclosure uses the composition for producing polyhydroxyalkanoic acid according to the present disclosure, a separate carbon source can also be applied regardless of the presence or absence of a carbon source contained in the composition for producing polyhydroxyalkanoic acid according to the present disclosure.

[0030] The sample containing the carbon source may be in a solid form (eg, powder, granules, gel, paste, etc.) or in a liquid form (eg, solution, suspension, etc.).

[0031] As a method for allowing Ideonella sakaiensis to act on a sample containing a carbon source (more specifically, a method for culturing Ideonella sakaiensis using a sample containing a carbon source), conventional methods and conditions can be adopted, for example, methods and conditions used for culturing Ideonella sakaiensis can be adopted. For example, when allowing Ideonella sakaiensis to act on a sample containing a carbon source, stirring, shaking, aeration, etc. can be performed as necessary. When Ideonella sakaiensis is allowed to act on a sample containing a carbon source, other components may be mixed in addition to Ideonella sakaiensis and the sample containing a carbon source. Examples of other components include components generally used in the culture of microorganisms. More specifically, examples of such components include nitrogen sources such as ammonium salts, nitrates, yeast extract, and peptone; inorganic salts such as sodium bicarbonate and calcium carbonate; vitamin sources; trace elements; and antibiotics. These can be used alone or in combination of two or more. The other components may be contained in the sample containing the carbon source.

[0032] The temperature at which Ideonella sakaiensis is allowed to act on a sample containing a carbon source is not particularly limited as long as Ideonella sakaiensis survives, and may be, for example, 15 to 42° C. The upper or lower limit of the range may be, for example, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41° C. More specifically, the temperature may be, for example, 25 to 39° C.

[0033] The time for which Ideonella sakaiensis is allowed to act on a sample containing a carbon source is not particularly limited, and may be, for example, 1 day to 1 month. The upper or lower limit of the range may be, for example, 7, 14, 21, or 28 days. More specifically, it may be, for example, 7 to 14 days.

[0034] In the method for producing polyhydroxyalkanoic acid according to the present disclosure, the ratio of Ideonella sakaiensis to the carbon source can be appropriately set and can be, for example, 0.0001:99.9999 to 90:10.

[0035] The method for producing polyhydroxyalkanoic acid of the present disclosure can further include extracting polyhydroxyalkanoic acid from Ideonella sakaiensis. Conventional methods and reaction conditions can be used for the extraction method.

[0036] In the method for producing polyhydroxyalkanoic acid according to the present disclosure, when PET is used as a carbon source, it is not necessary to treat PET at a high temperature to decompose the PET, for example, at the melting temperature of PET, more specifically, at 255° C. or higher.

[0037] In the method for producing polyhydroxyalkanoic acid according to the present disclosure, when PET is used as a carbon source, it is not necessary to treat the PET with a PET-degrading enzyme other than PETase derived from Ideonella sakaiensis in order to degrade the PET. Examples of the PET-degrading enzyme other than PETase derived from Ideonella sakaiensis include cutinase having PET degrading activity.

[0038] According to the present disclosure, polyhydroxyalkanoic acid can be produced. In addition, since Ideonella sakaiensis can degrade PET and produce polyhydroxyalkanoic acid, according to the present disclosure, polyhydroxyalkanoic acid can be produced from PET.

[0039] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes all arbitrary combinations of the constituent features described in this specification.

[0040] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0041] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, the experiments are performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass".

[0042] Amorphous PET (A-PET) granules were sterilized with 70% ethanol, air-dried in a clean bench for 60 minutes, and then dried in a desiccator (humidity 0%) for 24 hours. After that, 50 mL of minimal medium was dispensed into petri dishes (φ90) in a clean bench, and 10 g of sterilized A-PET granules or sterilized maltose solution was added as a carbon source to a final concentration of 1%. The composition of the minimal medium is as follows. The contents were 0.01% yeast extract, 0.02% sodium bicarbonate, 0.1% ammonium sulfate, 0.01% calcium carbonate, 0.1% vitamin mixture (0.25% thiamine hydrochloride, 0.005% biotin, and 0.05% vitamin B12), and 0.2% trace elements (0.5% MgSO4·7H2O, 0.05% CuSO4·5H2O, 0.05% MnSO4·5H2O, and 0.05% ZnSO4·7H2O) dissolved in 10 mM phosphate buffer (pH 7.4).

[0043] Ideonella sakaiensis NBRC 110686 precultured in nutrient medium (1.0% polypeptone, 0.2% yeast extract, and 0.1% MgSO4) was cultured at an initial OD 660 = 0.002, and then subjected to reciprocal shaking at 30°C and 50 rpm. After 216 hours, the culture solution was centrifuged (5000 x g, 10 minutes) to collect only the bacterial cells. The cells were then freeze-dried, and the dry bacterial weight was measured.

[0044] 2 ml of sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added to 10-30 mg of the obtained dried cells, and the mixture was sealed and heated at 100°C for 140 minutes to obtain methyl esters of polyhydroxyalkanoic acid decomposition products in the cells. 1 ml of distilled water was added and vigorously stirred. After leaving the mixture to stand and separating into two layers, the lower organic layer was taken out and its composition was analyzed by a capillary glass chromatograph. The gas chromatograph used was Shimadzu GC-2014, and the capillary column used was GL Science InertCap-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). The temperature conditions were as follows: the initial temperature was 100°C, and the temperature was raised at a rate of 8°C / min. Methyl octanoate was used as an internal standard substance, and the polyhydroxyalkanoic acid content in the cells was calculated from the peak area of ​​hydroxyalkanoic acid methyl ester.

[0045] The analysis results when sterilized A-PET granules were used as the carbon source are shown in Figure 2. When maltose was used as the carbon source, the PHA (more specifically, PHB) content per dry cell weight was 20.9% by weight, whereas when PET was used, it was 33.8% by weight.

Claims

1. A method for producing polyhydroxyalkanoic acid, comprising allowing Ideonella sakaiensis NBRC 110686 to act on a sample containing a carbon source.

2. The method according to claim 1 , wherein the carbon source is at least one selected from the group consisting of polyethylene terephthalic acid and sugars.

3. The method according to claim 1 or 2, wherein the polyhydroxyalkanoic acid has 3-hydroxybutanoic acid as a constituent unit.

4. A composition for producing polyhydroxyalkanoic acid, comprising Ideonella sakaiensis NBRC 110686.

5. The composition for producing a polyhydroxyalkanoic acid according to claim 4 , further comprising a carbon source.

6. The composition for producing a polyhydroxyalkanoic acid according to claim 5 , wherein the carbon source is at least one selected from the group consisting of polyethylene terephthalic acid and sugars.

7. The composition for producing a polyhydroxyalkanoic acid according to any one of claims 4 to 6, wherein the polyhydroxyalkanoic acid has 3-hydroxybutanoic acid as a constituent unit.

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