KR19990080695A - Biodegradable Poly-3-hydroxyalkanoate with Rubber Elasticity, Production Strain and Polymer Composition - Google Patents

Abstract

Pseudomonas sp., Deposited as KCTC 0406 BP. Polyhydroxyalkanoate copolymer comprising C ₃ to C ₅ monomers and / or C _{6 to} C ₁₄ monomers produced by culturing HJ-2 strains in a medium containing saturated and / or unsaturated carboxylic acids It has excellent biocompatibility, biodegradability and elasticity and can be used in agriculture, daily necessities, medical supplies, food packaging and medicine because it can adjust elasticity according to the type, concentration and culture conditions of substrate carbon source.

Description

Biodegradable Poly-3-hydroxyalkanoate, Producing Strains and Polymer Composition with Rubber Elasticity

[Industrial use]

The present invention relates to a polyhydroxyalkanoate excellent in biocompatibility and biodegradability, and more particularly, it is excellent in biocompatibility and biodegradability as well as containing various monomers in various compositions, which can freely control elasticity. The present invention relates to polyhydroxyalkanoate and its production strain which are useful in all fields requiring degradability and elasticity of devices, medical devices, medical supplies and the like.

[Prior art]

Due to its excellent durability, synthetic plastic and synthetic fiber products, which have been widely used in daily life, are disposed of after being used and severely polluted soils and rivers without inhibiting the growth of plants and degrading plants after decades of use. It is causing a problem. The cause of environmental pollution by plastics is the excellent durability of plastics that withstands the physical and chemical changes of the surroundings, including the decomposition of microorganisms.

In order to solve the environmental pollution problem caused by such plastics, biodegradable plastics decomposed by microorganisms have been developed. These biodegradable plastics are prepared by adding starch or metal complexes to existing synthetic plastics such as polyolefin or polyester, so that they can be easily collapsed by the action of decomposition and photooxidation of microorganisms. However, such biodegradable plastics easily decompose starch in the plastic components, but the synthetic plastic portion still remains undecomposed to contaminate the environment, and the metal complex portion used in the manufacture of these plastics is another environment. There is a problem that can be a source of contamination.

Recently, in order to solve the environmental pollution problem caused by synthetic plastics more fundamentally, research on bioplastics generated from living organisms has been actively conducted. These bioplastics, also called biopolymers, are natural high molecular materials obtained directly from microorganisms, animals or plants, and some of them have excellent mechanical durability like conventional synthetic plastics, and thus, materials such as general materials, packaging materials and agricultural films. Not only can it be used as a biodegradable but also has a very good biodegradability, and after a certain time in nature, it is completely decomposed into water and carbon dioxide. It is also used as a material.

Bioplastics known to date include pullulan, chitosan, xanthan, gellan, rhamsan, poly-beta-hydroxybutyrate, PHB), which is a kind of poly-beta-hydroxyalkanoates (hereinafter referred to as PHAs), has an intermediate property between polyester and polypropylene. It is known to have the greatest application potential. This PHB is biothermoplastics that are completely degraded by microorganisms in nature and naturally absorbed and degraded in vivo when used in vivo, and can completely solve environmental pollution problems.

In general, PHA is a storage material that some bacteria store as carbon and energy sources. In 1926, PHB, one of the PHAs, was first discovered in Bacillus megaterium by M. Lemoigne (Poivier et al. , Biotechnol. 13: 142-150, 1995). PHAs exist in cells in the form of inclusion bodies and are known to increase their accumulation rate under unbalanced conditions in which nitrogen (N), phosphorus (P) and sulfur (S) are limited. PHAs are short-chain-Length (SCL; C _{3 ~} C ₅ ) PHAs and medium-chains depending on the carbon number of 3-hydroxyalkanoates, the monomers that make up PHAs. -Length, MCL; C ₆ -C ₁₄ ) Long-Chain-Length (LCL) PHAs, which are divided into two groups of PHAs and which have monomers of 15 to 18 carbon atoms, have also been reported (Song et al. ., J. Microbiol Biotechnol 3:. . 123-128, 1993). This difference in length is thought to be due to the difference in affinity of the PHA synthase to 3-hydroxyalkyl-CoA, a precursor of PHAs.

Bacteria that biosynthesize PHAs found to date are over 90 genera and about 90 kinds of PHAs monomers have been found (Song et al. , J. Microbiol. Biotechnol. 3: 123-128, 1993). Among the representative bacteria that biosynthesize short-chain PHAs, Alcaligenes sp. And Bacillus spp., And particularly, Alcaligenes sp. Is known as a representative bacterium for biosynthesis of PHB homopolymer, which is one of short chain length PHA.

The melting point of PHB is about 180 ° C, which is higher than the medium chain length PHA of 40-60 ° C (Babu et al. , International symposium on bacterial polyhydroxyalkanoates. 48-54, 1996). A typical pathway for short-chain length PHA synthesis is by Alcaligenes spp., Which begins with the production of acetoacetyl-CoA from two acetyl-CoAs, and by the action of an enzyme. Biosynthesize PHB (Poivier et al. , Biotechnol. 13: 142-150, 1995). The biosynthetic pathway of PHB by Alcaligenes eutrophus is shown in Scheme 1 below.

Scheme 1

In A. eutrophus , which biosynthesizes PHB, PHB accumulates up to 80% of dry weight, and Chromobacterium violaceum is known to biosynthesize PHV homopolymer from valerate (Anderson et al. , Microbiol. Rev. 54: 450 -472, 1990; Steinbuchel et al. , FEMS Microbiol. Lett. 128: 219-228, 1995). In addition, in some bacteria, poly (3HB- co- 3HV), when incubated with an auxiliary substrate such as propionate, valerate, and γ-hydroxy butyrolactone in addition to the periodic matrix, Copolyesters such as poly (3HB- co- 4HB) are also known to synthesize.

Biosynthesis of heavy chain-length PHA is currently extensively conducted in Pseudomonas spp., Such as Pseudomonas oleovorans and Pseudomonas putida, and mainly in the process of β-oxidation and de novo fatty acid biosynthesis. Through biosynthesis of heavy chain length PHA consisting of monomers longer or shorter than a given substrate. The reaction for obtaining a precursor for PHA synthesis from hexanoate is shown in Scheme 2 below. The 3-hydroxyhexanoate monomer is synthesized through β-oxidation process, and the acetyl-CoA molecule produced at the same time is introduced into PHA through dinovo fatty acid biosynthesis.

Scheme 2

Unlike the short-chain PHAs, the medium chain length PHAs have low crystallinity and low melting point of about 40 to 60 ° C., and the longer the length of the monomer side chain, the stronger the rubber elastomer (Babu et al. , International symposium). on bacterial polyhydroxyalkanoates. 48-54, 1996).

Most bacteria biosynthesize either short or long chain PHAs, but some Pseudomonas spp. Are known to biosynthesize PHAs containing both short and medium chain monomers (Kato et al. , Appl. Microbiol Biotechnol). 45: 363-370, 1996; Lee et al. , Appl. Microbiol Biotechnol. 42: 901-909, 1995; Steinbuchel et al. , Appl. Microbiol Biotechnol. 37: 691-697, 1992). PHAs containing both short and medium chain length monomers have received a lot of attention because they may have various physical properties not known until now. In addition, short-chain length monomers are also available in a recombinant strain that transforms a poly (3HB) -synthetic gene of A. eutrophus that synthesizes poly (3HB) to P. oleovorans that biosynthesizes heavy-chain PHAs. And biosynthesis of PHAs with heavy chain length monomers, but these are physical mixtures (Timm et al. , Appl. Microbiol Biotechnol. 33: 296-301, 1990).

An object of the present invention is to provide a PHA having a desired elasticity and elasticity and its production strain by varying the composition ratio as well as varying monomers in PHA excellent in biodegradability and biocompatibility.

1 is Pseudomonas sp. Graph of cell growth and residual substrate count against elapsed time of batch feeding fermentation of heptanoate by HJ-2.

2 is Pseudomonas sp. By HJ-2 synthesized from heptanoate poly (3HB- co -3HV- co -3HHp) the screen is methanol gas chromatography of a sample program is obtained by the reaction.

3 is Pseudomonas sp. Graph showing the change of PHA composition with elapsed time of batch feed fermentation of heptanoate by HJ-2.

4 is Pseudomonas sp. By HJ-2 heptanoate synthesized from a poly-Eight thermal analysis measurement of the melting point of the (3HB- co -3HV- co -3HHp) FIG.

[Means for solving the problem]

In order to achieve the object of the present invention as described above the present invention is a group consisting of C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ and C ₁₄ monomers Pseudomonas sp., Deposited as KCTC 0406 BP, at the Genetic Bank of Korea Institute of Science and Technology, which produces a polyhydroxyalkanoate copolymer containing one or more monomers selected from the group as main units. Provide the HJ-2 strain.

The above monomers are preferably 3-hydroxybutylate of Formula 1, 3-hydroxyvalerate of Formula 2 and 3-hydroxyheptanoate of Formula 3.

[Formula 1]

[Formula 2]

[Formula 3]

A in Formula 1 is 5 to 100%, b in Formula 2 is 0 to 95%, and c in Formula 3 is 0 to 80%.

In addition, the above monomers are preferably 3-hydroxyoctanoate of Formula 4, 3-hydroxydecanoate of Formula 5 and 3-hydroxydodecanoate of Formula 6.

[Formula 4]

[Formula 5]

[Formula 6]

D in Formula 4 is 0 to 100%, e in Formula 5 is 0 to 100%, and f in Formula 6 is 0 to 100%.

The above-mentioned strains synthesize polyhydroxyalkanoate using a medium containing saturated and / or unsaturated carboxylic acids obtained by hydrolyzing an oil selected from the group consisting of vegetable oil, animal oil and shellfish oil. desirable.

The vegetable or animal oil is preferably selected from the group consisting of cooking oil, waste cooking oil, pork oil and bovine oil.

The above-mentioned oils, such as cooking oil, are mixed with distilled water, boiled, added with water as necessary, and kept well mixed, followed by pretreatment by filtering or drying the solid obtained by adding sodium hydroxide (NaOH) until tangled like soap. do.

Also in the present invention, two or more monomers selected from the group consisting of C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ and C ₁₄ monomers It provides a polyhydroxyalkanoate copolymer comprising a main unit.

The monomer is preferably a polyhydroxyalkanoate copolymer which is 3-hydroxybutylate of formula 1, 3-hydroxyvalerate of formula 2 and 3-hydroxyheptanoate of formula 3.

[Formula 1]

[Formula 2]

[Formula 3]

A in Formula 1 is 0 to 90%, b in Formula 2 is 0 to 90%, and c in Formula 3 is 10 to 95%.

In addition, the above monomers are preferably 3-hydroxyoctanoate of Formula 4, 3-hydroxydecanoate of Formula 5 and 3-hydroxydodecanoate of Formula 6.

[Formula 4]

[Formula 5]

[Formula 6]

D in Formula 4 is 0 to 55%, e in Formula 5 is 45 to 90%, and f in Formula 6 is 0 to 55%.

In addition, the above monomers include 3-hydroxybutylate of formula 1, 3-hydroxyoctanoate of formula 4, 3-hydroxydecanoate of formula 5 and 3-hydroxydodecanoate of formula 6 Is preferably.

[Formula 1]

[Formula 4]

[Formula 5]

[Formula 6]

A in Formula 1 is 5 to 85%, d in Formula 4 is 5 to 85%, e in Formula 5 is 5 to 85%, and f in Formula 6 is 5 to 85%.

In addition, the above monomers are preferably 3-hydroxybutylate of Formula 1, 3-hydroxyoctanoate of Formula 4 and 3-hydroxydodecanoate of Formula 6.

[Formula 1]

[Formula 4]

[Formula 6]

A in Formula 1 is 5 to 90%, d in Formula 4 is 5 to 90%, and f in Formula 6 is 5 to 90%.

In another embodiment of the present invention, Pseudomonas sp. It provides a method for producing polyhydroxyalkanoate comprising the step of culturing the HJ-2 strain in a medium containing saturated and / or unsaturated carboxylic acid to produce polyhydroxyalkanoate.

The saturated and / or unsaturated carboxylic acid described above is preferably selected from the group consisting of valerate, heptanoate, vegetable oil, animal oil and shellfish oil.

In still another aspect of the present invention, there is provided an elastic body comprising the above polyhydroxyalkanoate copolymer as a main component.

The elastic body is a medical rubber elastic body, and can be used for medical gloves, rubber bands, packaging materials or contraceptive devices.

And in the present invention, a polyhydroxyalkanoate air having an improved tensile strength, which comprises a step of stretching the film prepared using the polyhydroxyalkanoate copolymer described above in one or both directions before or after crystallization. Provided are methods for preparing coalescing.

In the present invention, there is also provided a molded article characterized in that it is in the form of a fiber, fabric or film comprising the polyhydroxyalkanoate copolymer described above.

In the present invention, the strains that can be grown by using dialkanoic acid in sludge are separated and Pseudomonas sp. Using HJ-2, this strain biosynthesizes PHAs from various carbon sources such as monoalkanioic acid and dialkanoic acid.

When producing the elastic body of the present invention, its properties vary depending on the composition and processing method of the polymer. As described above, various methods may be used to adjust the elasticity of the elastic body.

The first method is the same as the method of polymer composition control. Pseudomonas sp. When culturing HJ-2, materials having different elasticities are prepared by controlling the relative amounts of the repeater of the short chain length PHA and the repeater of the heavy chain length PHA by adjusting the concentration of carbon source, oxygen or nitrogen.

The second method is to process the obtained polymer. There are two methods of treating the obtained polymer before crystallization and after the crystallization is completed. One is to cut the polymer film into the desired shape, crystallize it for 4 to 5 hours, cut it into squares, and then use instron to stretch it to the maximum extent that it will not break to 200 to 400% of its original length. Another is to cut the desired film into the desired shape and then stretch it to the maximum extent that it will not break by 200-400% using Instron without crystallization. To manufacture.

The third method involves mixing a polymer with a sensitizer, such as a few percent benzophenone or a benzophenone derivative, and preparing the material prepared by the second or third method above at low temperature using ice or dry ice. Photocrosslinking produces crosslinked materials with different elasticities.

In addition, the film using the present invention can be produced as follows. First, the polymer is dissolved in chloroform, acetone, ethyl acetate, etc., and poured into a container such as glass, metal or Teflon with horizontal adjustment, and the solvent is slowly blown to prepare a film.

Second, the polymer mass is placed in a spacer having a desired thickness between two sheets of iron plate, for example, Teflon-coated iron plate, heated to room temperature to 150 ° C., and pressed at a pressure of 10 to 50 tons to press the film. Manufacture.

Third, the film produced by the first or second method described above is tensioned in one direction (uniaxially) or biaxially (before crystallization) to prepare a film. At this time, the degree of tension is from 100 to just before destruction.

Fourth, the film produced by the first or second film production method is crystallized, and then stretched in one or both directions to produce a film. At this time, the degree of tension is from 100 to just before destruction.

Fifth, a polymer is mixed with a sensitizer such as benzophenone or a benzophenone derivative of several percent to make a film by the above-described method, and irradiate ultraviolet rays.

In addition, the fiber (fiber) using the present invention is produced by stretching the obtained film in one direction and thinly torn in the form of a yarn in the tensioned direction.

EXAMPLE

Hereinafter, preferred examples and comparative examples of the present invention are described. However, the following examples are only examples of the present invention for the purpose of understanding the present invention, and the present invention is not limited to the following examples.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example

Strain Pseudomonas sp. Isolation and Identification of HJ-2

Pseudomonas sp. HJ-2 is gram positive, oxidase and catalase-positive, and has a rod-shaped morphology. Transluscent colonies were generated in agar medium, no fluorescent dyes were produced on King B medium, and yellowish green non-fluorescent diffuse pigments were generated on PCA (or NA). It also produces one or more polar flagella and grows at 42 ° C.

Another Pseudomonas sp. The characteristics of HJ-2 are summarized in Table 1 below.

Condition result Slime production from sucrose - β-galactosidase - Arginine dehydrolase + Lysine decarboxylase - Ornithine decarboxylase - Citrate utilization + H ₂ S production - Urease production - Tryptophan Deaminase - Indole production - VP reaction - Gelatin liquefaction - Oxidase + Acids of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibioz, arabinose, amigdaline - Denitrification + β-glucosidase - Availability of α-D-glucose, D-gluconate, caprate, adipate, malate, phenyl acetate +

The strain also contains citrate, succinate, Tween 40, Tween 80, L-arabinose, D-mannose, acetate, cis-aconitate, β-hydroxybutylate, p-hydroxy Phenylacetate, itaconate, α-keto glutarate, quinate, propionate, D, L-lactate, L-alanine, L-asparagine, L-asparatate, L- Glutamate, hydroxy L-proline, L-proline, L-serine, putrescine, D, L-carnitine, γ-aminobutylate, urocanate, inosine ), 2-aminoethanol.

Pseudomonas sp. Fatty acid profiles of HJ-2 are shown in Table 2 below.

C18: 1 w7c / w9t / w12t 33.2% C16: 0 24.6% C16: 1 w7c / C15: 0 Iso 2OH 21.5% C12: 0 2OH 7.4% C12: 0 3OH 3.9% C10: 0 3OH 3.1% C12: 0 3.0% C17: 0 cyclo 1.6% C14: 0 1.0% C18: 0 0.4% C19: 0 cyclo w8c 0.4%

As a result, Pseudomonas sp. HJ-2 was identified as Pseudomonas caryophylli belonging to RNA group II.

Pseudomonas sp. Used in this example. HJ-2 was obtained through enrichment cultures in sludge, and subcultured at weekly intervals in ½E ^* media plates using nonanoate as a carbon source for strain preservation. Pseudomonas sp. HJ-2 was deposited with KCTC 0406 BP on November 14, 1997, at the Genetic Bank of Korea Institute of Science and Technology. The composition of the ½ E ^* medium is shown in Table 1 below.

Pseudomonas sp. HJ-2 was incubated 1 L in a 2 L flask using a giant shaker in a constant temperature room at 30 ° C., and fermentation was carried out with a 3 L fermentation medium in a 5 L jar. The medium used for the flask and fermentation culture was ½E ^* medium, and the seed medium for the flask culture and the fermentation was added with the same carbon source as the type of each carbon source used in this example. %. Fermentation was carried out under the conditions of pH 7.0 ± 0.1, temperature 30.0 ± 1 ℃, air flow rate of 1.0vvm, 300rpm.

Composition of ½ E ^* Medium Badge Ingredient Flask Medium (g / ℓ) Fermentation medium (g / ℓ) NaNH ₄ HPO ₄ 4H ₂ O 1.75 1.75 K ₂ HPO ₄ 3H ₂ O 3.75 3.75 KH ₂ PO ₄ 1.85 1.85 0.1M MgSO ₄ 7H ₂ O 10 10 Mineral solution 5 5 Carbon source 5 5

The composition of the mineral solution is shown in Table 4 below.

Composition of mineral solution ingredient Concentration (g / 1M HCl 1ℓ) FeSO ₄ 7H ₂ O 2.78 MnCl ₂ 4H ₂ O 1.98 CoSO ₄ · 7H ₂ O 2.81 CaCl ₂ · 2H ₂ O 1.67 CuCl ₂ · 2H ₂ O 0.17 ZnSO ₄ · 7H ₂ O 0.29

How to measure biomass

Biomass was measured at 666 nm by diluting 1/5 of the stock solution using a spectrophotometer (Milton Roy spectronic 120011, U.S.A.).

Cell harvest and lyophilization

Cells obtained by flask culture and fermentation were obtained by centrifugation at 10,000 rpm for 10 minutes at 4 ° C. The obtained cells were frozen in a freezer and then dried cells were obtained using a freeze dryer.

PHAs Extraction and Purification

The lyophilized dry cells and sea sand were put together in a mortar and then put in a thimble filter to extract PHAs with a soxhlet extractor for 12 hours using chloroform. The extracted PHAs were purified using methanol. The above procedure was repeated two or three times to obtain purified PHAs.

Composition Analysis of PHAs

5 mg of purified PHAs, 1 ml of chloroform and 1 ml of methanol containing 15% H ₂ SO ₄ were added to a GC pyrex cap tube, and vortexed for 2 hours in a 105 ° C drying oven. Methanolysis during the reaction. After removing from the drying oven and cooling completely, 1 ml of distilled water was added and strongly vortexed to use the chloroform layer for analysis. The conditions of PHAs gas chromatography are summarized in Table 5.

Item Contents Model system Hewlett Packard 5890 series Ⅱ Detector Flame Ionization Detector (FID) Column Capillary HP-1, 25mID 0.2mm (narrow bore) Liquid phase 100% dimethyl polysiloxane (Gum) Solid support Chromosorb PAW DMCS Input and detection port temperature (Inj. & Det. Port temp.) 240/270 ℃ Carrier gas N ₂ Air / hydrogen flow rate 350 / 35ml / min Total flow 102ml / min Septum purge vent flow 1ml / min Column head pressure 1ml / min Auxiliary (make-up) gas flow 29 ml / min Initial temperature and time 80 ℃ / 4min Temp.up-grade rate 10 ℃ / min Final temperature and time 230 ℃ / 3min Solvent input (solvent & inj. Size) CHCl ₃ (chloroform) / 1 μl Injection port mode Split mode Split ratio 100 to 1

Example 1: PHA Synthesis from Sugar

Glucose and gluconate were each supplied as a single carbon source and Pseudomonas sp. The culture of 1 L of HJ-2 resulted in the biosynthesis of 100% PHB homopolymers in glucose and the synthesis of heavy chain length PHAs (3HO, 3HD, 3HDD) with PHB in gluconate. Pseudomonas sp. PHA synthesis results from sugar by HJ-2 are shown in Table 6 below.

Pseudomonas sp. Synthesis of PHA from Sugar by HJ-2 Carbon source (g / ℓ) DCW (g / ℓ) PHAs (wt%) PHA composition C ₄ C ₅ C ₆ C ₇ C ₈ C ₉ C ₁₀ C ₁₁ C ₁₂ Glucose (10) 1.21 5.73 100 Fructose (10) 0.98 17.85 29.23 60.87 9.9 Gluconate (10) 0.79 11.45 76.73 5.29 15.31 2.47

Example 2: PHA Synthesis from Monoalkanoic Acid

PHB was mainly produced from butylate, hexanoate, octanoate and decanoate with an even number of carbon atoms, while PHAs composed of 8.5% and 22.3% 3HO, respectively, were combined with octanoate and decanoate. . When mono-alkanoic acid with an odd number of carbon atoms was given as a single carbon source, 3 HV was mainly biosynthesized in a 5 carbon valelarate and a poly (3HB-co-3HV-co-3HHp) type PHAs in heptanoate having 7 carbon atoms. Non-nanoate with 9 carbon atoms biosynthesized 3HHp and 3HN heavy chain length PHAs (Table 5). From this, Pseudomonas sp. It can be seen that HJ-2 separates the number of carbon atoms from a given carbon source to biosynthesize the polymer of PHB mainly in the even carbon number and biosynthesize PHAs corresponding to the carbon number of the given carbon source in the odd carbon number. In particular, given that a carbon source having an odd carbon number does not biosynthesize PHAs having a length greater than the carbon number of a given carbon source, when monoalkanoic acid is used as a substrate, it is shorter than or equal to the length of the carbon source given by β-oxidation. It is thought that biosynthesis of PHAs having a length corresponding to and that does not undergo a dinovo fatty acid biosynthesis process that can biosynthesize PHAs capable of biosynthesizing a longer number of PHAs than a given length.

Pseudomonas sp. Synthesis of PHA from Monoalkanoic Acid by HJ-2 Carbon source (mM / ℓ) DCW (g / ℓ) PHAs (wt%) PHA composition C ₄ C ₅ C ₆ C ₇ C ₈ C ₉ C ₁₀ C ₁₁ C ₁₂ Butylate (56.75) 1.54 11.91 100 Balarate (48.96) 1.16 36.03 6.3 93.7 Hexanoate (43.04) 2.11 25.17 100 Heptanoate (38.40) 0.64 17.03 21.6 43.1 35.3 Octanoate (34.67) 2.38 22.82 91.5 <1 8.5 Nonano Eight (31.60) 0.53 12.57 <1 <1 22 78 Decanoate (29.02) 1.12 42.95 80.64 11.2 8.16

Example 2: Synthesis of PHA from dialkanoic acid

In dialkanoic acid, PHB homopolymers were biosynthesized when an odd carbon source was given as a single carbon source. That is, PHB homopolymers were biosynthesized when diheptanoate and dinonanoate were fed as a single carbon source and cultured in 1 L in a 2 L flask. On the other hand, when dioctanoate having an even number of carbons is given as a single carbon source, it can be seen that heavy chain length PHAs having a composition of 3HO, 3HD, and 3HDD are synthesized (Table 8).

Pseudomonas sp. Synthesis of PHA from Monoalkanoic Acid by HJ-2 Carbon source (mM / ℓ) DCW (g / ℓ) PHAs (wt%) PHA composition C ₄ C ₅ C ₆ C ₇ C ₈ C ₉ C ₁₀ C ₁₁ C ₁₂ Diheptanoate (31.22) 1.26 8.61 100 Dioctanoate (28.70) 0.73 14.11 20.79 64.20 15.01 Dino Nano Eight (26.56) 0.94 23.46 100 Didecanoate (24.72) 0.84 5.55 26.23 61.97 11.8

The results from dialkanoic acid show the opposite of the results from monoalkanoic acid. In monoalkanoic acids, PHB homopolymers are biosynthesized at even carbon sources, whereas in dialkanoic acid, PHB homopolymers are biosynthesized at odd carbon sources and even carbon atoms have longer carbon monomers than given substrates. PHAs having biosynthesis. Making PHAs with carbon numbers longer than the length of a given carbon source can be thought of as synthesizing PHAs through the dinovo fatty acid biosynthesis process (Scheme 2). The difference between monoalkanoic acid and dialkanoic acid is that there is one more -OH group in the side chain of dialkanoic acid, which causes β-oxidation in monoalkanoic acid and dinovoal in dialkanoic acid. It is believed that PHAs are biosynthesized through fatty acid biosynthesis. This is probably due to substrate specificity for a given carbon source of enzymes involved in biosynthesis of PHAs.

Example 3: Fermentation in Heptanoate

Heptanoate was given as a single carbon source and fed batch fermentation was performed at 30 ° C. and 300 rpm. In the batch feed fermentation, the amount of carbon source used was 5.2g / l, starting with 2.8g / l, and the substrate consumption in FIG. In Fig. 1,? Indicates Optical Density (OD) at 660 nm, and? Indicates residual substrate. GC results of the PHA composition analysis are shown in FIG. 2. 2, the composition was analyzed as 3HB, 3HV, 3HHp. By sampling 100ml by time to extract the PHAs to determine the monomer composition (Fig. 2). Regardless of the sampling time, all four samples had a monomer composition ratio of 3HB: 3HV: 3HHp = 50: 30: 20 at almost constant ratio. This is Pseudomonas sp. When HJ-2 makes PHA from heptanoate, it prefers which one of the PHA monomers is synthesized first and which one is not synthesized later, but always biosynthesizes each at a constant rate. In addition, since each monomer occupies a significant concentration of 3HB: 3HV: 3HHp = 50: 30: 20, it is considered that the ratio of monomers constituting the terpolymer is lower than that of the low-polymer terpolymer PHAs. The melting point of poly (3HB- co- 3HV- co- 3HHP) was 110.76 ° C (Fig. 4), which was lower than PHB of 180 ° C and 40-60 ° C. The heavy chain length is higher than that of PHAs.

Pseudomonas sp. Biosynthesis of PHB homopolymers when HJ-2 gave glucose, butylate, hexanoate, etc. as a single carbon source showed similarities to the synthesis of most short-chain PHA biosynthetic strains including A. eutrophus . Biosynthesis of 3HB and heavy chain length monomers (3HO, 3HD, 3HDD) is similar to the PHA synthesis of P. aeruginosa . In addition, 3HO biosynthesis in octanoate, 3HHp biosynthesis in heptanoate, and 3HN biosynthesis in nonanoate are similar to the PHA synthesis functions of general Pseudomonas including P. oleovorans . Finally, The biosynthesis of the poly (3HB- co -3HV- c o-3HHp ) in heptanoate is similar to the function of the Rhodococcus ruber biosynthesis of poly (3HB- co -3HV- co -3HHx) from hexanoate. Pseudomonas sp. HJ-2 has the characteristics seen in several strains. Also, as can be seen from the results of monoalkanoic acid and the results of dialkanoic acid, the PHA-biosynthetic pathway of this strain should be investigated. In other words, how to use the β-oxidation process and the dionov fatty acid biosynthesis process when biosynthesizing PHAs. Combining various carbon sources to produce PHAs composed of various monomers, and to study the properties of PHA according to its composition and its applicability. Pseudomonas sp. Research is also ongoing on optimal conditions for biosynthesis of specific PHAs from HJ-2.

Pseudomonas sp. PHA having a composition of monomers having various chain lengths using HJ-2 has excellent biocompatibility, biodegradability, and elasticity, and is more useful than conventional PHA by controlling elasticity according to the concentration of a carbon source as a substrate. It can be used in various medical technology fields. In particular, it is excellent in biocompatibility and can be used in place of the hardly decomposable rubber used in artificial breasts or condoms to solve the problem of hardly decomposability.

Claims (14)

C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ and C ₁₄ monomers comprising one or more monomers selected from the group consisting of Pseudomonas sp., Deposited as KCTC 0406 BP, at the Genetic Bank of Korea Institute of Science and Technology, which produces polyhydroxyalkanoate copolymers. HJ-2 strain. The Pseudomonas sp. According to claim 1, wherein the monomer is 3-hydroxybutylate of Formula 1, 3-hydroxyvalerate of Formula 2, and 3-hydroxyheptanoate of Formula 3. HJ-2 strain. [Formula 1] [Formula 2] [Formula 3] A in Formula 1 is 5 to 100%, b in Formula 2 is 0 to 95%, and c in Formula 3 is 0 to 80%. The Pseudomonas sp according to claim 1 or 2, wherein the monomer is 3-hydroxyoctanoate of Formula 4, 3-hydroxydecanoate of Formula 5 and 3-hydroxydodecanoate of Formula 6. . HJ-2 strain. [Formula 4] [Formula 5] [Formula 6] D in Formula 4 is 0 to 100%, e in Formula 5 is 0 to 100%, and f in Formula 6 is 0 to 100%. The method according to any one of claims 1 to 3, wherein the strain is a carbon source containing a saturated and / or unsaturated carboxylic acid obtained by hydrolyzing an oil selected from the group consisting of vegetable oil, animal oil and shellfish oil. Pseudomonas sp. To synthesize polyhydroxyalkanoate. HJ-2 strain. C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ and C ₁₄ monomers comprising two or more monomers selected from the group consisting of Polyhydroxyalkanoate copolymers. The polyhydroxyalkanoate copolymer according to claim 5, wherein the monomer is 3-hydroxybutylate of Formula 1, 3-hydroxy valerate of Formula 2 and 3-hydroxyheptanoate of Formula 3. . [Formula 1] [Formula 2] [Formula 3] A in Formula 1 is 0 to 90%, b in Formula 2 is 0 to 90%, and c in Formula 3 is 10 to 95%. The polyhydroxyalkanoate of claim 5, wherein the monomer is 3-hydroxyoctanoate of formula 4, 3-hydroxydecanoate of formula 5 and 3-hydroxydodecanoate of formula 6 Copolymer. [Formula 4] [Formula 5] [Formula 6] D in Formula 4 is 0 to 55%, e in Formula 5 is 45 to 90%, and f in Formula 6 is 0 to 55%. The method of claim 5, wherein the monomer is a 3-hydroxybutylate of formula 1, 3-hydroxyoctanoate of formula 4, 3-hydroxy decanoate of formula 5 and 3-hydroxy of formula 6 Polyhydroxyalkanoate copolymer which is dodecanoate. [Formula 1] [Formula 4] [Formula 5] [Formula 6] A in Formula 1 is 5 to 85%, d in Formula 4 is 5 to 85%, e in Formula 5 is 5 to 85%, and f in Formula 6 is 5 to 85%. The polyhydroxyalkanoate aerial of claim 5, wherein the monomer is 3-hydroxybutylate of Formula 1, 3-hydroxyoctanoate of Formula 4, and 3-hydroxydodecanoate of Formula 6. coalescence. [Formula 1] [Formula 5] [Formula 6] A in Formula 1 is 5 to 90%, d in Formula 4 is 5 to 90%, and f in Formula 6 is 5 to 90%. Pseudomonas sp. Culturing the HJ-2 strain in a medium comprising saturated and / or unsaturated carboxylic acids to produce polyhydroxyalkanoate; Method for producing a polyhydroxyalkanoate comprising the step. The method of claim 10, wherein the saturated and / or unsaturated carboxylic acid is selected from the group consisting of valerate, heptanoate, vegetable oil, animal oil, and shellfish oil. An elastic body comprising the polyhydroxyalkanoate copolymer of claim 5 as a main component. Stretching the film produced using the polyhydroxyalkanoate copolymer of claim 5 in one or both directions before or after crystallization; Method for producing a polyhydroxyalkanoate copolymer having an improved tensile strength comprising. A molded article characterized in the form of a fiber, fabric or film comprising the polyhydroxyalkanoate copolymer of claim 5.