U.S. patent application number 10/242696 was filed with the patent office on 2003-09-25 for process for producing polyhydroxyalkanoates by utilizing microorganisms.
Invention is credited to Honma, Tsutomu, Imamura, Takeshi, Kenmoku, Takashi, Kobayashi, Shin, Kobayashi, Toyoko, Suda, Sakae, Yano, Tetsuya.
Application Number | 20030180899 10/242696 |
Document ID | / |
Family ID | 27566939 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180899 |
Kind Code |
A1 |
Honma, Tsutomu ; et
al. |
September 25, 2003 |
Process for producing polyhydroxyalkanoates by utilizing
microorganisms
Abstract
A microbial polyhydroxyalkanoate which comprises one or more of
monomer units represented by Formula (1), 1 where R is at least one
selected from the group represented by any one of Formulas (2), (3)
and (4); 2 in Formula (2), R1 is selected from the group consisting
of hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and q is an integer of 1 to
8; in Formula (3), R2 is selected from the group consisting of
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and r is an integer of 1 to
8; in Formula (4), R3 is selected from the group consisting of
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and s is an integer of 1 to
8. The production method is also disclosed.
Inventors: |
Honma, Tsutomu; (Atsugi-shi,
JP) ; Kobayashi, Toyoko; (Yokohama-shi, JP) ;
Yano, Tetsuya; (Atsugi-shi, JP) ; Kobayashi,
Shin; (Kawasaski-shi, JP) ; Imamura, Takeshi;
(Chigasaki-shi, JP) ; Suda, Sakae; (Ushiku-shi,
JP) ; Kenmoku, Takashi; (Fujisawa-shi, JP) |
Correspondence
Address: |
FITZPATRICK CELLA HARPER & SCINTO
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
US
|
Family ID: |
27566939 |
Appl. No.: |
10/242696 |
Filed: |
September 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10242696 |
Sep 13, 2002 |
|
|
|
09745476 |
Dec 26, 2000 |
|
|
|
Current U.S.
Class: |
435/135 ;
528/272; 528/274 |
Current CPC
Class: |
C08G 63/6822 20130101;
C08G 63/06 20130101; C12P 7/625 20130101; C08G 63/6852 20130101;
Y10T 428/2907 20150115 |
Class at
Publication: |
435/135 ;
528/272; 528/274 |
International
Class: |
C12P 007/62; C08G
063/87 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 1999 |
JP |
11-371864 |
Dec 27, 1999 |
JP |
11-371867 |
Dec 27, 1999 |
JP |
11-371868 |
Dec 27, 1999 |
JP |
11-371869 |
Jan 31, 2000 |
JP |
2000-023024 |
Jan 31, 2000 |
JP |
2000-023025 |
Nov 28, 2000 |
JP |
2000-361323 |
Claims
What is claimed is:
1. A polyhydroxyalkanoate comprising one or more of monomer units
represented by Formula (1), 40where R is at least one selected from
the group represented by any one of Formulas (2), (3) and (4); 41in
formula (2), R1 is selected from the group consisting of hydrogen
atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and q is an integer of 1 to
8; in formula (3), R2 is selected from the group consisting of
hydrogen atom (h), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and r is an integer of 1 to
8; in formula (4), R3 is selected from the group consisting of
hydrogen atom (h), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and s is an integer of 1 to
8; provided that following R is not selected: when one kind of R is
selected: R being R1=H and q=2, R being R1=H and q=3 in Formula
(2), R being R2=halogen and r=2, or R being R2=--CN and r=3 and R
being R2=--NO.sub.2 and r=3 in Formula (3); when two kinds of R are
selected: a combination of R being R1=H and q=3 and 5 respectively
in Formula (2), a combination of R being R2=H and r=2 and 4
respectively, a combination of R being R2=H and r=2 and 6
respectively, and a combination of R being R2=halogen and r=2 and 4
respectively in Formula (3); when three kinds of R are selected: a
combination of R being R1=H and q=3, 5 and 7 respectively in
Formula (2), a combination of R being R2=H and r=1, 3 and 5
respectively, and a combination of R being R2=H and r=2, 4 and 6
respectively in Formula (3).
2. The polyhydroxyalkanoate according to claim 1 wherein the
monomer unit is 3-hydroxy-4-phenoxybutyric acid unit of Formula
(5). 42
3. The polyhydroxyalkanoate according to claim 1 wherein the
monomer unit is 3-hydroxy-5-phenoxyvaleric acid unit of Formula
(6). 43
4. The polyhydroxyalkanoate according to claim 1 wherein the
monomer unit is 3-hydroxy-5-(4-fluorophenyl)valeric acid unit of
Formula (7). 44
5. The polyhydroxyalkanoate according to claim 1 wherein the
monomer unit is 3-hydroxy-4-cyclohexylbutyric acid unit of Formula
(8). 45
6. The polyhydroxyalkanoate according to claim 1 wherein the
monomer unit is 3-hydroxy-5-phenoxyvaleric acid unit of Formula (6)
and 3-hydroxy-5-phenylvaleric acid unit of Formula (9). 46
7. The polyhydroxyalkanoate according to claim 1 wherein the
monomer unit is 3-hydroxy-4-phenylbutyric acid unit of Formula (10)
and 3-hydroxy-6-phenylhexanoic acid units of Formula (11). 47
8. The polyhydroxyalkanoate according to any one of claims 1 to 7
wherein a number average molecular weight is 10 to 200
thousands.
9. A process of producing a polyhydroxyalkanoate comprising the
step of: culturing a microorganism in a culture medium containing a
raw material alkanoate and an yeast extract, wherein the
microorganism produces a polyhydroxyalkanoate utilizing the
alkanoate.
10. The process according to claim 9, wherein the alkanoate is
represented by Formula (12), and the polyhydroxyalkanoate has one
or more of monomer units represented by Formula (13): 48wherein in
Formula (12), R is at least one selected from the group consisting
of Formulas (2), (3) and (4); 49wherein in Formula (2), R1 is
selected from the group consisting of hydrogen atom (H), halogen
atom, --CN, --NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5 and
--C.sub.3F.sub.7, and q is an integer of 1 to 8; in Formula (3), R2
is selected from the group consisting of hydrogen atom (H), halogen
atom, --CN, --NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5 and
--C.sub.3F.sub.7, and r is an integer of 1 to 8; in Formula (4), R3
is selected from the group consisting of hydrogen atom (H), halogen
atom, --CN, --NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5 and
--C.sub.3F.sub.7, and s is an integer of 1 to 8; 50wherein R' is
the selected R for the alkanoate or a group having the same R1, R2
or R3 as R but q=q.sub.0, q=q.sub.0-2, q=q.sub.0-4 q=q.sub.0-6; or
r=r.sub.0, r=r.sub.0-2, r=r.sub.0-4 or r=r.sub.0-6; or s=s.sub.0 or
s=s.sub.0-2, s=s.sub.0-4 or s=s.sub.0-6; wherein q.sub.0, r.sub.0,
and s.sub.0 are q, r, or s of the R, and q.sub.0-2, r.sub.0-2;
s.sub.0-2, q.sub.0-4, r.sub.0-4; s.sub.0-4, q.sub.0-6, r.sub.0-6;
or s.sub.0-6 being integers only more than 1.
11. The process according to claim 10 wherein the monomer unit is
of Formula (5), and the microorganism is cultured in a culture
medium containing 4-phenoxybutyric acid of Formula (14) and an
yeast extract, the microorganism produces
poly-3-hydroxy-4-phenoxybutyric acid utilizing 4-phenoxybutyric
acid. 51
12. The process according to claim 10 wherein the monomer unit is
of Formula (6), and the culture medium contains 5-phenoxyvaleric
acid of Formula (15) and an yeast extract, and the microorganism
produces poly-3-hydroxy-5-phenoxyvaleric acid utilizing
5-phenoxyvaleric acid. 52
13. The process according to claim 10 wherein the monomer unit is
of Formula (16), the culture medium contains
5-(4-fluorophenoxy)valeric acid of Formula (17) and yeast extract,
and the microorganism produces
poly-3-hydroxy-5-(4-fluorophenoxy)valeric acid utilizing
5-(4-fluorophenoxy)valeric acid. 53
14. The process according to claim 10 wherein the monomer unit is
of Formula (9), the culture medium contains 5-phenylvaleric acid of
Formula (18) and an yeast extract, and the microorganism produces
poly-3-hydroxy-5-phenylvaleric acid utilizing 5-phenylvaleric acid.
54
15. The process according to claim 10 wherein the monomer unit is
of Formula (7), the culture medium contains
5-(4-fluorophenyl)valeric acid of Formula (19) and an yeast
extract, and the microorganism produces
poly-3-hydroxy-5-(4-fluorophenyl)valeric acid utilizing
5-(4-fluorophenyl)valeric acid. 55
16. The process according to claim 10 wherein the monomer unit is
of Formula (8), the culture medium contains 4-cyclohexylbutyric
acid of Formula (20) and an yeast extract, and the microorganism
produces poly-3-hydroxy-4-cyclohexylbutyric acid utilizing
4-cyclohexylbutyric acid. 56
17. The process according to claim 10 wherein the monomer unit is
of Formulas (6) and (9), the culture medium contains
5-phenoxyvaleric acid of Formula (15), 5-phenylvaleric acid of
Formula (18) and an yeast extract, and the microorganism produces a
polyhydroxyalkanoate comprising 3-hydroxy-5-phenoxyvaleric acid and
3-hydroxy-5-phenylvaleric acid utilizing 5-phenoxyvaleric acid and
5-phenylvaleric acid. 57
18. The process according to claim 10 wherein the monomer unit is
of Formulas (10) and (11), the culture medium contains
6-phenylhexanoic acid of Formula (21) and an yeast extract, and the
microorganism produces a polyhydroxyalkanoate comprising
3-hydroxy-4-phenylbutyric acid and 3-hydroxy-6-phenylhexanoic acid
utilizing 6-phenylhexanoic acid. 58
19. The process according to claim 10 wherein the monomer unit is
of Formulas (6) and (22), the culture medium contains
7-phenoxyheptanoic acid of Formula (23) and an yeast extract, and
the microorganism produces a polyhydroxyalkanoate comprising
3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic
acid utilizing 7-phenoxyheptanoic acid. 59
20. The process according to claim 10 wherein the monomer unit is
of Formulas (5), (24) and (25), the culture medium contains
8-phenoxyoctanoic acid of Formula (26) and an yeast extract, and
the microorganism produces a polyhydroxyalkanoate comprising
3-hydroxy-4-phenoxybutyric acid, 3-hydroxy-6-phenoxyhexanoic acid
and 3-hydroxy-8-phenoxyoctanoic acid utilizing 8-phenoxyoctanoic
acid. 60
21. The process according to claim 10 wherein the monomer unit is
of Formulas (6), (22) and (27), the culture medium contains
11-phenoxyundecanoic acid of Formula (28) and an yeast extract, the
microorganism produces a polyhydroxyalkanoate comprising
3-hydroxy-5-phenoxyvaleric acid, 3-hydroxy-7-phenoxyheptanoic acid
and 3-hydroxy-9-phenoxynonanoic acid utilizing 11-phenoxyundecanoic
acid. 61
22. The process according to claim 9 wherein the step of culturing
is one step culture in a culture medium containing an alkanoate and
an yeast extract.
23. The process according to claim 9 wherein the step of culturing
is two step culture in a culture medium containing an alkanoate and
an yeast extract followed by culturing in a nitrogen-restricted
culture medium containing the alkanoate.
24. The process according to claim 9 which further comprises a step
of separation/refinement of the polyhydroxyalkanoate.
25. The process according to claim 9 wherein the microorganism
belongs to a genus Pseudomonas.
26. The process according to claim 25 wherein the microorganism is
at least one selected from the group consisting of Pseudomonas
cichorii YN2 (FERM BP-7375); Pseudomonas cichorii H45 (FERM
BP-7374); Pseudomonas putida P91 (FERM BP-7373); and Pseudomonas
jessenii P161 (FERM BP-7376).
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to novel polyhydroxyalkanoate
(PHA), as well as a method of producing such a novel PHA by
utilizing microorganisms.
[0003] 2. Related Background Art
[0004] Synthetic polymers derived from petroleum have been used as
plastics etc. for a long time. Recently, the treatment of the used
plastics has become one of serious social problems. These synthetic
polymers have advantages of hard-to-decompose have been used in the
place of metal or glass materials. On mass consumption and mass
disposal, however, this feature of hard-to-decompose makes them
accumulated in waste-disposal facilities, or when they are burned,
it causes increased carbon dioxide exhaust, and harmful substances
such as dioxin and endocrine-disruptors may be generated to cause
environmental pollution.
[0005] On the other hand, polyesters produced by microorganisms
(hereinafter referred to as "microbial polyesters") can be
biologically degraded to be incorporated in a natural recycling
system. Thus they would not remain in natural environment without
causing pollution, in contrast to the numerous usual synthetic
polymer compounds. Furthermore, since the biodegradability
dispenses with incinerating treatment, microbial polyesters are
effective from the standpoint of the prevention of air pollution
and global warming, and usable as plastics to maintain the
environment. In addition, their potential as soft materials for
medical use has been investigated (Japanese Patent Application
Laid-Open No. 5-159, Nos. 6-169980, 6-169988, 6-225921, etc.).
[0006] Heretofore, various bacteria have been reported to produce
and accumulate PHB or copolymers of other hydroxyalkanoic acids in
the cells (Handbook of Biodegradable Plastics, ed. by Biodegradable
Plastics Society, published by N.T.S., p. 178-197 (1995)).
Microbial PHA thus obtained is known to have various compositions
and structures depending on the class of microorganisms used,
medium composition, culture conditions, etc. during production, and
many studies related to the control of composition and structure of
PHA products have been conducted to improve PHA properties.
[0007] For example, Alcaligenes eutropus H16 ATCC No. 17699 and its
mutants can produce copolymers of 3-hydroxybutyric acid (3HB) and
3-hydroxyvaleric acid (3HV) at a various composition ratio by
varying carbon sources during culture (Published Japanese
Translation of PCT International Publication Nos. 6-15604, 7-14352,
8-19227, etc.).
[0008] Japanese Patent No. 2642937 discloses that Pseudomonas
oleovorans ATCC29347, when given acyclic aliphatic hydrocarbons as
a carbon source, produces PHA having a monomer unit of
3-hydroxyalkanoate of 6 to 12 carbon atoms.
[0009] Japanese Patent Application Laid-Open No. 5-74492 discloses
the method comprising contacting a microorganism of
Methylobacterium sp., Paracoccus sp., Alcaligenes sp., or
Pseudomonas sp. with a primary alcohol of 3 to 7 carbon atoms,
thereby allowing to produce a copolymer of 3HB and 3HV.
[0010] Japanese Patent Application Laid-Open Nos. 5-93049 and
7-265065 disclose that Aeromonas caviae can produce, by using oleic
acid and olive oil as carbon sources, a binary copolymer of 3HB and
3-hydroxyhexanoic acid (3HHx).
[0011] Japanese Patent Application Laid-Open No. 9-191893 discloses
that Comamonas acidovorans IFO13852 can produce, by using gluconic
acid and 1,4-butanediol as a carbon source, a polyester having
monomer units of 3HB and 4-hydroxybutyric acid.
[0012] Furthermore, certain microorganisms has been reported to
produce PHA having various substituents such as groups derived from
unsaturated hydrocarbons, ester group, allyl group, cyano group,
nitro group, groups derived from halogenated hydrocarbon, and
epoxide. Thus, there have been started several attempts to improve
the properties of microbial PHA by using such a technique. Examples
of microbial polyester having such substituents are described in
FEMS Microbiology Letters, 128 (1995) p.219-228, in detail.
Makromol. Chem., 191, 1957-1965, 1990, Macromolecules, 24,
5256-5260, 1991, and Chirality, 3, 492-494, 1991 report that
Pseudomonas oleovorans produces PHA comprising a monomer unit of
3-hydroxy-5-phenylvaleric acid (3HPV), and changes in polymer
properties probably due to the presence of the monomer unit of
3HPV.
[0013] As stated above, microbial PHA of various
compositions/structures can be obtained by varying the
microorganism, medium composition, culture conditions, etc. for
polymer production. Their physical properties, however, are still
insufficient for plastics. In order to further extend the
application field, it is important to investigate more extensively
the improvement of properties, and it is, therefore, essential to
develop and search PHA made of structurally various monomer units,
methods of producing them, as well as microorganisms capable of
efficiently producing the desired PHA.
[0014] On the other hand, those PHA having introduced substituents
in the side chains as described above, can be expected to be
developed as "functional polymer" having useful functions and
properties by selecting the substituent to be introduced according
to the desired properties, etc. It is also important to develop and
search PHA satisfying both functionality and biodegradability,
methods of producing them, as well as microorganisms capable of
efficiently producing desired PHA.
[0015] One example of such PHA having a substituent introduced in
side chains is PHA having phenoxy in side chains.
[0016] For example, Macromol. Chem. Phys., 195, 1655-1672 (1994)
reports that Pseudomonas oleovorans produces PHA containing units
of 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-9-phenoxynonanoic
acid, from 11-phenoxyundecanoic acid.
[0017] Macromolecules, 29, 3432-3435 (1996) also reports that
Pseudomonas oleovorans can be used to produce PHA containing
3-hydroxy-4-phenoxyburyr- ic acid and 3-hydroxy-6-phenoxyhexanoic
acid units from 6-phenoxyhexanoic acid, PHA containing
3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic
acid units from 8-phenoxyoctanoic acid, and PHA containing
3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic
acid units from 11-phenoxyunndecanoic acid. The polymer yield is as
follows.
[0018] Furthermore, Can. J. Microbiol., 41, 32-43 (1995) reports
that when given octanoic acid and p-cyanophenoxyhexanoic acid or
p-nitrophenoxyhexanoic acid as substrates, Pseudomonas oleovorans
ATCC29347 or Pseudomonas putida KT2442 can produce PHA containing a
monomer unit of 3-hydroxy-p-cyanophenoxyhexanoic acid or
3-hydroxy-p-nitrophenoxyhexanoic acid.
[0019] Japanese Patent No. 2989175 describes a homopolymer
consisting of 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P)
unit or 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DFP)P) unit, a
copolymer containing at least one of 3H5(MFP)P unit and 3H5(DFP)P
unit, Pseudomonas putida which can produce such polymers; and a
method of producing the above polymers by using a Pseudomonas
sp.
[0020] Such productions are conducted by "2-step culture" described
below. Culture period: step 1-24 hours; step 2-96 hours.
[0021] Substrates in each step and polymers obtained are as
follows.
[0022] (1) Polymer obtained: 3H5(MFP)P homopolymer
[0023] Substrates in step 1: citric acid, yeast extract
[0024] Substrates in step 2: monofluorophenoxyundecanoic acid
[0025] (2) Polymer obtained: 3H5(DFP)P homopolymer
[0026] Substrates in step 1: citric acid, yeast extract
[0027] Substrates in step 2: difluorophenoxyundecanoic acid
[0028] (3) Polymer obtained: 3H5(MFP)P copolymer
[0029] Substrates in step 1: octanoic or nonanoic acid, yeast
extract
[0030] Substrates in step 2: monofluorophenoxyundecanoic acid
[0031] (4) Polymer obtained: 3H5(MFP)P homopolymer
[0032] Substrates in step 1: octanoic or nonanoic acid, yeast
extract
[0033] Substrates in step 2: difluorophenoxyundecanoic acid
[0034] It describes that the microorganism can assimilate
substituted aliphatic acids of a medium chain length to produce a
polymer having phenoxy group substituted with 1 to 2 fluorine atoms
at the end of a side chain, and such a polymer has stereoregularity
and water repellency while keeping a high melting point and a good
processibility.
[0035] It has been reported a PHA containing a cyclohexyl group in
its monomer unit is expected to exhibit polymer properties
differing from a PHA containing an usual aliphatic hydroxyalkanoic
acid as a unit, as well as its production by Pseudomonas oleovorans
(Macromolecules, 30, 1611-1615 (1997)).
[0036] According to this report, Pseudomonas oleovorans is cultured
in a medium containing nonanoic acid (hereinafter referred to as
NA), and 4-cyclohexylbutyric acid (hereinafter referred to as CHBA)
or 5-cyclohexylvaleric acid (hereinafter referred to as CHVA) to
obtain PHA made of a cyclohexyl-containing unit and a unit derived
from nonanoic acid (each proportion is unknown).
[0037] By varying the ratio of CHBA to NA under the conditions that
the total concentration of substrates is 20 mM, the results shown
in Table 2 were obtained. In Table 2, CDW: Cell mass (dry weight)
(mg/L); PDW: polymer mass (dry weight) (mg/L); and Yield: PDW/CDW
(%).
[0038] In this case, however, the polymer yield per culture (w/v)
was insufficient, and nonanoic acid--derived aliphatic
hydroxyalkanoic acid units were present in the resultant PHA.
[0039] As described above, to produce PHA having various introduced
substituents in the side chain, as with the above Pseudomonas
oleovorans, an alkanoate having a substituent to be introduced has
been utilized not only as a polymer raw material but also as a
carbon source for growth.
[0040] Such a method to utilize an alkanoate having a substituent
to be introduced into the polymer, not only as a raw material for
the polymer but also as a carbon source for growth expects to
supply the carbon source and energy source as the acetyl-CoA formed
by .beta.-oxidation of the alkanoate. In such a method, however,
acetyl-CoA would not be formed by .beta.-oxidation unless the
substrate has a certain chain length, so that there is a serious
problem that the alkanoate available as the substrate for PHA is
limited. In general, p-oxidation generates a new substrate, of
which chain length is shorter by two methylene units at a time, and
they are incorporated as the monomer units of PHA, synthesized PHA
is often a copolymer consisting of monomer units each differing by
two methylene chains in the chain length. In the foregoing report,
the produced polymer is a copolymer consisting of three monomer
units, that is, 3-hydroxy-8-phenoxyoctanoic acid derived from the
substrate 8-phenoxyoctanoic acid, 3-hydroxy-6-phenoxyhexanoic acid
and 3-hydroxy-4-phenoxybutyric acid being metabolic by-products.
Thus, PHA consisting of a single monomer unit is hard to obtain by
this method. Furthermore, in the method depending on the acetyl-CoA
formed by .beta.-oxidation as the carbon and energy source, there
are such problems as slow growth rate of the microorganism, slow
synthesis of PHA, and low yield of PHA.
[0041] Thus, usually the microorganism is grown in a medium
containing a medium-length aliphatic acid such as octanoic acid and
nonanoic acid, etc. as a carbon source for growth in addition to
the alkanoate having a substituent to be introduced, and then PHA
is extracted.
[0042] The PHA produced by the above method, however, contains
monomer units having a substituent to be introduced and monomer
units derived from the carbon source for growth (for example,
3-hydroxyoctanoic acid and 3-hydroxynonanoic acid). The polymer of
such a medium chain length (mcl) monomer unit is adhesive at
ambient temperature, and, when mixed with the desired PHA,
significantly lowers the glass transition point (Tg). Thus, to
obtain a polymer being solid at ambient temperature, contamination
of mcl-monomer units is undesirable. In addition, the presence of
heterogeneous side chains is known to interfere with intramolecular
or intermolecular interactions due to the side chain structure, and
significantly affects crystallinity and orientation. In order to
improve the polymer properties and endowment of functions, a
mixture of such mcl-monomer units is a serious problem. One means
to solve this problem is to add a purification step to separate and
remove such "unintended" polymers of mcl-monomer units derived from
the carbon source for growth and to obtain PHA consisting only of a
monomer unit having a specific substituent. Nevertheless,
operations become troublesome and a significant decrease of the
yield is inevitable. A more important problem is the fact that, if
the intended monomer units form a copolymer with the unintended
monomer units, it is very difficult to remove the unintended
monomer units only. In particular, when the PHA containing monomer
units having such groups as the groups derived from unsaturated
hydrocarbons, ester groups, aryl group, cyano group, nitro group,
groups derived from halogenated hydrocarbons and epoxide as side
chain structure, mcl-monomer units often form a copolymer with the
intended monomer unit, so it is very difficult to remove
mcl-monomer units after the PHA synthesis.
SUMMARY OF THE INVENTION
[0043] The present invention can solve the above problems. The
object of the present invention is to provide a PHA containing
monomer units of various structures having substituents useful for
device materials, medical materials, etc. in the side chains.
Another object of the present invention is to provide a method of
producing such a PHA by utilizing microorganisms, especially a
method of producing PHA with little contamination of monomer units
and in a high yield. The other object of the present invention is
to provide novel PHA consisting only of the desired monomer units
without contamination of unintended monomer units, as well as a
method of producing such a PHA by utilizing microorganisms.
[0044] In order to solve the above problems, especially to develop
PHA having substituted or unsubstituted phenoxy group, phenyl group
and cyclohexyl group in the side chains, being useful as device
materials, medical materials, etc., the present inventor have
extensively searched for novel microorganisms capable of producing
and accumulating PHA in the cell, and a method of producing the
desired PHA by utilizing novel microorganisms.
[0045] Further, to develop a method of obtaining efficiently the
desired PHA without mixing of unintended monomer units, the present
inventors made extensive study and found that by culturing the
microorganism in a medium supplemented with yeast extract in
addition to an alkanoate having a desired atomic group, it is
possible to produce selectively only the desired PHA without being
mixed with unintended monomer units or with reduced incorporation
of unintended monomer units, then completed the present
invention.
[0046] Thus, the method of producing novel PHA of the present
invention is characterized by culturing a microorganism in a
culture medium containing an alkanoate and yeast extract, which
microorganism is capable of producing the object PHA by utilizing
the alkanoate in the medium as a low material. In particular, the
method of producing PHA of the present invention can be carried out
in accordance with the embodiments described below.
[0047] According to one aspect of the present invention, there is
provided a polyhydroxyalkanoate comprising one or more of monomer
units represented by Formula (1), 3
[0048] where R is at least one selected from the group represented
by any one of Formulas (2), (3) and (4); 4
[0049] in Formula (2), R1 is selected from the group consisting of
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and q is an integer of 1 to
8;
[0050] in Formula (3), R2 is selected from the group consisting of
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3.
--C.sub.2F.sub.5 and --C.sub.3F.sub.7. and r is an integer of 1 to
8;
[0051] in Formula (4), R3 is selected from the group consisting of
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5 and --C.sub.3F.sub.7, and s is an integer of 1 to
8;
[0052] provided that following R is not selected:
[0053] when one kind of R is selected:
[0054] R being R1=H and q=2, R being R1=H and q=3 in Formula
(2),
[0055] R being R2=halogen and r=2, or R being R2=--CN and r=3
and
[0056] R being R2=--NO.sub.2 and r=3 in Formula (3);
[0057] when two kinds of R are selected:
[0058] a combination of R being R1=H and q=3 and 5 respectively in
Formula (2),
[0059] a combination of R being R2=H and r=2 and 4
respectively,
[0060] a combination of R being R2=H and r=2 and 6 respectively,
and
[0061] a combination of R being R2=halogen and r=2 and 4
respectively in Formula (3);
[0062] when three kinds of R are selected:
[0063] a combination of R being R1=H and q=3, 5 and 7 respectively
in Formula (2),
[0064] a combination of R being R2=H and r=1, 3 and 5 respectively,
and a combination of R being R2=H and r=2, 4 and 6 respectively in
Formula (3).
[0065] According to another aspect of the present invention, there
is provided a process of producing a polyhydroxyalkanoate
comprising the step of:
[0066] culturing a microorganism in a culture medium containing a
raw material alkanoate and an yeast extract, wherein the
microorganism produces a polyhydroxyalkanoate utilizing the
alkanoate.
[0067] The present invention provides a method for producing
polyhydroxyalkanoate, which uses .omega.-substituted-straight-chain
alkanoic acid, of which terminal of a chain is substituted by any
one of 6-carbon ring atomic group of a substituted or unsubstituted
phenyl group, a substituted or unsubstituted phenoxy group, and a
substituted or unsubstituted cyclohexyl group, as the material and
also which contains corresponding
.omega.-substituted-3-hydroxy-alkanoic acid as the monomer units,
and also provides microorganisms suitable for selective production
of polyhydroxyalkanoate having 6-carbon ring atomic group in the
terminal of these side chains. Various polyhydroxyalkanoate, of
which production by microorganisms becomes first possible according
to the present invention, in an inorganic culture medium containing
the yeast extract and the .omega.-substituted-straight-chain
alkanoic acid as the material, a microorganism belonging to the
genus Pseudomonas, for example, is cultured to work on the
.omega.-substituted-straight-chain alkanoic acid as the material
allowing an efficient production. Therefore, polyhydroxyalkanoate
useful as a functional polymer having biodegradability can be
expected application thereof to various fields such as a device
material and a material for a medical treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1 is an .sup.1H-NMR spectrum of PHA collected from the
cultured cells of strain P91 in Example A-2.
[0069] FIG. 2 is an NMR spectrum of 5-phenoxy valeric acid obtained
in Example B-1.
[0070] FIG. 3A is a total ion chromatogram (TIC) of GC-MS
measurement of a methyl esterified compound of the monomer unit
composing PHA obtained in Example B-2, and
[0071] FIG. 3B is a mass spectrum of a main peak in the TIC.
[0072] FIG. 4 is an NMR spectrum of PHA obtained in Example
B-2.
[0073] FIG. 5A is a total ion chromatogram (TIC) of GC-MS
measurement of a methyl esterified compound of the monomer unit
composing PHA obtained in Example B-3, and
[0074] FIG. 5B is a mass spectrum of a main peak in the TIC.
[0075] FIG. 6 is an NMR spectrum of 5-(4-fluorophenoxy) valeric
acid obtained in Example C-1.
[0076] FIG. 7A is a total ion chromatogram (TIC) of GC-MS
measurement of a methyl esterified compound of the monomer unit
composing PHA obtained in Example C-2, and
[0077] FIG. 7B is a mass spectrum of a main peak in the TIC.
[0078] FIG. 8 is an NMR spectrum of PHA obtained in Example
C-2.
[0079] FIG. 9A is a total ion chromatogram (TIC) of GC-MS
measurement of a methyl esterified compound of the monomer unit
composing PHA obtained in Example C-3, and
[0080] FIG. 9B is a mass spectrum of a main peak in the TIC.
[0081] FIG. 10 is an .sup.1H-NMR spectrum of
poly-3-hydroxy-5-phenyl valeric acid produced by strain H45 in
Example D-5.
[0082] FIG. 11 is a .sup.13C-NMR spectrum of
poly-3-hydroxy-5-phenyl valeric acid produced by strain H45 in
Example D-5.
[0083] FIG. 12 is a DNA sequence of a 16s rRNA coding region of
Pseudomonas jessenii P161; FERM BP-7376.
[0084] FIG. 13 is an NMR spectrum of FPVA biosynthesized as
alkanoate as the material in Example E-1.
[0085] FIG. 14 is a .sup.1H-NMR spectrum of PHA obtained by the
production method of the present invention using FPVA as the
material in Example E-5.
[0086] FIG. 15 is a .sup.13C-NMR spectrum of PHA obtained by in
Example E-5 using FPVA as the material.
[0087] FIG. 16 is a .sup.1H-NMR spectrum of PHA consisting of
3-hydroxy-4-cyclohexyl butyric acid unit purified in Example
F-3.
[0088] FIG. 17 is a .sup.13C-NMR spectrum of PHA consisting of
3-hydroxy-4-cyclohexyl butyric acid unit purified in Example
F-3.
[0089] FIG. 18 is a mass spectrum of a 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) methyl ester obtained by GC-MS of the polymer produced
in Example G-1.
[0090] FIG. 19 is a mass spectrum of 3-hydroxy-7-phenoxyheptanoic
acid (3HPxHp) methyl ester obtained by GC-MS of the polymer
produced in Example G-1.
[0091] FIG. 20 is a mass spectrum of 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) methyl ester obtained by GC-MS of the polymer produced
in Example G-2.
[0092] FIG. 21 is a mass spectrum of 3-hydroxy-7-phenoxyheptanoic
acid (3HPxHp) methyl ester obtained by GC-MS of the polymer
produced in Example G-2.
[0093] FIG. 22 is a mass spectrum of 3-hydroxy-4-phenoxy butyric
acid (3HPxB) methyl ester obtained by GC-MS of the polymer produced
in Example H-1.
[0094] FIG. 23 is a mass spectrum of 3-hydroxy-6-phenoxy hexanoic
acid (3HPxHx) methyl ester obtained by GC-MS of the polymer
produced in Example H-1.
[0095] FIG. 24 is a mass spectrum of 3-hydroxy-8-phenoxy octanoic
acid (3HPxO) methyl ester obtained by GC-MS of the polymer produced
in Example H-1.
[0096] FIG. 25 is a mass spectrum of 3-hydroxy-4-phenoxy butyric
acid (3HPxB) methyl ester obtained by GC-MS of the polymer produced
in Example H-2.
[0097] FIG. 26 is a mass spectrum of 3-hydroxy-6-phenoxy hexanoic
acid (3HPxHx) methyl ester obtained by GC-MS of the polymer
produced in Example H-2.
[0098] FIG. 27 is a mass spectrum of 3-hydroxy-8-phenoxy octanoic
acid (3HPxO) methyl ester obtained by GC-MS of the polymer produced
in Example H-2.
[0099] FIG. 28 is a mass spectrum of 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) methyl ester obtained by GC-MS of the polymer produced
in Example I-1.
[0100] FIG. 29 is a mass spectrum of 3-hydroxy-7-phenoxyheptanoic
acid (3HPxHp) methyl ester obtained by GC-MS of the polymer
produced in Example I-1.
[0101] FIG. 30 is a mass spectrum of 3-hydroxy-9-phenoxy nonanoic
acid (3HPxN) methyl ester obtained by GC-MS of the polymer produced
in Example I-1.
[0102] FIG. 31 is a mass spectrum of 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) methyl ester obtained by GC-MS of the polymer produced
in Example I-2.
[0103] FIG. 32 is a mass spectrum of 3-hydroxy-7-phenoxyheptanoic
acid (3HPxHp) methyl ester obtained by GC-MS of the polymer
produced in Example I-2.
[0104] FIG. 33 is a mass spectrum of 3-hydroxy-9-phenoxy nonanoic
acid (3HPxN) methyl ester obtained by GC-MS of the polymer produced
in Example I-2.
[0105] FIG. 34 is a mass spectrum of 3-hydroxy-6-phenyl hexanoic
acid (3HPHx) methyl ester obtained by GC-MS of the polymer produced
in Example J-1.
[0106] FIG. 35 is a mass spectrum of 3-hydroxy-4-phenyl butyric
acid (3HPB) methyl ester obtained by GC-MS of the polymer produced
in Example J-2.
[0107] FIG. 36 is a mass spectrum of 3-hydroxy-6-phenyl hexanoic
acid (3HPHx) methyl ester obtained by GC-MS of the polymer produced
in Example J-2.
[0108] FIG. 37 is a mass spectrum of 3-hydroxy-5-phenyl valeric
acid (3HPV) methyl ester obtained by GC-MS of the polymer produced
in Example K-1.
[0109] FIG. 38 is a mass spectrum of 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) methyl ester obtained by GC-MS of the polymer produced
in Example K-1.
[0110] FIG. 39 is a mass spectrum of 3-hydroxy-5-phenyl valeric
acid (3HPV) methyl ester obtained by GC-MS of the polymer produced
in Example K-2.
[0111] FIG. 40 is a mass spectrum of 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) methyl ester obtained by GC-MS of the polymer produced
in Example K-2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0112] The present invention relates to a novel
polyhydroxyalkanoate (PHA) and a method of producing PHA.
[0113] The first embodiment in the method of producing PHA of the
present invention is a production method characterized by
[0114] incubating a microorganism in a medium containing yeast
extract and an alkanoate of Formula (12) 5
[0115] wherein R is at least one or more groups selected from group
represented by any of the following general Formula (2), (3), or
(4)),
[0116] extracting a polyhydroxyalkanoate (PHA) from cells of the
microorganism, and
[0117] obtaining the PHA having a monomer unit of Formula (13),
6
[0118] wherein R' is at least one or more groups selected from a
group selected as R in Formula (12);
[0119] a group having the corresponding R1, wherein q=q.sub.0-2,
q=q.sub.0-4, or q=q.sub.0-6, provided that the group selected as R
is of Formula (2), wherein q=q.sub.0;
[0120] a group having the corresponding R2, wherein r=r.sub.0-2,
r=r.sub.0-4, or r=r.sub.0-6, provided that the group selected as R
is of Formula (3), wherein r=r.sub.0; and
[0121] a group having the corresponding R3, wherein s=s.sub.0-2,
s=s.sub.0-4, or s=s.sub.0-6, provided that the group selected as R
is of Formula (4), wherein s=s.sub.0,
[0122] provided that q.sub.0-2, r.sub.0-2, or s.sub.0-2, q.sub.0-4,
r.sub.0-4, or s.sub.0-4, q.sub.0-6, r.sub.0-6, or s.sub.0-6 can be
only the integer of one or more), 7
[0123] wherein R1 is a group selected from hydrogen atom (H),
halogen atom, --CN, --NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5,
--C.sub.3F.sub.7, and q is selected from the integer of 1 to 8;
[0124] in Formula (3), R2 is a group selected from hydrogen atom
(H), halogen atom, --CN, --NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5,
--C.sub.3F.sub.7, and r is selected from the integer of 1 to 8;
and
[0125] in Formula (4), R3 is a group selected from hydrogen atom
(H), halogen atom, --CN, --NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5,
--C.sub.3F.sub.7, and s is selected from the integer of 1 to 8). In
particular, it is the production method characterized by obtaining
polyhydroxyalkanoate consisting of a monomer unit represented by
the above general Formula (13).
[0126] In this method, PHA containing the corresponding monomer
unit, as well as, in some cases, an accompanying secondary monomer
unit having shorter carbon chain can be produced by using a single
kind of alkanoate of Formula (12) as a raw material. As mentioned
above, a plurality of alkanoates of Formula (12) can also be used
as a raw material, and at that time, it is preferred to use an
appropriate number of alkanoates, in consideration of function and
property requisite for polymer to be produced. In general, the
above aim can be expected to fully achieve by using up to about
five different alkanoates of Formula (12) as a raw material.
Furthermore, to finely control functions and properties, five or
more of different raw materials can be utilized. For example, the
total of more than five different raw materials can be used which
consist of up to about three different alkanoates selected each
from alkanoate group represented by the above general Formula (2),
(3), and (4).
[0127] The substituent R1 on benzene ring in the general Formula
(2) and the substituent R2 on benzene ring in the general Formula
(3) can be selected from any of ortho-position (2- or 6-position),
meta-position (3- or 5-position), or para-position (4-position).
The resultant polyhydroxyalkanoate will contain a monomer unit
having the corresponding substituted benzene ring. Which isomer is
selected as a raw material is properly determined depending on
intended functions and properties. When differences in the above
functions and properties is not a critical problem, an alkanoate
having substituent at para-position (4-position) on benzene ring
can be more advantageously used similar to an unsubstituted
alkanoate with respect to yield and readiness to be incorporated
into polymer. Similarly, the location of substituent R3 on
cyclohexyl ring of the general Formula (4) can be selected from any
of 1-, 2-(or 6-), 3-(or 5-), and 4-position, and both cis- and
trans-configuration can be selected. The resultant
polyhydroxyalkanoate will contain monomer unit having the
corresponding substituted cyclohexyl ring. Which isomer is selected
as a raw material is properly determined depending on intended
functions and properties. When differences in the above functions
and properties is not a critical problem, an alkanoate having
substituents at 4-position on cyclohexyl ring can be more
advantageously used similar to an unsubstituted alkanoate with
respect to yield and readiness to be incorporated into polymer.
Polyhydroxyalkanoate produced by microorganisms, which has chiral
center at 3-position carbon atom of monomer unit, is generally a
polymer consisting only of R-body, that is, an isotactic polymer.
As a result, PHA produced by the present method becomes a polymer
having biodegradability.
[0128] According to the method of the present invention,
microorganisms can be cultured by two steps comprising the initial
culture in the medium containing alkanoate of Formula (12) and
yeast extract, and subsequent culture in the medium containing the
alkanoate and restricted nitrogen source. Microorganism can also be
cultured in one step in the medium containing alkanoate of Formula
(12) and yeast extract. Furthermore, the microorganism utilized is
preferably selected from those belonging to Pseudomonas sp. As
examples of advantageously available strains belonging to
Pseudomonas sp., Pseudomonas cichorii YN2 (FERM BP-7375),
Pseudomonas cichorii H45 (FERM BP-7374), Pseudomonas putida P91
(FERM BP-7373), and Pseudomonas jessenii P161 (FERM BP-7376) can be
shown, and it is more preferred to select any of the above four
strains.
[0129] Preferred mode of the invention in the first embodiment of
the method of producing polyhydroxyalkanoate of the present
invention will be individually and more definitely described
below.
[0130] The present inventors have succeeded in obtaining
microorganism capable of producing poly-3-hydroxy-4-phenoxybutyric
acid (PHPxB) homopolymer consisting of a monomer unit of
3-hydroxy-4-phenoxybutyric acid (3HPxB) of Formula (5): 8
[0131] when cultured in a medium containing yeast extract and
4-phenoxybutyric acid (PxBA) of Formula (14). 9
[0132] Thus, one mode included in the above first embodiment
according to the method of producing polyhydroxyalkanoates of the
present invention is a method characterized by having a process of
incubating a microorganism capable of producing PHPxB homopolymer
consisting of repeats units of 3HPxB monomer unit of Formula (5) by
utilizing PxBA in the medium containing PxBA of Formula (14) and
yeast extract.
[0133] There have been no reports of the production of
polyhydroxyalkanoate containing a monomer unit of 3HPxB by
microorganisms by using PxBA as a substrate, as well as the
production of polyhydroxyalkanoate of PHPxB homopolymer by
microorganisms. PHPxB obtained by the above method, therefore, is
new, and is encompassed in the invention of novel
polyhydroxyalkanoates, which the present invention provides.
[0134] The present inventors have also succeeded in obtaining
microorganism capable of producing a homopolymer consisting of
3-hydroxy-5-phenoxyvaleric acid (3HPxV) monomer unit of Formula
(6): 10
[0135] when cultured in a medium containing yeast extract and
5-phenoxyvaleric acid (PxVA) of Formula (15). 11
[0136] Thus, another one mode included in the above first
embodiment is a method characterized by having a process of
incubating a microorganism capable of producing
poly-3-hydroxy-5-phenoxyvaleric acid (PHPxV) homopolymer consisting
of repeats units of 3HPxV monomer unit of Formula (6) by utilizing
PxVA in a medium containing PxVA of Formula (15) and yeast
extract.
[0137] There have been no reports of the production of
polyhydroxyalkanoates containing a monomer unit of 3HPxV by
microorganisms by using PxVA as a substrate, as well as the
production of polyhydroxyalkanoates of PHPxV homopolymer by
microorganisms. PHPxV obtained by the above method, therefore, is
new, and is encompassed in the invention of novel
polyhydroxyalkanoates provided by the present invention.
[0138] The present inventors have also succeeded in obtaining
microorganism capable of producing a homopolymer consisting of
3-hydroxy-5-(fluorophenoxy)valeric acid (3HFPxV) monomer unit of
Formula (16): 12
[0139] when cultured in the medium containing yeast extract and
5-(4-fluorophenoxy)valeric acid (FPxVA) of Formula (17). 13
[0140] Thus, another mode included in the above first embodiment is
a method characterized by having a process of incubating a
microorganism capable of producing
poly-3-hydroxy-5-(fluorophenoxy)valeric acid (PHFPxV) homopolymer
consisting of repeats units of 3HFPxV monomer unit of Formula (16)
by utilizing FPxVA in a medium containing FPxVA of Formula (17) and
yeast extract.
[0141] The present inventors have also succeeded in obtaining
microorganism capable of producing a copolymer consisting of
3-hydroxy-5-phenoxyvaleric acid (3HPxV) and
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) of Formula (6) and (22),
respectively: 14
[0142] when cultured in a medium containing yeast extract
7-phenoxyheptanoic acid (PxHpA) of Formula (23). 15
[0143] Thus, another mode included in the above first embodiment is
a method characterized by having a process to culture a
microorganism capable of producing a polyhydroxyalkanoate copolymer
consisting of 3HPxV and 3HPxHp monomer units of Formula (6) and
(22), respectively, by utilizing PxHpA in the medium containing
PxHpA of Formula (23) and yeast extract.
[0144] The present inventors have also succeeded in obtaining a
microorganism capable of producing a copolymer consisting of
3-hydroxy-4-phenoxybutyric acid (3HPxB),
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx), and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO) of Formula (5), (24), and
(25), respectively: 16
[0145] when cultured in a medium containing yeast extract and
8-phenoxyoctanoic acid (PxOA) of Formula (26). 17
[0146] Thus, another mode included in the above first embodiment is
the method characterized by having a process of incubating a
microorganism capable of producing a polyhydroxyalkanoate copolymer
consisting of 3HPxB, 3HPxHx, and 3HPxO monomer units of Formula
(5), (24), and (25), respectively, by utilizing PxOA in the medium
containing PxOA of Formula (26) and yeast extract.
[0147] A microorganism capable of producing a copolymer consisting
of 3-hydroxy-5-phenoxy valeric acid (3HPxV), 3-hydroxy-7-phenoxy
heptanoic acid (3HPxHp), and 3-hydroxy-9-phenoxy nonanoic acid
(3HPxN) units, which are expressed by Formula, was successfully
obtained. 18
[0148] One more mode included in the above described first
embodiment is a method having a step of culturing of microorganisms
to produce a polyhydroxyalkanoate copolymer consisting of 3HPxV,
3HPxHp, and 3HPxN monomer units expressed by the above described
Formulae (6), (22), and (27) using PxUDA in a culture medium
containing PxUDA expressed by the above described Formula (28) and
yeast extract.
[0149] Furthermore, in addition to the methods described in the
above described series of specific forms in detail, by using an
alkanoate, of Formula (12), in which a side chain having a phenoxy
group is replaced with a desired group as a material of the monomer
component, PHA having various corresponding side chains can be
selectively produced using microorganisms. A production method for
polyhydroxyalkanoate using alkanoate of Formula (12) as the
material and having the monomer unit composition shown by Formula
(13) is also included in the above described first embodiment by
the production method for polyhydroxyalkanoate of the present
invention. 19
[0150] (R is at least one or more groups selected from groups
expressed by Formula (3)); 20
[0151] (R' is the group selected in the above described Formula
(12) as R)
[0152] and in the case where expressed by the following Formula (3)
and is the group of r=r.sub.0, the group selected as the R has a
corresponding R2 and at least one or more group selected from
groups of r=r.sub.0-2, q=r.sub.0-4, or r=r.sub.0-6.)
[0153] r.sub.0-2, r.sub.0-4, or r.sub.0-6 can be an integer value
of 1 or more; 21
[0154] (R2 is the group selected from a hydrogen atom (H), halogen
atom, --CN, --NO.sub.2, --CF.sub.3'-C.sub.2F.sub.5, and
--C.sub.3F.sub.7 and r is selected from integers of 1 to 8.)
[0155] In many cases, when PHA is produced by containing one kind
of alkanoate expressed by Formula (12) as the material and a
corresponding monomer unit, in some cases, a by-produced monomer
unit of which carbon chain accompanied is reduced. On the other
hand, as described above, for alkanoate as the material expressed
by Formula (12), a plurality of kinds can be used for culture. In
consideration of a function and a physical property necessary for a
polymer produced, it is preferable to use a proper number of kinds.
In general, by using 3 kinds, in maximum, of alkanoate expressed by
Formula (12) as the material, it is expected that the above
described purpose can be sufficiently achieved. In addition, in the
purpose to control finely functionality and the physical property,
many kinds of materials more than three can be used.
[0156] For the material, any one of a substitution position of R2
on a benzene ring Formula (3) can be selected from an ortho
position (position 2 or position 6), meta position (position 3 or
position 5), or para position (position 4). Polyhydroxyalkanoate
yielded is that containing the monomer unit having a corresponding
substituted phenoxy group. An isomer to be selected as the material
is determined appropriately according to objective functionality
and physical property. In the case where a difference in the above
described functionality and physical property are not become the
problem, normally, that having the substitution group in the para
position (position 4) on the benzene ring can be more preferably
used, in a point of yield or easy uptake into the polymer,
comparably to that not substituted. In polyhydroxyalkanoate
produced by such microorganisms, a carbon atom of the position 3 of
the monomer unit has the chiral in a center and in general, is the
polymer consisting of only R-body and hence, isotactic polymer.
Consequently, PHA produced by such method is the polymer having
biodegradability.
[0157] In addition, the inventors successfully obtained the
microorganism capable of producing a homopolymer consisting of
3-hydroxy-5-phenylvaleri- c acid (3HPV) monomer unit expressed by
Formula (9), when cultured in a culture medium containing
5-phenylvaleric acid (PVA), expressed by Formula (18), and yeast
extract. 22
[0158] In other words, an alternative mode included in the above
described first embodiment is a method characterized by having a
step of culturing a microorganism which can produce a
poly-3-hydroxy-5-phenylvaleric acid (PHPV) homopolymer consisting
of repeated units of 3HPV monomer units expressed by the above
described Formula (9), using PVA in a culture medium containing PVA
of Formula (18) and yeast extract.
[0159] The inventors also successfully obtained the microorganism
capable of producing the homopolymer consisting of
3-hydroxy-5-(4-fluorophenyl) valeric acid (3HFPV) monomer unit
expressed by Formula (7) when cultured in a culture medium
containing 5-(4-fluorophenyl) valeric acid (FPVA) of Formula (19),
and yeast extract. 23
[0160] In other words, an alternative mode included in the above
described first embodiment is a method characterized by having a
step of cultivation of a microorganism which can produce a
poly-3-hydroxy-5-(4-fluorophenyl) valeric acid (PHFPV) homopolymer
consisting of repeated units of 3HFPV monomer units of Formula (7),
using FPVA in a culture medium containing FPVA of Formula (19) and
yeast extract.
[0161] So far, there is no report about production of
polyhydroxyalkanoate, in which 3HFPV as the monomer unit, using
FPVA as a substrate by microorganisms. Also, there is no report
about production of polyhydroxyalkanoate being the homopolymer of
PHFPV by microorganisms. Consequently, PHFPV yielded by the above
described method is a new product and included in the invention a
new polyhydroxyalkanoate provided by the present invention.
[0162] The inventors also successfully obtained the microorganism
capable of producing the copolymer consisting of
3-hydroxy-4-phenylbutyric acid (3HPB) and
3-hydroxy-6-phenylhexanoic acid (3HPHx) units expressed by Formulas
(10) and (11) when cultured in a culture medium containing
6-phenylhexanoic acid (PHxA), expressed by Formula (21), and yeast
extract, 24
[0163] In other words, an alternative mode included in the above
described first embodiment is a method characterized by having a
step of cultivation of a microorganism to produce a
polyhydroxyalkanoate copolymer consisting of 3HPB and 3HPHx monomer
units expressed by the above described Formula (10) and (11), using
PHxA in a culture medium containing PHxA, expressed by the above
described Formula (21), and the yeast extract.
[0164] So far, there is no report about production of
polyhydroxyalkanoate containing 3HPB and 3HPHx monomer units, using
PHxA as a substrate by microorganisms. Also, there is no report
about production of polyhydroxyalkanoate consisting of 3HPB and
3HPHx monomer units by microorganisms. Consequently,
polyhydroxyalkanoate consisting of 3HPB and 3HPHx monomer units and
yielded by the above described method is a new product and included
in the invention a new polyhydroxyalkanoate provided by the present
invention.
[0165] Furthermore, in addition to method described in the above
described series of specific forms in detail, by using an
alkanoate, of Formula (12), in which a side chain having a phenyl
group is substituted to a desired group as a material of the
monomer component, PHA having various corresponding side chains can
be selectively produced using microorganisms. A production method
for polyhydroxyalkanoate using alkanoate of Formula (12) as the
material and having the monomer unit composition shown by Formula
(13) is also included in the above described first embodiment by
the production method for polyhydroxyalkanoate of the present
invention. 25
[0166] (In Formula (12), R is at least one or more groups selected
from groups expressed by the following general Formula (2).) 26
[0167] (In Formula (13), R' is the group selected as R in the above
described Formula (12),
[0168] and if the group selected as R is
[0169] expressed by Formula (2) and is the group of q=q.sub.0, has
the corresponding R1 and at least one or more group selected from
q=q.sub.0-2, q=q.sub.0-4, or q=q.sub.0-6. q.sub.0-2, q.sub.0-4, or
q.sub.0-6 can be the integer value of 1 or more.) 27
[0170] (Of Formula (2), the R1 is the group selected from the
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5, and --C.sub.3F.sub.7 and q is selected from
integers of 1 to 8.)
[0171] In many cases, when PHA is produced by containing one kind
of alkanoate expressed by Formula (12) as the material and the
corresponding monomer unit, in some cases, the by-product monomer
unit of which carbon chain accompanied is reduced. On the other
hand, as described above, for alkanoate as the material expressed
by Formula (12), the plurality of kinds can be used for culture. In
consideration of the function and the physical property necessary
for the polymer produced, it is preferable to use the proper number
of kinds. In general, by using 3 kinds, in maximum, of alkanoate
expressed by Formula (12) as the material, it is expected that the
above described purpose can be sufficiently achieved. In addition,
in the purpose to control finely the functionality and the physical
property, many kinds of materials more than three can be used.
[0172] For the material, any one of the substitution position of R1
on the benzene ring of Formula (2) can be selected from the ortho
position (position 2 or position 6), meta position (position 3 or
position 5), or para position (position 4). Polyhydroxyalkanoate
yielded is that containing the monomer unit having the
corresponding substituted phenyl group. The isomer to be selected
as the material is determined appropriately according to objective
functionality and physical property. In the case where the
difference in the above described functionality and physical
property does not become the problem, normally, that having the
substitution group in the para position (position 4) on the benzene
ring can be more preferably used, in the point of yield or easy
uptake into the polymer, comparably to that not substituted. In
polyhydroxyalkanoate produced by such microorganisms, the carbon
atom of the position 3 of the monomer unit has the chiral in the
center and in general, is the polymer consisting of only R-body,
and hence, the isotactic polymer. Consequently, PHA produced by
such method is the polymer having biodegradability.
[0173] In addition, the inventors found that
[0174] when by using 4-cyclohexyl butyric acid (CHBA) as the
substrate, microorganisms are cultured in the culture medium
containing CHBA and yeast extract to produce polyhydroxyalkanoate
to accumulate in cells; the monomer unit of polyhydroxyalkanoate
contains 3-hydroxy-4-cyclohexyl butyric acid (3HCHB) in the high
ratio and showed the sufficiently high yield and for
polyhydroxyalkanoate containing 3HCHB yielded, found that the PHCHB
homopolymer consisting of the repeated unit of 3HCHB monomer units
can be separated by carrying out purification treatment.
[0175] In other words, an alternative mode included in the above
described first embodiment is
[0176] the method of producing poly-3-hydroxy-4-cyclohexyl butyric
acid (PHCHB) consisting of 3HCHB monomer units and 28
[0177] the method of producing PHCHB homopolymer consisting of
3HCHB monomer units expressed by Formula (8) and characterized by
having the step of cultivation of microorganisms in the culture
medium containing CHBA expressed by Formula (20) and the yeast
extract. 29
[0178] So far, there is no report about production of
polyhydroxyalkanoate being the homopolymer of PHCHB by
microorganisms. Consequently, PHCHB yielded by the above described
method is a new product and included in the invention of a new
polyhydroxyalkanoate provided by the present invention.
[0179] Furthermore, in addition to method described in the above
described series of specific forms in detail,
[0180] by using an alkanoate, of Formula (12), in which a side
chain having a cyclohexyl group is substituted to the desired group
as a material of the monomer component, PHA having various
corresponding side chains can be selectively produced using
microorganisms. A production method for polyhydroxyalkanoate using
alkanoate of Formula (12) as the material and having the monomer
unit composition shown by Formula (13) is also included in the
above described first embodiment by the production method for
polyhydroxyalkanoate of the present invention. 30
[0181] (In Formula (12), R is at least one or more groups selected
from groups expressed by the following general Formula (4).) 31
[0182] (In Formula (13), R' is the group selected as R in the above
described Formula (12),
[0183] and if the group selected as R is
[0184] expressed by Formula (4) and is the group of s=s.sub.0, it
has the corresponding R3 and at least one or more group selected
from s=s.sub.0-2, s=s.sub.0-4, or s=s.sub.0-6. s.sub.0-2,
s.sub.0-4, or s.sub.0-6 can be the integer value of 1 or more.)
32
[0185] (Of Formula (4), the R3 is the group selected from the
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5, and --C.sub.3F.sub.7 and s is selected from
integers of 1 to 8.)
[0186] In many cases, when PHA is produced by containing one kind
of alkanoate expressed by Formula (12) as the material and the
corresponding monomer unit, in some cases, the by-produced monomer
unit of which carbon chain accompanied is reduced. On the other
hand, as described above, for alkanoate as the material expressed
by Formula (12), the plurality of kinds can be used for culture. In
consideration of the function and the physical property necessary
for the polymer produced, it is preferable to use the proper number
of kinds. In general, by using 3 kinds, in maximum, of alkanoate
expressed by Formula (12) as the material, it is expected that the
above described purpose can be sufficiently achieved. In addition,
in the purpose to control finely the functionality and the physical
property, many kinds of materials more than three can be used.
[0187] For the material, any one of the substitution positions of
R3 on the cyclohexyl ring of Formula (4) can be selected from
position 1, position 2 (or position 6), position 3 (or position 5),
and position 4. In addition, either cis configuration or trans
configuration can be selected. Polyhydroxyalkanoate yielded is that
containing the monomer unit having the corresponding substituted
cyclohexyl ring. The isomer to be selected as the material is
determined appropriately according to objective functionality and
physical property. In the case where the difference in the above
described functionality and physical property does not become the
problem, normally, that having the substitution group in the 4 on
the cyclohexyl ring can be more preferably used, in the point of
yield or easy uptake into the polymer, comparably to that not
substituted. In polyhydroxyalkanoate produced by such
microorganisms, the carbon atom of the position 3 of the monomer
unit has the chiral in the center and in general, is the polymer
consisting of only R-body and hence, the isotactic polymer.
Consequently, PHA produced by such method is the polymer having
biodegradability.
[0188] In addition, the inventors successfully obtained a
microorganism capable of producing copolymer consisting of
3-hydroxy-5-phenoxy valeric acid (3HPxV) monomer unit expressed by
Formula (6) and 3-hydroxy-5-phenyl valeric acid (HPV) monomer unit
expressed by Formula (9) when cultured in the culture medium
containing yeast extract and 5-phenoxy valeric acid (PxVA)
expressed by Formula (15), 33
[0189] and
[0190] 5-phenyl valeric acid (PVA) expressed by Formula 34
[0191] In other words, an alternative mode included in the above
described first embodiment is the method of producing a
poly-(3-hydroxy-5-phenoxy valeric acid/3-hydroxy-5-phenyl valeric
acid) copolymer consisting of repeated units of the 3HPxV monomer
unit and the 3HPV monomer units expressed by the above described
Formula (6) and (9), using PxVA and PVA and characterized by having
the step of cultivation of microorganisms in the culture medium
containing PxVA and PVA expressed by the above described Formula
(15), (18), and the yeast extract.
[0192] So far, there is no report about production of
polyhydroxyalkanoate containing 3HPxV and 3HPV monomer units, using
PxVA and PVA as a substrate by microorganisms. Also, there is no
report about production of polyhydroxyalkanoate consisting of 3HPxV
and 3HPV monomer units by microorganisms. Consequently,
polyhydroxyalkanoate consisting of 3HPxV and 3HPV monomer units and
yielded by the above described method is a new product and included
in the invention the new polyhydroxyalkanoate provided by the
present invention.
[0193] Furthermore, in addition to method described in the above
described specific forms in detail,
[0194] by using a plurality of kinds of alkanoate, as the material,
selected from the following alkanoate of Formula (12),
polyhydroxyalkanoate containing a plurality of kinds of monomer
units having various corresponding side chains can be selectively
produced using microorganisms. In other words, the first embodiment
of the production method for polyhydroxyalkanoate of the present
invention includes the specific form of production method for
polyhydroxyalkanoate containing a plurality of kinds of monomer
units having various corresponding side chains by using a plurality
of kinds of alkanoate of as the material.
[0195] In other words, the production method is characterized in
that a microorganism is cultured in a culture medium containing
yeast extract and the alkanoate expressed by Formula (12): 35
[0196] (In Formula (12), R is at least one or more group selected
from groups expressed by any one of the following general Formula
(2), general Formula (3), or general Formula (4)), and the
microorganism is extracted to obtain polyhydroxyalkanoate expressed
by Formula (13) having at least one of the monomer units
represented by Formula (13): 36
[0197] (In Formula (13), R1 is the group selected as R in the above
described Formula (12),
[0198] and at least one or more groups selected from the groups
[0199] having the corresponding R1 and being q=q.sub.0-2,
q=q.sub.0-4, or q=q.sub.0-6, if the group selected as R is
expressed by Formula (2) and is the group of q=q.sub.0, having the
corresponding R2 and being r=r.sub.0-2, r=r.sub.0-4, or
r=r.sub.0-6, if the group selected as R is expressed by Formula (3)
and is the group of r=r.sub.0, and
[0200] having the corresponding R3 and being s=s.sub.0-2,
s=s.sub.0-4, or s=s.sub.0-6, if the group selected as R is
expressed by Formula (4) and is the group of s=s.sub.0.
[0201] q.sub.0-2, r.sub.0-2, or s.sub.0-2, or q.sub.0-4, r.sub.0-4,
or s.sub.0-4, or q.sub.0-6, r.sub.0-6, or s.sub.0-6, can be the
integer value of 1 or more.) 37
[0202] (Of Formula (2), the R1 is the group selected from the
hydrogen atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5, and --C.sub.3F.sub.7 and q is selected from
integers of 1 to 8; of Formula (3) the R2 is the group selected
from the hydrogen atom (H), halogen atom, --CN, --NO.sub.2,
--CF.sub.3, --C.sub.2F.sub.5, and --C.sub.3F.sub.7 and r is
selected from integers of 1 to 8; of Formula (4) the R3 is the
group selected from the hydrogen atom (H), halogen atom, --CN,
--NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5, and --C.sub.3F.sub.7 and
s is selected from integers of 1 to 8.) Specifically, the
production method is characterized in obtaining
polyhydroxyalkanoate consisting of monomer units holding side
chains corresponding to at least one alkanoate as the material. In
other words, the production method for polyhydroxyalkanoate having
monomer units characterized by having at least one or more side
chain structure, in which side chain structure of monomer units
corresponds to each alkanoate, particularly, consisting of each
monomer units derived from each alkanoate, is that in which a
plurality of kinds of alkanoate is used as the material, as monomer
units, which can be taken in polyhydroxyalkanoate, according to a
plurality of kinds of alkanoate expressed by Formula (12)
selected.
[0203] In this method, by using one kind of the alkanoate as the
material expressed by Formula (12), PHA containing the
corresponding monomer unit and in some cases, the by-produced
monomer unit of, which carbon chain accompanied is reduced, can
produced. On the other hand, as described above, for the alkanoate
as the material expressed by Formula (12), the plurality of kinds
can be used for culture. In consideration of the function and the
physical property necessary for the polymer produced, it is
preferable to use the proper number of kinds. In general, by using
about 5 kinds, in maximum, of alkanoate expressed by Formula (12)
as the material, it is expected that the above described purpose
can be sufficiently achieved. In addition, in the purpose to
control finely the functionality and the physical property, many
kinds of materials more than five can be used. For example, it is
possible to contain all three of the above described general
Formula (2), general Formula (3), or general Formula (4), select
three about kinds in maximum, and sum them to use more than 5 kinds
of materials.
[0204] The substitution group R1 on the benzene ring in the general
Formula (2) and the substitution group R2 on the benzene ring in
the general Formula (3) can be selected from any one of the ortho
position (position 2 or position 6), meta position (position 3 or
position 5), and para position (position 4). Polyhydroxyalkanoate
yielded is that containing the monomer unit having a corresponding
substituted benzene ring. An isomer to be selected as the material
is determined appropriately according to objective functionality
and physical property. In the case where a difference in the above
described functionality and physical property are not become the
problem, normally, that having the substitution group in the para
position (position 4) on the benzene ring can be more preferably
used, in the point of yield or easy uptake into the polymer,
comparably to that not substituted. Similarly, the substitution
group R3 on the cyclohexyl ring in the general Formula (4) can be
selected from any one of the position 1, position 2 (or position
6), position 3 (or position 5), and position 4. In addition, either
cis configuration or trans configuration can be selected.
Polyhydroxyalkanoate yielded is that containing the monomer unit
having the corresponding substituted cyclohexyl ring. The isomer to
be selected as the material is determined appropriately according
to objective functionality and physical property. In the case where
a difference in the above described functionality and physical
property are not become the problem, normally, that having the
substitution group in the position 4 on the cyclohexyl ring can be
more preferably used, in the point of yield or easy uptake into the
polymer, comparably to that not substituted. In
polyhydroxyalkanoate produced by such microorganisms, the carbon
atom of the position 3 of the monomer unit has the chiral in a
center and in general, is the polymer consisting of only R-body and
hence, isotactic polymer. Consequently, PHA produced by such method
is the polymer having biodegradability.
[0205] As described in detail showing representative specific forms
in the production method for polyhydroxyalkanoate of the present
invention, polyhydroxyalkanoate having various corresponding side
chains as the monomer components can be selectively produced by
using microorganisms by using a derivative, which was made by
substituting the side chain of alkanoate by the desired group, as
the material and therefore, the present invention also provides an
invention of polyhydroxyalkanoate, obtained by such method, having
a monomer composition expressed by the following general Formula
(1). In other words, a new polyhydroxyalkanoate according to the
present invention is polyhydroxyalkanoate consisting of monomer
units expressed by Formula (1). 38
[0206] (In Formula (1), R is at least one or more group selected
from groups expressed by any one of the following general Formula
(2), general Formula (3), or general Formula (4)). 39
[0207] (Of Formula (2), R1 is the group selected from the hydrogen
atom (H), halogen atom, --CN, --NO.sub.2, --CF.sub.3,
--C.sub.2F.sub.5, and --C.sub.3F.sub.7 and q is selected from
integers of 1 to 8; of Formula (3), the R2 is the group selected
from the hydrogen atom (H), halogen atom, --CN, --NO.sub.2,
--CF.sub.3, --C.sub.2F.sub.5, and --C.sub.3F.sub.7 and r is
selected from integers of 1 to 8; of Formula (4) the R3 is the
group selected from the hydrogen atom (H), halogen atom, --CN,
--NO.sub.2, --CF.sub.3, --C.sub.2F.sub.5, and --C.sub.3F.sub.7 and
s is selected from integers of 1 to 8.
[0208] Here, as R in the above described general Formula (1),
[0209] if one kind of group is selected,
[0210] in Formula (2), the group of q=2 in R1=H and the group of
q=3 in R1=H
[0211] in Formula (3), the group of r=2 in R2=halogen atom, the
group of r=3 in R2=--CN, and the group of r=3 in R2=--NO.sub.2
[0212] is eliminated from alternatives,
[0213] if two kind of group is selected,
[0214] in Formula (2), a combination of two groups of q=3 and 5 in
R1=H,
[0215] in Formula (3), the combination of two groups of r=1 and 3
in R2=H, the combination of two groups of r=2 and 4 in R2=H, the
combination of two groups of r=2 and 6 in R2=H, and the combination
of two groups of r=2 and 4 in R2=halogen atom
[0216] is eliminated from alternatives,
[0217] if three kind of group is selected,
[0218] in Formula (2), the combination of three groups of q=3, 5,
and 7 in R1=H,
[0219] in Formula (3), the combination of three groups of r=1, 3,
and 5 in R2=H and the combination of three groups of r=2, 4, and 6
in R2=H
[0220] is eliminated from alternatives.)
[0221] Polyhydroxyalkanoate of the present invention, as described
above, can contain one kind of monomer unit expressed by the
general Formula (1) and also contain a plurality of kinds. In
consideration of a function and a physical property necessary for
the objective polymer, it is preferable to use a proper number of
kinds of monomer units. In general, by selecting 10 kinds of
monomer units, in total, expressed by Formula (1), it is expected
that the above described purpose can be sufficiently achieved. In
addition, in the purpose to control finely functionality and the
physical property, many kinds of monomer units more than 10 can be
contained in configuration.
[0222] For example, when polyhydroxyalkanoate is produced by
containing a plurality of kinds of monomer units expressed by such
general Formula (1), in addition to monomer units corresponding to
alkanoate as the material, in some cases, a by-produced monomer
unit of which carbon chain accompanied is reduced. Therefore, the
present invention include that in which even if alkanoate itself is
about 5 kinds as the material, each yields monomer unit of two
kinds or more including the by-produced monomer unit and as the
results, PHA contains monomer units of 10 kinds or more in total.
In addition, for example, the present invention include that in
which contains all of three kinds of the above described general
Formula (2), general Formula (3), and general Formula (4), of which
about 3 kinds in maximum are selected each, and becomes about 10
kinds of alkanoate as the material in total to yield PHA containing
monomer units of 10 kinds or more containing a little numbers of
the by-produced monomer unit.
[0223] Any one of the substitution position of R1 on a benzene ring
Formula (2) and the substitution position of R2 on a benzene ring
Formula (3) can be selected from the ortho position (position 2 or
position 6), meta position (position 3 or position 5), and para
position (position 4). Polyhydroxyalkanoate yielded is that
containing the monomer unit having a corresponding substituted
benzene group. An isomer to be selected as the material is
determined appropriately according to objective functionality and
physical property. In the case where a difference in the above
described functionality and physical property are not become the
problem, normally, that having the substitution group in the para
position (position 4) on the benzene ring can be more preferably
used, in a point of yield or easy uptake into the polymer,
comparably to that not substituted. Similarly, for the substitution
position of R3 on the cyclohexyl ring of the general Formula (4),
any one of position 1; position 2, (or, position 6), position 3
(or, position 5), and position 4 can be selected and in addition,
either cis configuration or trans configuration can be selected. In
polyhydroxyalkanoate produced is that containing the monomer unit
having the corresponding substituted cyclohexyl ring. The isomer to
be selected is determined appropriately according to objective
functionality and physical property. In the case where a difference
in the above described functionality and physical property are not
become the problem, normally, that having the substitution group in
the position 4 on the cyclohexyl ring can be more preferably used,
in a point of yield or easy uptake into the polymer, comparably to
that not substituted. In polyhydroxyalkanoate produced by such
microorganisms, the carbon atom of the position 3 of the monomer
unit has the chiral in a center and in general, is the polymer
consisting of only R-body and hence, isotactic polymer.
Consequently, PHA producible by such method using microorganisms is
the polymer having biodegradability.
[0224] The production method for PHA of the present invention is
characterized in that in cultivation of microorganisms, by adding
the yeast extract to alkanoate of Formula (12) as the material in
the culture medium, content ratio of the objective monomer unit in
PHA is increased very high or PHA consists of the objective monomer
unit only in PHA produced by microorganisms to accumulate. An
effect to enhance prioritization of a specified monomer unit is
realized by adding only yeast extract to the culture medium as a
carbon source other than alkanoate used as the material of PHA.
[0225] As an example of use of the yeast extract in the culture
medium in production of PHA by microorganisms, the method described
on the Japanese Patent Application Laid-Open No.5-49487 and using
microorganisms belonging to the genus Rhodobacter is exemplified.
However, this conventional method is the method for production of
common PHB and PHV by using hydroxyalkanoic acid having no
substitution group as the monomer unit. It has been known that a
biosynthetic path of the objective PHA of the present invention is
an independent path from the biosynthetic path to produce PHB and
PHV. In the Japanese Patent Application Laid-Open No. 5-49487,
there is no mention about the effect of the yeast extract in the
PHA biosynthetic path of the object of the present invention. In
addition, it has been evidently described about the effect of the
yeast extract that for PHB and PHV commonly produced by
microorganisms, addition of the yeast extract shows the effect to
increase in PHA accumulation simply in cells and the yeast extract
is not added for cell proliferation. The present invention carries
out production and accumulation of PHA as well as proliferation by
coexistence of alkanoate of Formula (12) with the yeast extract and
thus, the yeast extract expresses a quite different effect. In
addition, prioritization, being the effect of the present
invention, of the specified monomer unit was never mentioned and
therefore, in the composition of PHA produced by microorganisms,
the effect, found in the present invention, of prioritization of
the specified monomer unit having phenoxy group, phenyl group, and
cyclohexyl group as the substitution group was not shown.
[0226] As an example using the yeast extract for PHA production by
microorganisms, the method, described on the above described
Japanese Patent No. 2989175, using Pseudomonas putida is
exemplified. The production method for PHA disclosed in this patent
uses the two-step cultivation and it is disclosed that PHA
accumulation is carried out in only the cultivation of a second
step under limitation of a nutrient source other than carbon
source. In this point, this method quite differs in configuration
and effect from the method of the present invention, in which
cultivation is one step only in the culture medium containing
alkanoate of Formula (12) and the yeast extract to perform
biosynthesis and accumulation of the desired PHA. The effect of the
yeast extract in Japanese Patent No. 2989175 simply aims
proliferation of microorganisms used for cultivation of the second
step in the first step cultivation in using the two-step
cultivation and it has been evidently described that the first step
cultivation is carried out under a nutrient rich condition. Here,
the substrate of PHA does not coexist with the first step
cultivation. The effect of the yeast extract in the two-step
cultivation of the present invention is that doing production and
accumulation of PHA as well as cell proliferation in the first step
cultivation by coexisting of alkanoate of Formula (12) and the
yeast extract in the first step cultivation, and the effect
expressed by the yeast extract in the first step cultivation is
quite different. In addition, in Japanese Patent No. 2989175, any
one of citric acid, octanoic acid, nonanoic acid coexists as the
carbon source in the first step cultivation and hence, it also
differs in configuration from the present invention, in which
alkanoate of Formula (12) and the yeast extract coexist.
[0227] As an example of a report of a microorganism, which can
produce PHA, containing 3HPxB in the present invention as the
monomer unit, to accumulated in the cell, there is the method using
Pseudomonas oleovorans described on Macromolecules 29, 3432-3435,
1996. However, the method using Pseudomonas oleovorans uses only
8-phenoxyoctanoic (PxOA) as the substrate and therefore, it has an
essential difference in which it cannot produce acetyl-Co A by
p-oxidation, for example, of the present invention and it quite
differs from the method using PxBA as the substrate together with
the yeast extract. For PHA biosynthesized, the copolymer, which
consists of three kinds of monomers of 3-hydroxy-8-phenoxy octanoic
acid derived from PxOA as the substrate, 3-hydroxy-6-phenoxy
hexanoic acid being the by-product derived from a metabolite, and
3HPxB, is produced. In contrast, in the present invention, use of
the yeast extract allows production of PHA containing only 3HPxB
derived from PxBA as the monomer unit containing the phenoxy group.
PHA itself to be produced differs clearly between the above
described reported and the present invention. In addition, there is
no report of the production of PHA, containing 3HPxB as the monomer
unit, by microorganisms using PxBA as the substrate. In addition,
there is no report of the production of PHA, containing only 3HPxB
as the monomer unit containing the phenoxy group, by microorganisms
using PxBA as the substrate.
[0228] The production method of the present invention will be
described below in detail.
[0229] Microorganisms used for the present invention may be any
microorganisms, which is microorganisms capable of production of
PHA from the alkanoate to accumulate using the alkanoate of Formula
(12) as the substrate. According to the inventors' study, bacteria
of Pseudomonas is good and among them, we found that Pseudomonas
cichorii YN2, FERM BP-7375; Pseudomonas cichorii H45, FERM BP-7374;
Pseudomonas putida P91, FERM BP-7373; and Pseudomonas jessenii P161
FERM BP-7376 are preferable microorganisms. Also using cells other
than these strains, by cultivation using the alkanoate as the
substrate, by carrying out screening of bacteria belonging to, for
example, the genus Pseudomonas, microorganisms usable for the
production method for PHA, of the present invention, can be
obtained. For example, as bacteria belonging to the genus
Pseudomonas, using Pseudomonas oleovorans is possible. In addition
to microorganisms belonging to the genus Pseudomonas, use of
microorganisms, which belongs to the genera Aeromonas, Comamonas,
and Burkholderia and, by using the alkanoate as the material
(substrate), produce PHA containing corresponding
3-hydroxyalkanoate as the monomer unit, is possible. However, in
consideration of productivity, the above described 4 strains can be
recommended as the most preferable strains.
[0230] The details of strains YN2, H45, P91, and P161 will be
listed below.
[0231] <Bacteriological Properties of Strain YN2>
[0232] Cultivation temp.: 30.degree. C.
[0233] Morphology:
[0234] Cell form: rod, 0.8 .mu.m.times.(1.5 to 2.0) .mu.m
[0235] Gram staining: negative
[0236] Spore formation: negative
[0237] Mobility: motile
[0238] Form of colony: circular, entire smooth margin, low convex,
smooth surface, glossy, translucent
[0239] Physiological properties:
[0240] Catalase: positive
[0241] Oxidase: positive
[0242] O/F test: non-fermentable
[0243] Nitrate reduction: negative
[0244] Indole production: positive
[0245] Glucose acidification: negative
[0246] Arginine dihydrolase: negative
[0247] Urease: negative
[0248] Esculin hydrolysis: negative
[0249] Gelatin hydrolysis: negative
[0250] .beta.-galactosidase: negative
[0251] Substrate assimilation:
[0252] Glucose: positive
[0253] L-arabinose: positive
[0254] D-mannose: negative
[0255] D-mannitol: negative
[0256] N-acetyl-D-glucosamine: negative
[0257] Maltose: negative
[0258] Potassium gluconate: positive
[0259] n-capric acid: positive
[0260] Adipic acid: negative
[0261] dl-malic acid: positive
[0262] Sodium citrate: positive
[0263] Phenyl acetate: positive
[0264] Production of fluorescence on King's B agar: positive
[0265] Growth in 4% NaCl: positive (weak)
[0266] Accumulation of poly-p-hydroxy butyric acid: negative
(determined by staining with Sudan Black a colony grown on a
nutrient agar)
[0267] Hydrolysis of Tween 80: positive
[0268] <Bacteriological Properties of Strain H45>
[0269] Morphological characteristics:
[0270] Cell shape and size: rod, 0.8 .mu.m.times.(1.0 to 1.2)
.mu.m
[0271] Cell polymorphism: absent
[0272] Mobility: positive
[0273] Spore formation: negative
[0274] Gram staining: negative
[0275] Form of colony: circular, entire margin smooth, low convex,
smooth surface, glossy, and cream color
[0276] Physiological properties:
[0277] Catalase: positive
[0278] Oxidase: positive
[0279] O/F test: oxidative
[0280] Nitrate reduction: negative
[0281] Indole production: negative
[0282] Glucose acidification: negative
[0283] Arginine dihydrolase: negative
[0284] Urease: negative
[0285] Esculin hydrolysis: negative
[0286] Gelatin hydrolysis: negative
[0287] .beta.-galactosidase: negative
[0288] Production of fluorescence on King's B agar: positive:
[0289] Growth in 4% NaCl: negative
[0290] Accumulation of poly-p-hydroxy butyric acid: negative
[0291] Substrate assimilation:
[0292] Glucose: positive
[0293] L-arabinose: negative
[0294] D-mannose: positive
[0295] D-mannitol: positive
[0296] N-acetyl-D-glucosamine: positive
[0297] Maltose: negative
[0298] Potassium gluconate: positive
[0299] n-capric acid: positive
[0300] Adipic acid: negative
[0301] dl-malic acid: positive
[0302] Sodium citrate: positive
[0303] Phenyl acetate: positive
[0304] <Bacteriological Properties of Strain P91>
[0305] Morphological characteristics:
[0306] Cell shape and size: rod, 0.6 .mu.m.times.1.5 .mu.m
[0307] Cell polymorphism: absent
[0308] Mobility: positive
[0309] Spore formation: negative
[0310] Gram staining: negative
[0311] Form of colony: circular, entire margin smooth, low convex,
smooth surface, glossy, creamy color
[0312] Physiological properties:
[0313] Catalase: positive
[0314] Oxidase: positive
[0315] O/F test: oxidative
[0316] Nitrate reduction: negative
[0317] Indole production: negative
[0318] Glucose acidification: negative
[0319] Arginine dihydrolase: positive
[0320] Urease: negative
[0321] Esculin hydrolysis: negative
[0322] Gelatin hydrolysis: negative
[0323] .beta.-galactosidase: negative
[0324] Production of fluorescence on King's B agar: positive:
[0325] Substrate assimilation:
[0326] Glucose: positive
[0327] L-arabinose: negative
[0328] D-mannose: negative
[0329] D-mannitol: negative
[0330] N-acetyl-D-glucosamine: negative
[0331] Maltose: negative
[0332] Potassium gluconate: positive
[0333] n-capric acid: positive
[0334] Adipic acid: negative
[0335] dl-malic acid: positive
[0336] Sodium citrate: positive
[0337] Phenyl acetate: positive
[0338] <Bacteriological Properties of Strain P161>
[0339] Morphological characteristics:
[0340] Cell shape and size: coccus, diameter 0.6 .mu.m or bacillus
0.6 .mu.m.times.1.5 to 2.0 .mu.m
[0341] Cell polymorphism: elongation
[0342] Mobility: positive
[0343] Spore formation: negative
[0344] Gram staining: negative
[0345] Form of colony: circular, entire margin smooth, low convex,
smooth surface, and light yellow
[0346] Physiological properties:
[0347] Catalase: positive
[0348] Oxidase: positive
[0349] O/F test: oxidative
[0350] Nitrate reduction: positive
[0351] Indole production: negative
[0352] Glucose acidification: negative
[0353] Arginine dihydrolase: positive
[0354] Urease: negative
[0355] Esculin hydrolysis: negative
[0356] Gelatin hydrolysis: negative
[0357] .beta.-galactosidase: negative
[0358] Production of fluorescence on King's B agar: positive
[0359] Substrate assimilation:
[0360] Glucose: positive
[0361] L-arabinose: positive
[0362] D-mannose: positive
[0363] D-mannitol: positive
[0364] N-acetyl-D-glucosamine: positive
[0365] Maltose: negative
[0366] Potassium gluconate: positive
[0367] n-capric acid: positive
[0368] Adipic acid: negative
[0369] dl-malic acid: positive
[0370] Sodium citrate: positive
[0371] Phenyl acetate: positive
[0372] From the above described bacteriological properties,
according to identification based on the Bergey's Manual of
Systematic Bacteriology, Vol. 1 (1984) and Bergey's Manual of
Determinative Bacteriology, 9th Ed. (1994), strains YN2 and H45 are
identified to belong to Pseudomonas cichorii and strain P91 to
Pseudomonas putida, respectively. Therefore, we gave names these
strains as Pseudomonas cichorii YN2, Pseudomonas cichorii H45, and
Pseudomonas putida P91.
[0373] On the other hand, though strain P161 was identified as to
belong to the genus Pseudomonas, taxonomic identification was
impossible on the basis of bacteriological properties. Then, in
order to attempt identification based on genetic criteria, the DNA
sequence, as shown in the FIG. 12, of 16s rRNA of P 161 was
determined (SEQ ID NO:1) to test the homology with the DNA
sequences of 16s rRNA of known microorganisms of genus Pseudomonas.
As the result, a very high homology was found between sequences of
the strain P161 and Pseudomonas jessenii. In addition, a high
similarity was found between bacteriological properties of
Pseudomonas jessenii, described on System. Appl. Microbiol.
20:137-149 (1997) and System. Appl. Microbiol. 22:45-58 (1999), and
bacteriological properties of the strain P161. From the above
described results, the strain P161 can be justifiably identified as
Pseudomonas jessenii and hence, we named the strain P161 as
Pseudomonas jessenii P161.
[0374] The strains YN2, H45, P91 and P161 are deposited in National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, the Japanese Ministry of International
Trade and Industry by receiving identification code of FERM
BP-7375, FERM BP-7374, FERM BP-7373, and FERM BP-7376,
respectively.
[0375] By culturing these microorganisms using a material for
introducing the desired monomer unit and a culture medium
containing an alkanoate of the general Formula (12) and yeast
extract, the objective PHA can be produced.
[0376] For normal cultivation microorganisms used for the
production method for PHA of the present invention, for example,
cultivation for preparation of a preservation of cell strain and
keeping of cell number and active condition, excluding that
influencing badly to growth and survival of microorganisms, any
kinds of culture medium, for example, a general culture medium and
an synthetic culture medium to which the nutrient source has been
added, can be used. A cultivation condition such as temperature,
aeration, and stirring is properly adjusted in accordance with the
microorganism used.
[0377] On the other hand, in the case where production and
accumulation of PHA is carried out by microorganisms, as the
culture medium for PHA production, an inorganic culture medium
containing at least alkanoate of the corresponding general Formula
(12) can be used. It is the feature that in this case, as carbon or
energy source other than alkanoate as the material of PHA, only the
yeast extract is added.
[0378] As the inorganic culture medium used for the above described
culture method, any one containing necessary components, such as a
phosphor source (for example, phosphate salt) and nitrogen source
(for example, ammonium salt and nitrate salt), for proliferation of
microorganisms can be used. For example, as a representative
inorganic culture medium, can be exemplified by MSB medium, E
medium (J. Biol. Chem. 218: 97-106 (1956)), and M9 medium.
[0379] The composition of the M9 culture medium used in Examples of
the present invention is as follows.
1 Na.sub.2HPO.sub.4: 6.2 g KH.sub.2PO.sub.4: 3.0 g NaCl: 0.5 g
NH.sub.4Cl: 1.0 g (for one liter culture medium; pH 7.0)
[0380] As the cultivation condition, shaking culture and stirring
culture under 15 to 40.degree. C., preferably 20 to 35.degree. C.,
and aerobic condition are exemplified.
[0381] For cultivation steps, any method, used for normal
microorganisms, such as batch system, fluidized batch system,
semi-continuous culture, continuous culture, and reactor type can
be used. A multi-step system by connecting a plurality of these
steps may be utilized.
[0382] For example, as the method including the two steps of
culture steps, in the first step, as the carbon source for cell
proliferation, employing the inorganic culture medium containing
about 0.1 wt % to 1.0 wt % of the yeast extract and about 0.01 wt %
to 0.5 wt % of alkanoate of Formula (12), cultivation is conducted
from the late stage of the logarithmic growth period to the point
of a standing state period and in the second step, cells after
completion of cultivation in the first step are collected by a
centrifugation followed by a further cultivation in the inorganic
culture medium containing about 0.01 wt % to 0.5 wt % of alkanoate
of Formula (12) as the material and lacking any nitrogen source and
after completion of cultivation, cells are collected to extract
desired PHA.
[0383] There is the method in which cultivation is conducted in the
inorganic culture medium to which about 0.1 wt % to 1.0 wt % of the
yeast extract and about 0.01 wt % to 0.5 wt % of alkanoate of
Formula (12) and at the point from the late stage of the
logarithmic growth period to a standing state period, cells are
collected to extract desired PHA.
[0384] In this case, a concentration of the yeast extract to be
added to the culture medium is properly chosen in accordance with
the kind of alkanoate of Formula (12), species and genus of the
microorganism, a density of cells, or culture method. Normally, it
is preferable to choose from a range from about 0.1 wt % to 1.0 wt
% of content ratio in the culture medium in order to add it. On the
other hand, for the yeast extract, any one of commercialized yeast
extract generally used for cultivation of microorganisms can be
preferably used. In addition, in replacement to the yeast extract,
that prepared by pulverizing a lyophilized yeast product, which
naturally contains the components of the yeast extract, can be also
used. On the other hand, the concentration of alkanoate of Formula
(12) as the material is properly chosen in accordance with species
and genus of the microorganism, a density of cells, or culture
method. Normally, it is preferable to choose from a range from
about 0.01 wt % to 0.5 wt % of content ratio in the culture medium
in order to add it.
[0385] In the case where any one of the strains YN2, H45, P91 and
P161 previously described are used, in replacing to the yeast
extract, a middle chain fatty acid of C.sub.6 to C.sub.12 (for
example, octanoic acid, nonanoic acid, and the like), for example,
is used as the carbon source for cell proliferation, PHA yielded is
that in which the monomer unit derived from the middle chain fatty
acid, which has been added, is mixed. Specifically, the case
employing the method is exemplified, in which employing the
inorganic culture medium, in which as the carbon source for cell
proliferation, the middle chain fatty acid such as octanoic acid,
nonanoic acid, and the like are added and also alkanoate of Formula
(12) is added as the material, cultivation is conducted from the
late stage of the logarithmic growth period to the point of a
standing state period, cells are collected by centrifugation and
then, the middle chain fatty acid and alkanoate of Formula (12) are
added to carry out further cultivation in the inorganic culture
medium lacking the nitrogen source. Or, the case employing the
method is exemplified, in which the concentration of the nitrogen
source is limited to {fraction (1/10)} in the inorganic culture
medium, cultivation is carried out in the culture medium to which
the middle chain fatty acid and alkanoate of Formula (12) are
added, cells are collected in the period from the late stage of the
logarithmic growth period to the point of a standing state period
to extract the desired PHA. In the case where these methods, in
which the middle chain fatty acid is added to the culture medium as
the carbon source for cell proliferation, is applied, PHA yielded
is becomes PHA, to which the monomer unit, derived from the middle
chain fatty acid is added as the carbon source for cell
proliferation, has been mixed.
[0386] In contrast, in the present invention, as described above,
by cultivation of the above described microorganisms in the culture
medium containing the yeast extract, alkanoate of Formula (12), and
no other carbon source, the desired PHA, which contains a little
amount of or no amount of any unnecessary monomer unit other than
the monomer unit derived from the objective alkanoate of Formula
(12), is produced and accumulated.
[0387] Collection of PHA from cells in the method according to the
present invention is most conveniently carried out by extraction
using such organic solvent as chloroform normally used. However, in
an environment, in which the organic solvent is difficultly used,
the method to collect PHA by removing cell components other than
PHA by treatment with such surfactant as SDS, treatment with such
enzyme as lysozyme, and treatment with such reagent as EDTA, sodium
hypochlorite, and ammonium can be used.
[0388] The cultivation of microorganisms, production of PHA by
microorganisms and accumulation in cells, and collection of PHA
from cells are not restricted to the above described methods.
[0389] For example, and microorganisms used for production method
of PHA, according to the present invention, microorganisms, other
than the above described 4 bacterial strains, having productivity
of PHA production, according to the present invention and similar
to that of these 4 bacterial strains, can be used.
[0390] By using the above described methods, PHA having repeated
units expressed by Formula (1) can be yielded. It is preferable
that a number average molecular weight of this PHA is at least
10000 or more and a range of 10000 to 200000 is more preferable. In
other words, to get stably a desired characteristic as a polymer,
specifically, it is preferable that the PHA has repetition number
to make the number average molecular weight to at least about 10000
to make characteristics, such as a glass transition temperature,
softening point, melting point, crystallinity, and orientation
which are designated by a structure of the monomer unit composing
the monomer, to a specified range. On the other hand, for
processing and the like, in consideration of convenience of
processing such as dissolving operation, the number average
molecular weight is preferably under about 200000. Usually, the
range from 10000 to 100000 is more preferable. The number average
molecular weight of PHA yielded by the production method according
to the present invention is 10000 or more and about 20000 or more
and is within the range expectable sufficiently of a stable
expression of a physical property of the above described
polymer.
EXAMPLES
[0391] Specific example will be presented below and the present
invention will be explained in more detail. These specific examples
are examples of the best mode according to the present invention.
However, the present invention is not restricted to the following
specific examples.
[0392] A:
[0393] An example, in which the production method for
polyhydroxyalkanoate according to the present invention is applied
to the production of polyhydroxyalkanoate: poly-3-hydroxy-4-phenoxy
butyric acid (PHPxB) consisting of the monomer unit, which is
derived from 3-hydroxy-4-phenoxy butyric acid (3HPxB) expressed by
Formula (5) using 4-phenoxy butyric acid (PxBA) of Formula (14) as
the material, will be shown.
Example A-1
[0394] The strain P 91 was inoculated in a 200 ml of the M9 culture
medium containing the yeast extract of 0.5% and PxBA of 0.1% and
subjected to shaking-cultivation at 30.degree. C. and 125
strokes/min. After 24 hours, cells were collected by
centrifugation, suspended again in a 200 ml of the M9 culture
medium containing a 0.1% PxBA and lacking the nitrogen source
(NH.sub.4Cl), and subjected to the shaking-cultivation at
30.degree. C. and 125 strokes/min. After 24 hours, cells were
collected by centrifugation, washed with cold methanol once, and
subjected to freeze-dry.
[0395] This lyophilized pellet was suspended in chloroform of 100
ml and PHA was extracted by stirring at 60.degree. C. for 20 hours.
An extracted fluid was filtered using a membrane filter with a pore
size of 0.45 .mu.m followed by concentration by a rotary evaporator
and then, concentrated fluid was precipitated again in cold
methanol and precipitation only was collected and dried in vacuo to
yield PHA. PHA yielded was subjected to methanolysis by a routine
method followed by analysis by a gas chromatography mass
spectrometry apparatus (GC-MS, Shimadzu QP-5050, EI method) and a
methyl esterified product of the PHA monomer unit was identified.
On the other hand, the molecular weight of this PHA was measured by
gel permeation chromatography (GPC; Toso, HLC-8020, column: Polymer
Laboratory, PLgel-MIXED-C (5 .mu.m), solvent: chloroform,
polystyrene converted molecular weight.)
[0396] Table 3 shows the result of identification and an average
molecular weight. It is known that PHA yielded contains only
monomer unit derived from 3-hydroxy-4-phenoxy butyric acid
expressed by Formula (5) as the monomer unit and is a
poly-3-hydroxy-4-phenoxy butyric acid.
Example A-2
[0397] The strain P 91 was inoculated in a 200 ml of the M9 culture
medium containing the yeast extract of 0.5% and PxBA of 0.2% and
subjected to shaking-cultivation at 30.degree. C. and 125
strokes/min. After 48 hours, cells were collected by
centrifugation, washed with cold methanol once, and subjected to
freeze-drying.
[0398] This lyophilized pellet was suspended in chloroform of 100
ml and PHA was extracted by stirring at 60.degree. C. for 20 hours.
An extracted fluid was filtered using a membrane filter with a pore
size of 0.45 .mu.m followed by concentration by a rotary evaporator
and then, concentrated fluid was precipitated again in cold
methanol and precipitation only was collected and dried in vacuo to
yield PHA. PHA yielded was subjected to methanolysis by a routine
method followed by analysis by a gas chromatography mass
spectrometry apparatus (GC-MS, Shimadzu QP-5050, EI method) and a
methyl esterified product of the PHA monomer unit was identified.
Table 4 shows the result of identification. It is known that PHA
yielded contains only monomer unit derived from 3-hydroxy-4-phenoxy
butyric acid expressed by Formula (5) as the monomer unit and is a
poly-3-hydroxy-4-phenoxy butyric acid.
Example A-3
[0399] PHA collected from cultured cells of the strain P 91 was
analyzed by using a nuclear magnetic resonance apparatus (FT-NMR:
Bruker DPX400) under the following conditions.
[0400] Nuclear Species for Measurement: 1H, Solvent Used: Heavy
Chloroform (Containing TMS)
[0401] FIG. 1 shows the result of measurement and a Table 5 shows
the result of analysis (attribute) of each signal. Table 5 shows
the result of 3-hydroxy-4-phenoxy butyric acid described below.
From the result, PHA contains only monomer unit derived from
3-hydroxy-4-phenoxy butyric acid expressed by Formula (5) and is
confirmed that it is poly-3-hydroxy-4-phenoxy butyric acid.
[0402] B:
[0403] The example, in which the production method for
polyhydroxyalkanoate according to the present invention is applied
to the production of polyhydroxyalkanoate: poly-3-hydroxy-5-phenoxy
valeric acid (PHPxV) consisting of the monomer unit, which is
derived from 3-hydroxy-5-phenoxy valeric acid (HPxVA) expressed by
Formula (6) using 5-phenoxy valeric acid (PxVA) of Formula (15) as
the material, will be shown.
Example B-1
Synthesis of PxVA
[0404] Dehydrated acetone of 240 ml was put in a 3-necked
round-bottom flask, potassium iodide (0.06 mol), potassium
carbonate (0.11 mol), and phenol (0.07 mol) were added to stir
sufficiently. In this solution, 5-bromo valeric acid ethyl ester
(0.06 mol) was dropped in a nitrogen atmosphere and refluxed at
60.+-.5.degree. C. to react for 24 hours. After completion of the
reaction, a reaction solution was exsiccated to condensation using
an evaporator, and dissolved again in methylene chloride, and water
was added to the solution to separate and an organic layer was
dehydrated by using magnesium sulfate anhydride followed by
exsiccation for condensation using the evaporator. Hot methanol was
added to a dried matter (reactant) yielded to dissolve and cooled
slowly to precipitate again, resulting in a yield of 5-phenoxy
valeric acid ethyl ester (PxVA). At this point, the yield ratio of
this ester to 5-bromo valeric acid ethyl ester was 72 mol %.
[0405] Reactant (ester) yielded was dissolved in ethanol water (9:1
(v/v)) to make 5 wt %, potassium hydroxide of 10 times mol was
added to react at 0 to 4.degree. C. for 4 hours to carry out
hydrolysis of the ester. This reaction solution was added to 0.1
mol hydrochloric acid aqueous solution of 10 times volume and then,
precipitation was collected by filtration. The precipitation
(reactant) collected was dried under a reduced pressure under a
room temperature for 36 hours. The dried matter yielded was
dissolved in a small volume of hot methanol, the solution was
gradually cooled to precipitate again, and the precipitation was
dried under a reduced pressure under the room temperature for 24
hours resulting in yield of 5-phenoxy valeric acid, the objective
compound. The yield ratio of this objective compound to 5-bromo
valeric acid ethyl ester was 53 mol %.
[0406] The analysis of the compound yielded was conducted by the
nuclear magnetic resonance apparatus (NMR) under the following
conditions.
[0407] <Instruments Used>
[0408] FT-NMR: Bruker DPX400
[0409] .sup.1H resonance frequency: 400 MHZ
[0410] <Condition of Measurement>
[0411] Nuclear species for measurement: 1H
[0412] Solvent used: CDCl.sub.3
[0413] Reference: sealed in a capillary tube TMS/CDCl.sub.3
[0414] Temp. for measurement: room temp.
[0415] FIG. 2 shows a chart of the spectrum and Table 6 shows the
result of identification.
[0416] From the result, synthesis of the desired PxVA was
confirmed.
Example B-2
Production of PHPxV Homopolymer by Strain P91
[0417] The strain P 91 was inoculated in a 200 ml of the M9 culture
medium containing the yeast extract (DIFCO made) of 0.5 wt % and
PxVA of 0.1 wt % and subjected to shaking-cultivation at 30.degree.
C. and 125 strokes/min. After 24 hours, cells were collected by
centrifugation, suspended again in 200 ml of the M9 culture medium
containing 0.1% PxVA and lacking any nitrogen source (NH.sub.4Cl),
and further subjected to the shaking cultivation at 30.degree. C.
and 125 strokes/min. After 24 hours, cells were collected by
centrifugation, washed with cold methanol once, and subjected to
freeze-drying and weighing.
[0418] This lyophilized pellet was suspended in chloroform of 100
ml and PHA was extracted by stirring at 60.degree. C. for 20 hours.
The extracted fluid was filtered using a membrane filter with the
pore size of 0.45 .mu.m followed by concentration by the rotary
evaporator and then, concentrated fluid was precipitated again in
cold methanol and precipitation only was collected and dried in
vacuo to yield PHA and weigh. Table 7 shows the yields of cells and
the polymer.
[0419] A composition of PHA yielded was analyzed according to the
following steps. PHA sample of 5 mg was put in an egg-shaped flask
of 25 ml volume, methanol of 2 ml containing chloroform of 2 ml and
sulfuric acid of 3% (v/v) was added to reflux at 100.degree. C. for
3.5 hours, and water was added to separate, and then, the organic
layer was analyzed by the gas chromatography mass spectrometry
apparatus (GC-MS, Shimadzu QP-5050, DB-WAXETR (J & W Co. made),
EI method) and a methyl esterified product of the PHA monomer unit
was identified. As the result, there was a single main peak. From
the mass spectrum thereof, it was known as methyl esterified
compound of 3-hydroxy-5-phenoxy valeric acid. In addition, other
small components had no relation with the monomer unit of PHA.
FIGS. 3A and 3B show the total ion chromatogram (TIC) of GC-MS and
the mass spectrum of the main peak.
[0420] The polymer yielded was subjected to NMR analysis under the
following condition.
[0421] <Instruments Used>
[0422] FT-NMR: Bruker DPX400
[0423] .sup.1H resonance frequency: 400 MHz
[0424] <Condition of Measurement>
[0425] Nuclear species for measurement: 1H
[0426] Solvent used: CDCl.sub.3
[0427] Reference: sealed in a capillary tube TMS/CDCl.sub.3
[0428] Temp. for measurement: room temp.
[0429] FIG. 4 shows the chart of the spectrum and Table 8 shows the
result of identification.
[0430] In addition, the molecular weight of the PHA yielded was
measured by gel permeation chromatography (GPC; Toso, HLC-8020,
column: Polymer Laboratory, PLgel-MIXED-C (5 .mu.m), solvent:
chloroform, polystyrene conversion). The result was Mn=70000 and
Mw=121000.
[0431] As the above described result, according to the present
invention, the homopolymer of poly-3-hydroxy-5-phenoxy valeric acid
using PxVA as the material and production method thereof were
shown.
Example B-3
Production of PHPxV Homopolymer by the Strain H45
[0432] The strain H45 was inoculated in a 200 ml of the M9 culture
medium containing the yeast extract (DIFCO made) of 0.5 wt % and
PxVA of 0.1 wt % and subjected to shaking-cultivation at 30.degree.
C. and 125 strokes/min. After 24 hours, cells were collected by
centrifugation, washed with cold methanol once, and subjected to
freeze-drying and weighing.
[0433] This lyophilized pellet was suspended in chloroform of 100
ml and PHA was extracted by stirring at 60.degree. C. for 20 hours.
The extracted fluid was filtered using a membrane filter with the
pore size of 0.45 .mu.m followed by concentration by the rotary
evaporator and then, concentrated fluid was precipitated again in
cold methanol and precipitation only was collected and dried in
vacuo to yield PHA and weigh. Table 9 shows the yields of cells and
the polymer.
[0434] The composition of PHA yielded was analyzed according to the
following steps. PHA sample of 5 mg was put in the egg-shaped flask
of 25 ml volume, methanol of 2 ml containing chloroform of 2 ml and
sulfuric acid of 3% (v/v) was added to reflux at 100.degree. C. for
3.5 hours, and water was added to separate, and then, the organic
layer was analyzed by the gas chromatography mass spectrometry
apparatus (GC-MS, Shimadzu QP-5050, DB-WAXETR (J & W Co. made),
EI method) and the methyl esterified product of the PHA monomer
unit was identified. As the result, there was a single main peak.
From the mass spectrum thereof, it was known as methyl esterified
compound of 3-hydroxy-5-phenoxy valeric acid. In addition, other
small components had no relation with the monomer unit of PHA.
FIGS. 5A and 5B show the total ion chromatogram (TIC) of GC-MS and
the mass spectrum of the main peak.
[0435] In addition, the molecular weight of the PHA yielded was
measured by gel permeation chromatography (GPC; Toso, HLC-8020,
column: Polymer Laboratory, PLgel-MIXED-C (5 .mu.m), solvent:
chloroform, polystyrene conversion.) The result was Mn=64000 and
Mw=116000.
[0436] As the above described result, according to the present
invention, the homopolymer of poly-3-hydroxy-5-phenoxy valeric acid
using PxVA as the material and production method thereof were
shown.
[0437] C:
[0438] The example, in which the production method for
polyhydroxyalkanoate according to the present invention is applied
to the production of polyhydroxyalkanoate:
poly-3-hydroxy-5-(4-fluorophenoxy) valeric acid (PHFPxV) consisting
of the monomer unit, which is derived from
3-hydroxy-5-(4-fluorophenoxy) valeric acid (HFPxVA) expressed by
Formula (16) using 5-(4-fluorophenoxy) valeric acid (FPxVA) of
Formula (17) as the material, will be shown.
Example C-1
Synthesis of FPxVA
[0439] Sodium iodide (0.06 mol), potassium carbonate (0.11 mol),
and 4-fluorophenol (0.07 mol) were added to 240 ml of dehydrated
acetone in a three-neck round-bottom flask and the mixture was
stirred fully. To the solution was dripped 5-bromovaleric acid
ethyl ester (0.06 mol) in a nitrogen atmosphere and the mixture was
refluxed at 60.+-.5.degree. C. and allowed to react for 24 hours.
After the completion of reaction, the reactant solution was
concentrated to dryness using an evaporator. The residue was
dissolved in methyl chloride again and water added, followed by
separation. The separated organic solvent layer was dehydrated with
anhydrous magnesium sulfate and concentrated to dryness using an
evaporator to obtain the reactant.
[0440] Hot water was added to dissolve the obtained reactant and
the solution gradually cooled to precipitate 5-(4-fluorophennoxy)
valeric acid ethyl ester again. Here, the yield rate of the
compound to 5-bromo valeric acid ethyl ester was 68 mol %.
[0441] The obtained reactant (ester) was dissolved in ethanol-water
(9:1 (v/v)) to make a 5 wt % solution, 10 times the mol of
potassium hydroxide added, and the mixture allowed to react at 0 to
4.degree. C. for 4 hours to hydrolyze the ester.
[0442] The reactant solution was added in 10 times the volume of
0.1 M hydrochloric acid and the precipitate collected by
filtration. The collected precipitate (the reactant) was dried
under decreased pressure at room temperature for 36 hours. The
dried reactant was dissolved in a small amount of hot ethanol and
the solution gradually cooled for reprecipitation. The precipitate
was dried under decreased pressure at room temperature for 24 hours
to obtain the goal compound 5-(4-fluorophenoxy) valeric acid
expressed as Equation (17). The yield rate of this compound to
5-bromo valeric acid ethyl ester was 49 mol %.
[0443] The obtained compound was analyzed using NMR under the
following conditions.
[0444] <Instrument>
[0445] FT-NMR: Bruker DPX400
[0446] .sup.1H resonance frequency: 400 MHZ
[0447] <Measurement Conditions>
[0448] Nuclide: .sup.1H
[0449] Solvent: CDCl.sub.3
[0450] Reference: TMS/CDCl.sub.3 sealed into a capillary
[0451] Temperature: Room temperature
[0452] FIG. 6 shows the .sup.1H-NMR spectrum chart and Table 10
indicates the results of identification.
[0453] From the above-mentioned results, it was confirmed that the
goal FPxVA was synthesized.
Example C-2
Production of PHFPxV Homopolymer Using Strain P91
[0454] The strain P91 was inoculated in 200 ml of M9 medium
containing 0.5 wt % yeast extract (DIFCO) and 0.1 wt % FPxVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation and suspended again in 200 ml of M9 medium
containing 0.1 wt % FPxVA without a nitrogen source (NH.sub.4Cl),
followed by shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, washed with cold methanol once, lyophilized, and
weighed.
[0455] The lyophilized pellet was suspended in 100 ml of chloroform
and stirred at 60.degree. C. for 20 hours to extract PHA. The
extract solution was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain PHA. The obtained PHA was weighed. Table
11 shows the yields of the bacterial cell and polymer.
[0456] The obtained PHA was analyzed for composition as follows: 5
mg of the PHA sample was put in a 25-ml eggplant-type flask, 2 ml
of chloroform and 2 ml of methanol containing 3% (v/v) sulfuric
acid added, the mixture refluxed at 100.degree. C. for 3.5 hours,
and water added for separation. After separation, the organic
solvent layer was analyzed using gas chromatography-mass
spectrometry (GC-MS, Shimadzu QP-5050, column: DB-WAXETR (J&W
Co.), EI method) to identify the methyl ester of PHA monomer unit.
Consequently, only one main peak was shown and identified as the
methyl ester of 3-hydroxy-5-(4-fluorophenoxy) valeric acid by mass
spectrometry. The other trace components were unrelated to the PHA
monomer unit. FIGS. 7A and 7B show the TCI and mass spectrum of the
methyl ester of 3-hydroxy-5-(4-fluorophenoxy) valeric acid.
[0457] The molecular weight of the obtained PHA was determined
using gel permeation chromatography (GPC; Toso HLC-8020, column:
Polymer Laboratory PLgel MIXED-C (5 .mu.m), solvent: chloroform,
polystyrene conversion): Mn=68000 and Mw=120000.
[0458] The obtained PHA was further analyzed for structure using
nuclear magnetic resonance (NMR) under the following
conditions.
[0459] <Instrument>
[0460] FT-NMR: Bruker DPX400
[0461] .sup.1H Resonance frequency: 400 MHz
[0462] <Measurement Conditions>
[0463] Nuclide: .sup.1H
[0464] Solvent: CDCl.sub.3
[0465] Reference: TMS/CDCl.sub.3 sealed in capillary
[0466] Temperature: Room temperature
[0467] FIG. 8 shows the .sup.1H-NMR spectrum chart and Table 12 the
results of the identification.
[0468] Consequently, the poly-3-hydroxy-5-(4-fluorophenoxy)
valerate homopolymer using FPxVA as the material and its production
method according to this invention have been shown.
Example C-3
Production of PHFPxV Homopolymer Using Strain H45
[0469] The cells of strain H45 were inoculated in 200 ml of M9
medium containing 0.5 wt % yeast extract (DIFCO) and 0.1 wt % FPxVA
and subjected to shake culture at 30.degree. C. and at 125
strokes/min. After 24 hours, the bacterial cells were collected by
centrifugation, washed with cold methanol once, lyophilized, and
weighed.
[0470] The lyophilized pellet was suspended in 100 ml of chloroform
and stirred at 60.degree. C. for 20 hours to extract PHA. The
extract was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain and weigh PHA. Table 13 shows the yields
of the bacterial cells and polymer.
[0471] The obtained PHA was analyzed for composition as follows: 5
mg of the PHA sample was put in a 25-ml eggplant-type flask, 2 ml
of chloroform and 2 ml of methanol containing 3% (v/v) sulfuric
acid added, the mixture refluxed at 100.degree. C. for 3.5 hours,
and water added for separation. After separation, the organic
solvent layer was analyzed using gas chromatography-mass
spectrometry (GC-MS, Shimadzu QP-5050, column: DB-WAXETR (J&W
Co.), EI method) to identify the methyl ester of PHA monomer unit.
Consequently, only one main peak was shown and identified as the
methyl ester of 3-hydroxy-5-(4-fluorophenoxy) valeric acid by mass
spectrometry. FIGS. 9A and 9B show the GC-MS total ion chromatogram
(TIC) and mass spectrum of the main peak.
[0472] The molecular weight of the obtained PHA was determined
using gel permeation chromatography (GPC; Toso HLC-8020, column:
Polymer Laboratory PLgel MIXED-C (5 .mu.m), solvent: chloroform,
polystyrene conversion): Mn=67000 and Mw=119000.
[0473] Consequently, the poly-3-hydroxy-5-(4-fluorophenoxy)
valerate homopolymer using FPxVA as the material and its production
method according to this invention have been shown.
[0474] D:
[0475] There are given examples of the production method for
polyhydroxyalkanoate according to this invention applied to the
production of polyhydroxyalkanoate composed of the monomer unit
derived from 3-hydroxy-5-phenyl valeric acid (HPVA) expressed as
Equation (9), for which the material is 5-phenyl valeric acid (PVA)
expressed as Equation (18): poly-3-hydroxy-5-phenyl valerate
(PHPV).
Example D-1
[0476] The strain H45 was inoculated in 200 ml of medium M9
containing 0.5% yeast extract (Difco Co.) and 0.05% PVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, washed with cold methanol once, and
lyophilized.
[0477] The lyophilized pellet was suspended in 100 ml of chloroform
and stirred at 60.degree. C. for 20 hours to extract PHA. The
extract solution was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain PHA. The obtained PHA was subjected to
methanolysis by the usual method and analyzed using gas
chromatography-mass spectrometry (GC-MS, Shimadzu QP-5050, EI
method) to identify the methyl ester of the PHA monomer unit. The
molecular weight of the PHA was determined using gel permeation
chromatography (GPC: Toso HLC-8020, column: Polymer
Laboratory-PLgel-MIXED-C-5 .mu.m, solvent; chloroform,
polystyrene-converted molecular weight). Table 14 shows the results
of identification, average molecular weight, and the yield and
yield rate of the lyophilized pellet and collected polymer.
[0478] As shown in Table 14, the polymer, extracted and collected
from the bacterial cells has been confirmed to contain only the
monomer unit derived from 3-hydroxy-5-phenyl valeric acid expressed
as Equation (9) as the PHA monomer unit.
Example D-2
[0479] The strain H45 was inoculated in 200 ml of M9 medium
containing 0.5% yeast extract (Difco Co.) and 0.1% PVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, suspended again in 200 ml of medium M9 containing
0.2% PVA without a nitrogen source (NH.sub.4Cl), and subjected to
shake culture at 30.degree. C. and at 125 strokes/min. After 24
hours, the bacterial cells were collected by centrifugation, washed
with cold methanol once, and lyophilized.
[0480] The lyophilized pellet was suspended in 100 ml of chloroform
and stirred at 60.degree. C. for 20 hours to extract PHA. The
extract solution was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain PHA. The obtained PHA was subjected to
methanolysis by the usual method and analyzed using gas
chromatography-mass spectrometry (GC-MS, Shimadzu QP-5050, EI
method) to identify the methyl ester of PHA monomer unit. The
molecular weight of the PHA was determined using gel permeation
chromatography (GPC: Toso HLC-8020, column: Polymer
Laboratory-PLgel-MIXED-C-5 .mu.m, solvent: chloroform,
polystyrene-converted molecular weight). Table 15 shows the results
of identification, average molecular weight, and the yield and
yield rate of lyophilized pellet and collected polymer.
[0481] As shown in Table 15, the polymer, extracted and collected
from the bacterial cells has been confirmed to contain only the
monomer unit derived from 3-hydroxy-5-phenyl valeric acid expressed
as Equation (9), as the PHA monomer unit.
Example D-3
[0482] The strain P91 was inoculated in 200 ml of M9 medium
containing 0.5% yeast extract (Difco Co.) and 0.1% PVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, suspended again in 200 ml of M9 medium containing
0.1% PVA without a nitrogen source (NH.sub.4Cl), and subjected to
shake culture at 30.degree. C. and at 125 strokes/min. After 24
hours, the bacterial cells were collected by centrifugation, washed
with cold methanol once, and lyophilized.
[0483] The dried pellet was suspended in 100 ml of chloroform and
stirred at 60.degree. C. for 20 hours to extract PHA. The extract
solution was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain PHA. The PHA was subjected to
methanolysis by the usual method and analyzed using gas
chromatography-mass spectrometry (GC-MS, Shimadzu QP-5050, EI
method) to identify the methyl ester of PHA monomer unit. Table 16
shows the results of identification and the yield and yield rate of
the lyophilized pellet and collected polymer.
[0484] As shown in Table 16, the polymer, extracted and collected
from the bacterial cells has been confirmed to contain only the
monomer unit derived from 3-hydroxy-5-phenyl valeric acid expressed
as Equation (9), as the PHA monomer unit.
Example D-4
[0485] The strain P161 was inoculated in 200 ml of medium M9
containing 0.5% yeast extract (Difco Co.) and 0.1% PVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, suspended again in 200 ml of M9 medium containing
0.1% PVA without a nitrogen source (NH.sub.4Cl), and subjected to
shake culture at 30.degree. C. and at 125 strokes/min. After 24
hours, the bacterial cells were collected by centrifugation, washed
with cold methanol once, and lyophilized.
[0486] This lyophilized pellet was suspended in 100 ml of
chloroform and stirred at 60.degree. C. for 20 hours to extract
PHA. The extract solution was filtered with a 0.45-.mu.m membrane
filter and concentrated using a rotary evaporator. The concentrate
was reprecipitated in cold methanol and the precipitate was
collected and vacuum-dried to obtain PHA. The obtained PHA was
subjected to methanolysis by the usual method and then analyzed
using gas chromatography-mass spectrometry (GC-MS, Shimadzu QP5050,
EI method) to identify the methyl ester of PHA monomer unit. The
molecular weight of the obtained PHS was determined by gel
permeation chromatography (GPC; Toso-HLC-8020, column: Polymer
Laboratory-PLgel-Mixed-C-5 .mu.m, solvent: chloroform,
polystyrene-concerted molecular weight). Table 17 shows the results
of identification, average molecular weight, and the yield and
yield rate of lyophilized pellet and collected polymer.
[0487] As shown in Table 17, the polymer, extracted and collected
from the bacterial cells has been confirmed to contain only the
monomer unit derived from 3-hydroxy-5-phenyl valeric acid,
expressed as Equation (9), as the PHA monomer.
Example D-5
[0488] PHPV, produced through the strain H45 was analyzed using
nuclear magnetic resonance (FT-NMR: Bruker DPX400) under the
following conditions: nuclide: .sup.1H and .sup.13C, solvent: heavy
chloroform (containing TMS). FIG. 10 shows the .sup.1H-NMR spectrum
chart and Table 18 lists the identification of each peak. FIG. 11
shows the .sup.13C-NMR spectrum chart and Table 19 lists the
identification of each peak.
[0489] Consequently, the polymer, extracted and collected from the
bacterial cells, has been shown to be poly-3-hydroxy-5-phenyl
valeric acid containing only the monomer unit derived from
3-hydroxy-5-phenyl valeric acid expressed as Equation (9), as the
PHA monomer unit.
[0490] There are given examples of the production method for
polyhydroxyalkanoate according to this invention applied to the
production of polyhydroxyalkanoate consisting of the monomer unit
derived from 3-hydroxy-5-(4-fluorophenyl) valeric acid (HFPVA)
expressed as Equation (7) for which the material is
5-(4-fluorophenyl) valeric acid (FPVA) expressed as Equation (19):
poly-3-hydroxy-5-(4-fluorophenyl) valerate (PHFPV).
Example E-1
[0491] The substrate FPVA was synthesized first through Grignard
reaction as shown in Macromolecules, 29, 1762-1766 (1996) and 27,
45-49 (1994): 5-bromo valeric acid was dissolved in anhydrous
tetrahydrofuran (THF) and a 3M methylmagnesiumchloride THF solution
was added by dripping at -20.degree. C. in an argon atmosphere.
After stirring for 15 minutes, a THF solution of
1-bromo-4-fluorobenzene and magnesium was further dripped and a
0.1M Li.sub.2CuCl.sub.4 THF solution added (the temperature was
kept at -20.degree. C.). The temperature of the reaction solution
was returned to the room temperature. The solution was stirred
overnight, then poured in 20% ice-cooled sulfuric acid and stirred.
The water layer was collected and saturated with sodium chloride,
followed by extraction with ether. The extract was further
extracted with 100 ml of deionized water including 50 g of
potassium hydroxide and oxidized with 20% sulfuric acid, followed
by collection of the precipitate.
[0492] This precipitate was analyzed using nuclear magnetic
resonance (FT-NMR: Bruker DPX400) under the following conditions:
nuclide: 1H and 13C, solvent: heavy chloroform (containing TMS).
FIG. 13 and Table 20 indicate the results of the analysis.
Example E-2
[0493] The strain H45 was inoculated in 200 ml of M9 medium
containing 0.5% yeast extract (Difco Co.) and 0.1% FPVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, washed with cold methanol once, and
lyophilized.
[0494] This lyophilized pellet was suspended in 100 ml of
chloroform and stirred at 60.degree. C. for 20 hours to extract
PHA. The extract solution was filtered with a 0.45-.mu.m membrane
filter and concentrated using a rotary evaporator. The concentrate
was reprecipitated in cold methanol and the precipitate was
collected and vacuum-dried to obtain PHA. The obtained PHA was
subjected to methanolysis by the usual method and analyzed using
gas chromatography-mass spectrometry (GC-MS, Shimadzu QP-5050, EI
method) to identify the methyl ester of PHA monomer unit. Table 21
shows the results.
Example E-3
[0495] The P91 strain was inoculated in 200 ml of M9 medium
containing 0.5% yeast extract (Difco Co.) and 0.1% FPVA and
subjected to shake culture at 30.degree. C. and at 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, suspended again in 200 ml of M9 medium containing
0.1% FPVA without a nitrogen source (NH.sub.4Cl), and subjected to
shake culture at 30.degree. C. and 125 strokes/min. After 24 hours,
the bacterial cells were collected by centrifugation, washed with
cold methanol once, and lyophilized.
[0496] The lyophilized pellet was suspended in 100 ml of chloroform
and stirred at 60.degree. C. for 20 hours to extract PHA. The
extract solution was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain PHA. The obtained PHS was subjected to
methanolysis by the usual method and analyzed using gas
chromatography-mass spectrometry (GC-MS, Shimadzu QP-5050, EI
method) to identify the methyl ester of PHS monomer unit. Table 22
lists the results.
Example E-4
[0497] The P161 strain was inoculated in 200 ml of M9 medium
containing of 0.5% yeast extract (Difco Co.) and 0.1% FPVA and
subjected to shake culture at 30.degree. C. and 125 strokes/min.
After 24 hours, the bacterial cells were collected by
centrifugation, suspended again in 200 ml of M9 medium containing
0.1% FPVA without a nitrogen source (NH.sub.4Cl), and subjected to
shake culture at 30.degree. C. and 125 strokes/min. After 24 hours,
the bacterial cells were collected by centrifugation, washed with
cold methanol once, and lyophilized.
[0498] The lyophilized pellet was suspended in 100 ml of chloroform
and stirred at 60.degree. C. for 20 hours to extract PHA. The
extract solution was filtered with a 0.45-.mu.m membrane filter and
concentrated using a rotary evaporator. The concentrate was
reprecipitated in cold methanol and the precipitate was collected
and vacuum-dried to obtain PHA. The obtained PHA was subjected to
methanolysis by the usual method, and analyzed using gas
chromatography-mass spectrometry (GC-MS, Shimadzu QP-5050, EI
method) to identify the methyl ester of PHA monomer unit. Table 23
shows the results.
Example E-5
[0499] We analyzed PHFPV derived from strain H45, using nuclear
magnetic resonance spectrometer (FT-NMR: Bruker DPX400) under the
following conditions: measured nuclides, .sup.1H, .sup.13C: used
solvent, heavy chloroform (containing TMS). The results are shown
in FIG. 14, Table 24, FIG. 15 and Table 25.
[0500] F:
[0501] Following is an example of the process of the invention for
producing polyhydroxyalkanoate consisting of
3-hydroxy-4-cyclohexylbutyri- c acid (3-HCHBA) monomer unit
(Formula 8) by using 4-cyclohexylbutyric acid (CHBA, Formula 20) as
a raw material.
Example F-1
Production of PHA Containing 3-hydroxy-4-cyclohexylbutyric Acid as
a Monomer Unit by Strain YN2 (One-Step Culture)
[0502] Colonies of strain YN2 grown on an M9 agar medium
supplemented with 0.1% yeast extract were inoculated in 200 ml of a
liquid M9 medium supplemented with 0.5% yeast extract and 0.1%
4-cyclohexylbutyric acid, and then cultured at 30.degree. C. After
24 hours, the cells were collected by centrifugation, washed with
methanol and then lyophilized.
[0503] After weighing, the lyophiled pellet was suspended in 100 ml
of chloroform to extract PHA at 60.degree. C. for 20 hours with
mixing. Then the mixture was filtered through a 0.45 .mu.m filter,
the filtrate was concentrated by an evaporator. Cold methanol was
added to the concentrate to reprecipitate the polymer material. The
polymer was then vacuum-dried at room temperature and weighed.
Table 26 shows the weight of the obtained lyophilized pellet and
collected polymer and the yield of the polymer (CDW: cell mass (dry
weight), PDW: polymer (dry weight)).
[0504] In the above mentioned prior art (Table 2), the yield of PHA
comprised of 3-HCHBA unit was 89.1 mg/l culture. On the other hand,
the result of the above example of the invention shows that the
yield is 2.5 times higher than that. Moreover, the yield per dried
cell mass was improved significantly.
[0505] The composition of the obtained PHA was analyzed as
follows:
[0506] About 10 mg of PHA was put into a 25 ml eggplant type flask
and dissolved in 2 ml of chloroform, then 2 ml of methanol
containing 3% sulfuric acid was added to the solution and reacted
for 3,5 hours under reflux at 100.degree. C. Upon completion of the
reaction, 10 ml of deionized water was added to the solution and
shaken vigorously for 10 minutes. Subsequently, the separated lower
chloroform layer was taken out and dried over magnesium sulfate,
The methylesterified PHA components in the chloroform layer were
identified by using a gas chromatograph-mass spectrometer (GC-MS,
Shimadzu QP-5050, EI method). As a result, 98% of the PHA monomer
units were 3-HCHBA of Formula (8), and 2% of them were
3-hydroxybutyric acid. A small amount of cyclohexyl methanol was
also present.
[0507] As shown above, according to the production process of the
invention, PHA containing 3-HCHBA at a significantly high level can
be obtained. The effect of the yeast extract addition in the
process of the invention was verified. The production process was a
highly efficient method of high yield per unit volume of culture or
weight of cell mass. Thus, it was verified that the present
production process is highly efficient in both the high content of
3-HCHBA unit and the high yield.
[0508] Production of PHA containing 3-hydroxy-4-cyclohexylbutyric
acid as a monomer unit by strain H45 (one-step culture).
[0509] Colonies of strain H45 grown on an M9 agar medium
supplemented with 0.1% yeast extract were inoculated in 200 ml of a
liquid M9 medium supplemented with 0.5% yeast extract and 0.1%
4-cyclohexylbutyric acid, and then cultured at 30.degree. C. After
24 hours, the cells were collected by centrifugation, washed with
methanol and then lyophilized.
[0510] After weighing, the lyophiled pellet was suspended in 100 ml
of chloroform to extract PHA at 60.degree. C. for 20 hours with
mixing. Then the mixture was filtered through a 0.45 .mu.m filter,
the filtrate was concentrated by an evaporator. Cold methanol was
added to the concentrate to reprecipitate the polymer material. The
polymer was then vacuum dried at room temperature and weighed.
Table 27 shows the weight of the obtained lyophilized pellet (CDW)
and collected polymer (PDW) and the yield of the polymer.
[0511] In the above mentioned prior art (Table 2), the yield of PHA
comprised of a 3-HCHBA unit was 89.1 mg/l culture. On the other
hand, the result of the above example of the invention shows that
the yield is 1.3 times higher than that. Moreover, the yield per
unit dried cell mass was improved significantly.
[0512] The composition of the obtained PHA was analyzed as
follows:
[0513] About 10 mg of PHA was put into a 25 ml eggplant type flask
and dissolved in 2 ml of chloroform, then 2 ml of methanol
containing 3% sulfuric acid was added to the solution and reacted
for 3,5 hours under reflux at 100.degree. C. Upon completion of the
reaction, 10 ml of deionized water was added to the solution and
shaken vigorously for 10 minutes. Subsequently, the separated lower
chloroform layer was taken out and dried over magnesium sulfate,
The methylesterified PHA components in the chloroform layer were
identified by using a gas chromatograph-mass spectrometer (GC-MS,
Shimadzu QP-5050, EI method). As a result, 97% of the PHA monomer
units were 3-HCHBA of Formula (8), and 3% of them were
3-hydroxybutyric acid. A small amount of cyclohexyl methanol was
also present.
[0514] As shown above, according to the production process of the
invention, PHA containing 3-HCHBA at a significantly high level can
be obtained by strain H45 as well.
[0515] Production of PHA containing 3-hydroxy-4-cyclohexylbutyric
acid as a monomer unit by strain P161 (one-step culture)
[0516] Colonies of strain H45 grown on an M9 agar medium
supplemented with 0.1% yeast extract were inoculated in 200 ml of a
liquid M9 medium supplemented with 0.5% yeast extract and 0.1%
4-cyclohexylbutyric acid, and then cultured at 30.degree. C. After
24 hours, the cells were collected by centrifugation, washed with
methanol and then lyophilized.
[0517] After weighing, the lyophiled pellet was suspended in 100 ml
of chloroform to extract PHA at 60.degree. C. for 20 hours with
mixing. Then the mixture was filtered through a 0.45 .mu.m filter,
the filtrate was concentrated by an evaporator. Cold methanol was
added to the concentrate to reprecipitate the polymer material. The
polymer was then vacuum dried at room temperature and weighed.
Table 28 shows the weight of the obtained lyophilized pellet and
collected polymer and the yield of the polymer.
[0518] In the above mentioned prior art (Table 2), the yield of PHA
comprised of 3-HCHBA unit was 89.1 mg/l culture. On the other hand,
the result of the above example of the invention shows that the
yield is 1.5 times higher than that. Moreover, the yield per dried
cell mass was improved significantly.
[0519] The composition of the obtained PHA was analyzed as
follows:
[0520] About 10 mg of PHA was put into a 25 ml eggplant type flask
and dissolved in 2 ml of chloroform, then 2 ml of methanol
containing 3% sulfuric acid was added to the solution and reacted
for 3,5 hours under reflux at 100.degree. C. Upon completion of the
reaction, 10 ml of deionized water was added to the solution and
shaken vigorously for 10 minutes. Subsequently, the separated lower
chloroform layer was taken out and dried over magnesium sulfate,
The methylesterified PHA components in the chloroform layer were
identified by using a gas chromatograph-mass spectrometer (GC-MS,
Shimadzu QP-5050, EI method). As a result, 94% of the PHA monomer
units were 3-HCHBA of Formula (8), and 6% of them were
3-hydroxybutyric acid. A small amount of cyclohexyl methanol was
also present.
[0521] As shown above, according to the production process of the
invention, PHA containing 3-HCHBA at a significantly high level can
be obtained by strain P161 as well.
Example F-2
[0522] Production of PHA Containing 3-hydroxy-4-cyclohexylbutyric
Acid Units Using Strain YN2 (Two Step Cultivation).
[0523] Strain YN2 colonies grown on an M9 agar medium containing
0.1% yeast extract were inoculated in an M9 liquid medium (200 mL)
containing 0.5% yeast extract and 0.1% 4-cyclohexylbutyric acid,
and then cultured at 30.degree. C. After 24 hours, the cells were
collected by centrifugation. Subsequently, the cells were
transferred into a fresh M9 medium containing 0.1%
4-cyclohexylbutyric acid but free from NaCl and NH.sub.4Cl, and
cultured for 21 hours at 30.degree. C. Subsequently, the cells were
washed once with methanol and lyophilized.
[0524] This lyophilized pellet was weighed, and then the polymer
was collected through the same process as that conducted in Example
F-1. This polymer was vacuum dried at room temperature and weighed.
The obtained amounts of the lyophilized pellet (CDW) and the
collected polymer (PDW) and the yield are shown in Table 29.
[0525] While the yielded amount of PHA containing the units derived
from 3-hydroxy-4-cycrohexylbutyric acid in the above mentioned
ordinary report (Table 2) was 89.1 mg per liter of the culture
medium, the example of the invention resulted in obtaining
approximately 3.2 times of the yielded amount. And the yield per
dry cell mass is also improved significantly.
[0526] The composition of the obtained PHA was evaluated with the
same process as that conducted in Example of the invention F-1.
Consequently, 99% of them were derived from units of
3-hydroxy-4-cyclohexylbutyric acid expressed as Formula (8) and 1%
of them were units of 3-hydroxybutyric acid. A little amount of
cyclohexyl methanol was also existed.
[0527] The molecular weight of the obtained polymer was determined
as Mn=49000 and Mw=100000, with using GPC (TOSOH HLC-8020, column:
Polymer laboratory PLgel MIXED-C (5 .mu.m), solvent: chloroform,
conversion into polystyrene).
[0528] The above mentioned results show that the production process
using this invention enables obtaining PHA containing significantly
high level of units derived from 3-hydroxy-4-cyclohexylbutyric acid
expressed as the equation (8). The effect of adding yeast extract
to the medium in the process of this invention was verified.
Additionally, the yield per unit medium and the yield per unit cell
mass were sufficiently improved. Thus the method of the invention
is verified to be a highly efficient production method in both the
high content of 3-hydroxy-4-cyclohexylbutyr- ic acid unit in the
polymer and the high yield.
[0529] Example F-1 and this Example were compared and then it was
verified that the obtained PHA can also contain a significantly
high level of 3-hydroxy-4-cyclohexylbutyric acid unit by using a
process where the cells are cultured in a mineral medium containing
yeast extract and 4-cyclohexylbutyric acid, and then transferred
and cultured in a mineral medium not containing yeast extract.
Example F-3
[0530] Purification and NMR Analysis of PHA Consisting of
3-hydroxy-4-cyclohexylbutyric Acid Units
[0531] The following purifying process was conducted in order to
remove PHB (poly 3-hydroxybutyric acid) component that was mixed in
and the component considered as cyclohexylmethanol from the polymer
obtained in Example F-2.
[0532] The polymer was suspended in acetone, and extraction was
conducted for 24 hours at 60.degree. C. Supernatant liquid was
collected with centrifugation and extraction from the precipitated
segment with acetone was conducted again. This operation was
repeated 5 times, and then the collected supernatant liquid was
condensed and dried thoroughly with an evaporator. The thoroughly
dried sample was dissolved in a small amount of chloroform, and
then precipitated again in cold methanol. This operation was
repeated 3 times, and then the obtained polymer was vacuum dried.
The obtained dried polymer was weighed as 281 mg.
[0533] .sup.1H-NMR and .sup.13C-NMR analyses of the polymer were
conducted (FT-NMR: Bruker DPX 400, used solvent heavy chloroform
(containing TMS). The chart of .sup.1H-NMR is shown in FIG. 16, the
assignments are in Table 30, and the chart of .sup.13C-NMR is in
FIG. 17, and the assignments are in Table 31.
[0534] From this evaluation, it is judged that PHB (poly
3-hydroxylbutyric acid) component that was mixed in and the
component considered as cyclohexylmethanol were removed and PHA
consisting of 3-hydroxy-4-cyclohexylbutyric acid was collected.
[0535] G:
[0536] One example in which the production process of
polyhydroalkanoate described in this invention is applied for
production of the following compounds: polyhydroxyalkanoate
consisting of the monomer units including
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-5-phenoxyvaleric acid (3HPxV) with using
7-phenoxyheptanoic acid as a material, which is the copolymer
consisting of 3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-5-phenoxyvaleric acid (3HPxV).
Example G-1
[0537] Production of the P(HPxV/HPxHp) Polymer with Using Strain
YN2 (Yeast Extract--One Step Cultivation)
[0538] Strain YN2 was inoculated into 200 mL of M9 medium
containing 0.5% yeast extract (produced by Difco) and 0.1%
7-phenoxyheptanoic acid (PxHpA), and shake-cultured with 125
strokes per minute and at 30.degree. C. After 64 hours, cells were
collected by centrifugation, washed once with cold methanol,
lyophilized and weighed.
[0539] This lyophilized pellet was suspended into 100 mL of acetone
and the polymer was extracted through mixing for 72 hours at the
room temperature (23.degree. C.). The extract was filtrated through
a membrane-filter of 0.45 .mu.m pore size and then concentrated by
a rotary evaporator. Subsequently, the concentrate was
reprecipitated in cold methanol and then the precipitate was
collected. The obtained polymer was vacuum-dried and weighed.
[0540] The molecular weight of the obtained polymer was determined
by gel permeation chromatography (GPC: Toso HLC-8020, column:
Polymer laboratory-PLgel-MIXED-C-5 .mu.m, solvent: chloroform,
molecular weight converted in polystyrene).
[0541] The composition of the obtained polymer unit was analyzed
with the following process: the 5 mg of the polymer sample was put
into the 25 mL eggplant shaped flask, 2 mL of chloroform and 2 mL
of methanol containing 3% sulfuric acid (v/v) were added to the
solution, and reflux for 3.5 hours at 100.degree. C. was conducted.
Separating occurred by adding water to the solution. Then the
organic layer was analyzed with a gas chromatograph-mass
spectrometer (GC-MS, Shimadzu QP-5050, column: DB-WAXETR (produced
by J&W), EI method) and identification for methylesterified
compounds of PHA monomer unit was conducted. The yield rate of the
cells and the polymer and results of analysis of monomer units are
shown in Table 32. The mass spectra of 3-hydroxy-5-phenoxyvaler- ic
acid (3HPxV) methyl ester and 3-hydroxy-7-phenoxyheptanoic acid
(3HPxHp) methyl ester, which were obtained using GC-MS, are shown
in FIG. 18 and FIG. 19, respectively.
[0542] Consequently, it was suggested that using strain YN2, PHA
copolymer consisting of only 2 units of 3-hydroxy-5-phenoxyvaleric
acid (3HPxV) and 3-hydroxy-7-phenoxyheptanoic acid (3HPxHp), with
7-phenoxyheptanoic acid as a substrate, could be produced.
Example G-2
Production of P(HPxV/HPxHp) Polymer by Using Strain H45 (Yeast
Extract Single-Step Culture)
[0543] Strain H45 was inoculated in a 200 ml M9 culture medium
containing 0.5% yeast extract (produced by Difco Co.) and 0.1%
7-phenoxyheptanoic acid (PxHpA), and shake-cultured at 30.degree.
C., 125 stroke/min. After 64 hr, cells were collected by
centrifugation, washed once with cold methanol, lyophilized and
weighed.
[0544] This lyophilized pellet was suspended in 100 ml of acetone,
and a polymer was extracted by mixing for 72 hr at a room
temperature (23.degree. C.). The fluid extract was filtered by a
membrane filter of 0.45 .mu.m pore size, and condensed by a rotary
evaporator. The condensed fluid was reprecipitated in cold
methanol, and then the precipitate was collected and vacuum-dried
to obtain a polymer to be weighed.
[0545] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0546] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
the cells and polymer, and the result of analysis of monomer unit
are shown in Table 33. The mass spectrum of
3-hydroxy-5-phenoxyvaleic acid (3HPxV) methyl ester and
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) methyl ester, which were
obtained by GC-MS measurement, are shown in FIG. 20 and FIG. 21,
respectively.
[0547] From the above result, it was shown that strain H45 can
produce PHA copolymer composed of only two units of
3-hydroxy-5-phenoxyvaleic acid (3HPxV) and
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) by using
7-phenoxyheptanoic acid as substrate.
[0548] H:
[0549] An example of the production method of polyhydroxyalkanoate
in the present invention by using 8-phenoxyoctanoic acid (PxOA) as
the raw material is shown here, where this method was applied to
the production of a polyhydroxyalkanoate which is composed of
monomer units derived from three kinds of substances including
3-hydroxy-4-phenoxybutyric acid (3HPxB),
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO), and is a copolymer
composed of 3-hydroxy-4-phenoxybutyric acid (3HPxB),
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO).
Example H-1
Production of P(HPxB/HPxHx/HPxO) Polymer by Using Strain YN2 (Yeast
Extract, Single-Step Culture)
[0550] Strain YN2 was inoculated in 200 ml of an M9 medium
containing 0.5% yeast extract (produced by Difco Co) and 0.1%
8-phenoxyoctanoic acid (PxOA), and shaking cultured at 30.degree.
C., 125 stroke/min. After 24 hr, cells were collected by
centrifugation, washed once with cold methanol, lyophilized and
weighed.
[0551] This lyophilized pellet was suspended in 100 ml of acetone,
and the polymer was extracted by mixing for 72 hr at a room
temperature (23.degree. C.). The liquid extract was filtered by a
membrane filter of 0.45 .mu.m pore size, and condensed by a rotary
evaporator. The concentrate was reprecipitated in cold methanol,
and then the precipitate was collected and vacuum-dried to obtain a
polymer to be weighed.
[0552] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0553] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
the cells and polymer, and the result of analysis of monomer unit
are shown in Table 34. The mass spectrum of
3-hydroxy-4-phenoxybutyric acid (3HPxB) methyl ester,
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO) methyl ester, which were
obtained by GC-MS measurement, are shown in FIG. 22, FIG. 23 and
FIG. 24, respectively.
[0554] From the above result, it was shown that strain YN2 can
produce PHA copolymer composed of only three units of
3-hydroxy-4-phenoxybutyric acid (3HPxB),
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO) by using 8-phenoxyoctanoic
acid as substrate.
Example H-2
Production of P(HPxB/HPxHx/HPxO) Polymer by Using Strain H45 (Yeast
Extract Single-Step Culture)
[0555] strain H45 was inoculated on M9 culture medium, 200 ml in
volume, including 0.5% yeast extract (produced by Difco Co) and
0.1% 8-phenoxyoctanoic acid (PxOA), and its shaking culture was
done at 30.degree. C., 125 stroke/min. After 24 hr, cells were
collected by centrifugation, washed once with cold methanol,
lyophilized and weighed.
[0556] This lyophilized pellet was suspended in 100 ml of acetone,
and a polymer was extracted by mixing for 72 hr at a room
temperature (23.degree. C.). The fluid extract was filtered by a
membrane filter with 0.45 .mu.m pore diameter, and was condensed by
a rotary evaporator. The condensed fluid was reprecipitated in cold
methanol, and then the precipitate alone was collected and
vacuum-dried to obtain a polymer to be weighed.
[0557] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0558] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
the cells and polymer, and the result of analysis of monomer unit
are shown in Table 35. The mass spectrum of
3-hydroxy-4-phenoxybutyric acid (3HPxB) methyl ester,
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO) methyl ester, which were
obtained by GC-MS measurement, are shown in FIG. 25, FIG. 26 and
FIG. 27, respectively.
[0559] From the above result, it was shown that strain H45 can
produce PHA copolymer composed of only three units of
3-hydroxy-4-phenoxybutyric acid (3HPxB),
3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) and
3-hydroxy-8-phenoxyoctanoic acid (3HPxO) by using 8-phenoxyoctanoic
acid as substrate.
[0560] I:
[0561] An example of the production method of polyhydroxyalkanoate
in the present invention by using 11-phenoxyundecanoic acid (PxUDA)
as the raw material is shown here, where this method was applied to
the production of a polyhydroxyalkanoate which is composed of
monomer units derived from three kinds of substances including
3-hydroxy-5-phenoxyvaleric acid (3HPxV),
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-9-phenoxynonanoic acid (3HPxN), and is a copolymer
composed of 3-hydroxy-5-phenoxyvaleric acid (3HPxV),
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-9-phenoxynonanoic acid (3HPxN).
Example I-1
Production of P(HPxN/HPxHp/HPxV) Polymer by Using Strain YN2 (Yeast
Extract, Single-Step Culture)
[0562] Strain YN2 was inoculated in 200 ml of an M9 medium
containing 0.5% yeast extract (produced by Difco Co) and 0.1%
11-phenoxyundecanoic acid (PxUDA), and cultured at 30.degree. C.
with shaking at 125 stroke/min. After 64 hr, the cells were
collected by centrifugation, washed once with cold methanol,
lyophilized and weighed.
[0563] This lyophilized pellet was suspended in 100 ml of acetone,
and the polymer was extracted by mixing for 72 hr at a room
temperature (23.degree. C.). The extract was filtered by a membrane
filter of 0.45 .mu.m pore size and was condensed by a rotary
evaporator. The condensate was reprecipitated in cold methanol, and
then the precipitate was collected and vacuum-dried to obtain a
polymer to be weighed.
[0564] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0565] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was subjected to the analysis by
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
the cells and polymer, and the result of analysis of the monomer
unit are shown in Table 36. The mass spectrum of
3-hydroxy-5-phenoxyvaleric acid (3HPxV) methyl ester,
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-9-phenoxynonanoic acid (3HPxN) methyl ester, which were
obtained by GC-MS measurement, are shown in FIG. 28, FIG. 29 and
FIG. 30, respectively.
[0566] From the above result, it was shown that strain YN2 can
produce PHA copolymer composed of only three units of
3-hydroxy-5-phenoxyvaleric acid (3HPxV),
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-9-phenoxynonanoic acid (3HPxN) by using
11-phenoxyundecanoic acid as substrate.
Example I-2
[0567] Production of P(HPxN/HPxHp/HPxV) Polymer by Using Strain H45
(Yeast Extract, Single-Step Culture)
[0568] Strain H45 was inoculated in 200 ml of an M9 medium
containing 0.5% yeast extract (produced by Difco Co) and 0.1%
11-phenoxyundecanoic acid (PxUDA), and cultured at 30.degree. C.,
with shaking at 125 stroke/min. After 64 hr, the cells were
collected by centrifugation, washed once with cold methanol,
lyophilized and weighed.
[0569] This lyophilized pellet was suspended in 100 ml of acetone,
and the polymer was extracted by mixing for 72 hr at a room
temperature (23.degree. C.). The extract was filtered by a membrane
filter of 0.45 .mu.m pore size and was condensed by a rotary
evaporator. The condensed fluid was reprecipitated in cold
methanol, and then the precipitate was collected and vacuum-dried
to obtain a polymer to be weighed.
[0570] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0571] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
the cells and polymer, and the result of analysis of monomer unit
are shown in Table 37. The mass spectrum of
3-hydroxy-5-phenoxyvaleric acid (3HPxV) methyl ester,
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-9-phenoxynonanoic acid (3HPxN) methyl ester, which were
obtained by GC-MS measurement, are shown in FIG. 31, FIG. 32 and
FIG. 33, respectively.
[0572] From the above result, it was shown that strain H45 can
produce PHA copolymer composed of only three units of
3-hydroxy-5-phenoxyvaleric acid (3HPxV),
3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) and
3-hydroxy-9-phenoxynonanoic acid (3HPxN) by using
11-phenoxyundecanoic acid as substrate.
[0573] J:
[0574] An example of the production method of polyhydroxyalkanoate
in the present invention by using 6-phenylhexanoic acid (PHxA) as
the raw material is shown here, where this method was applied to
the production of a polyhydroxyalkanoate composed of monomer units
derived from 3-hydroxy-6-phenylhexanoic acid (3HPHx), that is,
poly-3-hydroxy-6-phenyl- hexanoic acid (PHPHx), or to the
production of a polyhydroxyalkanoate which is composed of monomer
units derived from 3-hydroxy-6-phenylhexanoi- c acid (3HPHx) and
3-hydroxy-4-phenylbutyric acid (3HPB), and is a copolymer composed
of 3-hydroxy-6-phenylhexanoic acid (3HPHx) and
3-hydroxy-4-phenylbutyric acid (3HPB).
Example J-1
Production of PHPHx Polymer by Using Strain YN2 (Yeast Extract,
Single-Step Culture)
[0575] Strain YN2 was inoculated in 200 ml of an M9 medium
containing 0.5% yeast extract (produced by Difco Co) and 0.1%
6-phenylhexanoic acid (PHxA), and cultured at 30.degree. C. with
shaking at 125 stroke/min. After 27 hr, cells were collected by
centrifugation, washed once with cold methanol, lyophilized and
weighed.
[0576] This lyophilized pellet was suspended in 100 ml of acetone,
and the polymer was extracted by mixing for 72 hr at room
temperature (23.degree. C.). The extract was filtered by a membrane
filter of 0.45 .mu.m pore size, and was condensed by a rotary
evaporator. The condensed fluid was reprecipitated in cold
methanol, and then the precipitate alone was collected and
vacuum-dried to obtain a polymer to be weighed.
[0577] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0578] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water and fractional
separation of the fluid, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
cells and polymer, and the result of analysis of monomer unit are
shown in Table 38. The mass spectrum of 3-hydroxy-6-phenylhexano-
ic acid (3HPHx) methyl ester obtained by GC-MS measurement is shown
in FIG. 34.
[0579] From the above result, it was shown that strain YN2 can
produce PHA polymer consisted of 3-hydroxy-6-phenylhexanoic acid
(3HPHx) alone by using 6-phenylhexanoic acid as substrate.
Example J-2
[0580] Production of P(HPHx/HPB) Polymer by Using Strain H45 (Yeast
Extract Single-Step Culture)
[0581] Strain H45 was inoculated in 200 ml of an M9 culture medium
containing 0.5% yeast extract (produced by Difco Co) and 0.1%
6-phenylhexanoic acid (PHxA), and was cultured at 30.degree. C.,
with shaking at 125 stroke/min. After 27 hr, cells were collected
by centrifugation, washed once with cold methanol, lyophilized and
weighed.
[0582] This lyophilized pellet was suspended in 100 ml of acetone,
and a polymer was extracted by mixing for 72 hr at a room
temperature (23.degree. C.). The extract was filtered by a membrane
filter of 0.45 .mu.m pore size, and was condensed by a rotary
evaporator. The condensed fluid was reprecipitated in cold
methanol, and then the precipitate alone was collected and
vacuum-dried to obtain a polymer to be weighed.
[0583] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0584] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
cells and polymer, and the result of analysis of monomer unit are
shown in Table 39. The mass spectrum of 3-hydroxy-4-phenylbutyric
acid (3HPB) methyl ester and 3-hydroxy-6-phenylhexanoic acid
(3HPHx), which were obtained by GC-MS measurement, are shown in
FIG. 35 and FIG. 36, respectively.
[0585] From the above result, it was shown that strain H45 can
produce PHA copolymer composed only of 3-hydroxy-4-phenylbutyric
acid (3HPB) and 3-hydroxy-6-phenylhexanoic acid (3HPHx) by using
6-phenylhexanoic acid as substrate.
[0586] K:
[0587] An example of the production method of polyhydroxyalkanoate
in the present invention by using both 5-phenylvaleric acid (PVA)
and 5-phenoxyvaleric acid (PxVA) as the raw materials is shown
here, where this method was applied to the production of a
polyhydroxyalkanoate which is composed of monomer units of
3-hydroxy-5-phenylvaleric acid (3HPV) and
3-hydroxy-5-phenoxyvaleric acid (3HPxV), and is a copolymer
composed of 3-hydroxy-5-phenylvaleric acid (3HPV) and
3-hydroxy-5-phenoxyvaleric acid (3HPxV).
Example K-1
[0588] Production of P(HPV/HPxV) Polymer by Using Strain YN2 (Yeast
Extract, Single-Step Culture)
[0589] Strain YN2 was inoculated in 200 ml of an M9 culture medium
containing 0.5% yeast extract (produced by Difco Co), 0.05%
5-phenylvaleric acid (PVA) and 0.05% 5-phenoxyvaleric acid (PxVA),
and cultured at 30.degree. C., with shaking at 125 stroke/min.
After 24 hr, cells were collected by centrifugation, washed once
with cold methanol, lyophilized and weighed.
[0590] This lyophilized pellet was suspended in 100 ml of acetone,
and the polymer was extracted by mixing for 72 hr at room
temperature (23.degree. C.). The extract was filtered by a membrane
filter with 0.45 .mu.m pore size, and was concentrated by a rotary
evaporator. The concentrate was reprecipitated in cold methanol,
and then the precipitate was collected and vacuum-dried to obtain a
polymer to be weighed.
[0591] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0592] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water and fractional
separation of the fluid, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
cells and polymer, and the result of analysis of monomer unit are
shown in Table 40. The mass spectrum of 3-hydroxy-5-phenylvaleri- c
acid (3HPV) methyl ester and 3-hydroxy-5-phenoxyvaleric acid
(3HPxV), which were obtained by GC-MS measurement, are shown in
FIG. 37 and FIG. 38, respectively.
[0593] From the above result, it was shown that strain YN2 can
produce PHA copolymer composed only of 3-hydroxy-5-phenylvaleric
acid (3HPv) and 3-hydroxy-5-phenoxyvaleric acid (3HPxV)
corresponding to 5-phenylvaleric acid and 5-phenoxyvaleric acid as
substrates.
Example K-2
[0594] Production of P(HPV/HPxV) Polymer by Using Strain H45 (Yeast
Extract, Single-Step Culture)
[0595] Strain H45 was inoculated into 200 ml of an M9 culture
medium containing 0.5% yeast extract (produced by Difco Co), 0.05%
5-phenylvaleric acid (PVA) and 0.05% 5-phenoxyvaleric acid (PxVA),
and cultured at 30.degree. C. with shaking at 125 stroke/min. After
24 hr, cells were collected by centrifugation, washed once with
cold methanol, lyophilized and weighed.
[0596] This lyophilized pellet was suspended in 100 ml of acetone,
and a polymer was extracted by mixing for 72 hr at room temperature
(23.degree. C.). The extract was filtered by a membrane filter of
0.45 .mu.m pore size, and was concentrated by a rotary evaporator.
The concentrate was reprecipitated in cold methanol, and then the
precipitate was collected and vacuum-dried to obtain a polymer to
be weighed.
[0597] The molecular weight of the polymer obtained was measured by
gel permeation chromatography (GPC: Toso, HLC-8020; Column: Polymer
Laboratory, PLgel MIXED-C, 5 .mu.m; Solvent: chloroform;
Polystyrene-converted molecular weight).
[0598] The unit composition of the polymer obtained was analyzed as
follows: Five milligram of polymer sample put into a 25 ml
eggplant-type flask was added with 2 ml of chloroform and 2 ml of
methanol containing 3% (v/v) sulfuric acid, and was refluxed for
3.5 hr at 100.degree. C. After addition of water for phase
separation, the organic layer was analyzed by a
gas-chromatograph-mass spectrometer (GC-MS: Shimadzu QP-5050;
Column: DB-WAXETR (produced by J&W Co); EI method) to identify
the methyl esterified compound of PHA monomer unit. The yield of
cells and polymer, and the result of analysis of monomer unit are
shown in Table 41. The mass spectrum of 3-hydroxy-5-phenylvaleric
acid (3HPV) methyl ester and 3-hydroxy-5-phenoxyvaleric acid
(3HPxV), which were obtained by GC-MS measurement, are shown in
FIG. 39 and FIG. 40, respectively.
[0599] From the above result, it was shown that strain H45 can
produce PHA copolymer composed only of 3-hydroxy-5-phenylvaleric
acid (3HPV) and 3-hydroxy-5-phenoxyvaleric acid (3HPxV)
corresponding to 5-phenylvaleric acid and 5-phenoxyvaleric acid as
substrates.
2TABLE 1 Cell Polymer (Dry weight) (Dry weight) Yield Carbon source
(Alkanoate) (mg/l) (mg/l) (%) 6-Phenoxyhexanoic acid 950 100 10.5
8-Phenoxyoctanoic acid 820 90 11 11-Phenoxyundecanoic acid 150 15
10
[0600]
3TABLE 2 NA:CHBA CDW PDW Yield Unit 5:5 756.0 89.1 11.8 NA, CHBA
1:9 132.8 19.3 14.5 NA, CHBA CDW: Cell dry weight (mg/l) PDW:
Polymer dry weight (mg/l) Yield: PDW/CDW (%)
[0601]
4 TABLE 3 P. putida P91 Cell (Dry weight) 520 mg/l Polymer (Dry
weight) 14 mg/l Polymer (Dry weight)/Cell (Dry weight) 2.7% Polymer
molecular weight Mn = 42,000 Mw = 84,000 Monomer unit composition
(area ratio) 3-hydroxy butyric acid 0% 3-hydroxy valeric acid 0%
3-hydroxy hexanoic acid 0% 3-hydroxy heptanoic acid 0% 3-hydroxy
octanoic acid 0% 3-hydroxy nonanoic acid 0% 3-hydroxy decanoic acid
0% 3-hydroxy-4-phenoxy butyric acid 100%
[0602]
5 TABLE 4 P. putida P91 Cell (Dry weight) 500 mg/l Polymer (Dry
weight) 8 mg/l Polymer (Dry weight)/Cell (Dry weight) 1.4% Monomer
unit composition (area ratio) 3-hydroxy butyric acid 0% 3-hydroxy
valeric acid 0% 3-hydroxy hexanoic acid 0% 3-hydroxy heptanoic acid
0% 3-hydroxy octanoic acid 0% 3-hydroxy nonanoic acid 0% 3-hydroxy
decanoic acid 0% 3-hydroxy-4-phenoxy butyric acid 100%
[0603]
6TABLE 5 .sup.1H NMR Spectrum Resonance frequency: 400 MHz .delta.
(ppm) Assignment 0.8 to 1.6 Impurity 2.71 d; 2H, a 3.97 d; 2H, c
5.47 m; 1H, b 6.79 d; 2H, f, h 6.90 t; 1H, g 7.19 t; 2H, e, i m:
multiplet, t: triplet, d: doublet
[0604]
7TABLE 6 .sup.1H NMR Spectrum Resonance frequency: 400 MHz Chemical
Integral Shift/ppm value/H type Identification 1.562 broad Impurity
1.863 4 m c, d 2.474 2 t b 3.994 2 t c 6.905 2 t h, j 6.964 1 t i
7.28 2 t g, k 9.35 broad --COOH m: multiplet, t: triplet, d:
doublet
[0605]
8TABLE 7 Dry cell Dry polymer Yield (mg/l) (mg/l) (dry polymer/dry
cell, %) 750 45 6.0
[0606]
9TABLE 8 .sup.1H NMR Spectrum Resonance frequency: 400 MHz Chemical
Integral Shift/ppm value/H type Identification 1.562 broad Impurity
2.009 2 m d 2.585 2 d b 3.9 2 m e 5.365 1 m c 6.81 2 m h, j 6.89 1
t i 7.21 2 t g, k m: multiplet, t: triplet, s: singlet, d:
doublet
[0607]
10TABLE 9 Dry cell Dry polymer Yield (mg/l) (mg/l) (dry polymer/dry
cell, %) 850 95 11.2
[0608]
11TABLE 10 .sup.1H NMR Spectrum Resonance frequency: 400 MHz
Chemical Shift/ppm Integral/H type Identification 1.85 4 m c, d
2.46 2 t b 3.95 2 t e 6.83 2 t h, j 6.97 2 t g, k 10.15 broad
--COOH m: multiplet, t: triplet, d: doublet
[0609]
12TABLE 11 Dry cell Dry polymer Yield (mg/l) (mg/l) (dry
polymer/dry cell, %) 700 35 5.0
[0610]
13TABLE 12 .sup.1H NMR Spectrum Resonance frequency: 400 MHz
Chemical Shift/ppm Integral/H type Identification to 1.55 Impurity
2.00 2 m d 2.59 2 d b 3.86 2 m c 5.36 1 m c 6.74 2 m h, j 6.90 2 t
g, k m: multiplet, t: triplet, d: doublet
[0611]
14TABLE 13 Dry cell Dry polymer Yield (mg/l) (mg/l) (dry
polymer/dry cell, %) 830 72 8.7
[0612]
15 TABLE 14 P. cichorii H45 Cell (Dry weight) 1050 mg/l Polymer
(Dry weight) 310 mg/l Polymer (Dry weight)/Cell (Dry weight) 30%
Polymer molecular weight Mn = 1.5 .times. 10.sup.5 Mw = 1.8 .times.
10.sup.5 Monomer unit composition (area ratio) 3-hydroxy butyric
acid 0% 3-hydroxy valeric acid 0% 3-hydroxy hexanoic acid 0%
3-hydroxy heptanoic acid 0% 3-hydroxy octanoic acid 0% 3-hydroxy
nonanoic acid 0% 3-hydroxy decanoic acid 0% 3-hydroxy-5-phenyl
valeric acid 100%
[0613]
16 TABLE 15 P. cichorii H45 Cell (Dry weight) 800 mg/l Polymer (Dry
weight) 320 mg/l Polymer (Dry weight)/Cell (Dry weight) 40% Polymer
molecular weight Mn = 9.7 .times. 10.sup.4 Mw = 2.1 .times.
10.sup.5 Monomer unit composition (area ratio) 3-hydroxy butyric
acid 0% 3-hydroxy valeric acid 0% 3-hydroxy hexanoic acid 0%
3-hydroxy heptanoic acid 0% 3-hydroxy octanoic acid 0% 3-hydroxy
nonanoic acid 0% 3-hydroxy decanoic acid 0% 3-hydroxy-5-phenyl
valeric acid 100%
[0614]
17 TABLE 16 P. putida P91 Cell (Dry weight) 880 mg/l Polymer (Dry
weight) 96 mg/l Polymer (Dry weight)/Cell (Dry weight) 11% Monomer
unit composition (area ratio) 3-hydroxy butyric acid 0% 3-hydroxy
valeric acid 0% 3-hydroxy hexanoic acid 0% 3-hydroxy heptanoic acid
0% 3-hydroxy octanoic acid 0% 3-hydroxy nonanoic acid 0% 3-hydroxy
decanoic acid 0% 3-hydroxy-5-phenyl valeric acid 100%
[0615]
18 TABLE 17 P. jessenii P161 Cell (Dry weight) 650 mg/l Polymer
(Dry weight) 410 mg/l Polymer (Dry weight)/Cell (Dry weight) 63%
Polymer molecular weight Mn = 4.9 .times. 10.sup.4 Mw = 9.2 .times.
10.sup.4 Monomer unit composition (area ratio) 3-hydroxy butyric
acid 0% 3-hydroxy valeric acid 0% 3-hydroxy hexanoic acid 0%
3-hydroxy heptanoic acid 0% 3-hydroxy octanoic acid 0% 3-hydroxy
nonanoic acid 0% 3-hydroxy decanoic acid 0% 3-hydroxy-5-phenyl
valeric acid 100%
[0616]
19TABLE 18 .sup.1H NMR Spectrum Resonance frequency: 400 MHz
.delta. (ppm) Assignment 0.9 to 1.7 Broad peak .fwdarw. Impurities
1.9 m; 2H, --CH.sub.2 .fwdarw. d 2.4 to 2.6 m; 4H, --CH.sub.2
.times. 2 .fwdarw. b, e 5.2 to 5.3 m; 1H, --OCH .fwdarw. c 6.9 to
7.0 m; 3H, .fwdarw. Benzene proton .fwdarw. h, i, j 7.1 m; 2H,
.fwdarw. Benzene proton .fwdarw. g, k 7.3 s; Solvent (CDCl.sub.3)
m: multiplet, s: singlet
[0617]
20TABLE 19 .sup.13C-NMR Spectrum Resonance frequency: 100 MHz
.delta. (ppm) Assignment 31.8 --CH.sub.2 .fwdarw. d 35.8 --CH.sub.2
.fwdarw. e 39.4 --CH.sub.2 .fwdarw. b 70.9 --CH .fwdarw. c 77.1 to
77.7 Solvent (CDCl.sub.3) 126.5 --CH (benzene ring) .fwdarw. i
128.7 to 128.9 --CH (benzene ring) .fwdarw. g, h, j, k 141.3 C
(benzene ring) .fwdarw. f 169.7 Carbonyl --C.dbd.O .fwdarw. a
[0618]
21TABLE 20 Chemical shift/ppm type Identification 1.67 m c, d 2.39
t b 2.62 t e 6.97 t h, j 7.12 t g, k 10.7 broad COOH m: multiplet,
t: triplet
[0619]
22 TABLE 21 P. cichorii H45 Cell (Dry weight) 1310 mg/l Polymer
(Dry weight) 270 mg/l Polymer (Dry weight)/Cell (Dry weight) 21%
Monomer unit composition (area ratio) 3-hydroxy butyric acid 0%
3-hydroxy valeric acid 0% 3-hydroxy hexanoic acid 0% 3-hydroxy
heptanoic acid 0% 3-hydroxy octanoic acid 0% 3-hydroxy nonanoic
acid 0% 3-hydroxy decanoic acid 0% 3-hydroxy-5-(4-fluorophenyl)
valeric acid 100%
[0620]
23 TABLE 22 P. putida P91 Cell (Dry weight) 430 mg/l Polymer (Dry
weight) 17 mg/l Polymer (Dry weight)/Cell (Dry weight) 4% Monomer
unit composition (area ratio) 3-hydroxy butyric acid 0% 3-hydroxy
valeric acid 0% 3-hydroxy hexanoic acid 0% 3-hydroxy heptanoic acid
0% 3-hydroxy octanoic acid 0% 3-hydroxy nonanoic acid 0% 3-hydroxy
decanoic acid 0% 3-hydroxy-5-(4-fluorophenyl) valeric acid 100%
[0621]
24 TABLE 23 P. jessenji P161 Cell (Dry weight) 780 mg/l Polymer
(Dry weight) 330 mg/l Polymer (Dry weight)/Cell (Dry weight) 42%
Monomer unit composition (area ratio) 3-hydroxy butyric acid 0%
3-hydroxy valeric acid 0% 3-hydroxy hexanoic acid 0% 3-hydroxy
heptanoic acid 0% 3-hydroxy octanoic acid 0% 3-hydroxy nonanoic
acid 0% 3-hydroxy decanoic acid 0% 3-hydroxy-5-(4-fluorophenyl)
valeric acid 100%
[0622]
25TABLE 24 .sup.1H-NMR Spectrum Resonance frequency: 400 MHz
.delta. (ppm) Assignment 0.9 to 1.7 Broad peak .fwdarw. Impurities
1.8 to 1.9 m; 2H, --CH.sub.2 .fwdarw. d 2.4 to 2.6 m; 4H,
--CH.sub.2 .times. 2 .fwdarw. b, e 5.2 to 5.3 m; 1H, --OCH .fwdarw.
c 6.9 to 7.0 t; 2H, Proton at o-benzene .fwdarw. h, j 7.1 t; 2H,
Proton at m-benzene .fwdarw. g, k 7.3 s; Solvent (CDCl.sub.3) m:
multiplet, t: triplet, s: singlet
[0623]
26TABLE 25 .sup.13C-NMR Spectrum Resonance frequency: 100 MHz
.delta. (ppm) Assignment 31.0 --CH.sub.2 .fwdarw. d 35.9 --CH.sub.2
.fwdarw. e 39.4 --CH.sub.2 .fwdarw. b 70.5 --CH .fwdarw. c 77.1 to
77.7 Solvent (CDCl.sub.3) 115.5, 115.7 --CH at o-benzene .fwdarw.
h, i 130.0 --CH at m-benzene .fwdarw. g, k 136.3 C at p-benzene
.fwdarw. f 160.5, 163.0 --C at with F substitution .fwdarw. i 169.7
Carbonyl --C.dbd.O .fwdarw. a
[0624]
27TABLE 26 CDW PDW Yield 1100 225 20.5 CDW: cell dry weight (mg/l)
PDW: Polymer dry weight (mg/l) Yield rate: PDW/CDW (%)
[0625]
28TABLE 27 CDW PDW Yield 800 120 15.0 CDW: Cell dry weight (mg/l)
PDW: Polymer dry weight (mg/l) Yield rate: PDW/CDW (%)
[0626]
29TABLE 28 CDW PDW Yield 750 130 17.3 CDW: Cell dry weight (mg/l)
PDW: Polymer dry weight (mg/l) Yield rate: PDW/CDW (%)
[0627]
30TABLE 29 CDW PDW Yield 1100 285 25.9 CDW: Cell dry weight (mg/l)
PDW: Polymer dry weight (mg/l) Yield rate: PDW/CDW (%)
[0628]
31TABLE 30 .sup.1H Spectrum Resonans frequency: 400 MHz .delta.
(ppm) Assignment 0.9 to 1.8 m; 11H, --CH.sub.2 .times. 5 .fwdarw.
f, g, h, i, j --CH .fwdarw. e 1.5 to 1.7 m; 2H, --CH.sub.2 .fwdarw.
d 2.5 to 2.6 dd; 2H, --CH.sub.2 .fwdarw. b (further splitting due
to remote H--H coupling with hexyl group) 5.2 to 5.3 m; 1H, --OCH
.fwdarw. c d: doublet, dd: double doublet
[0629]
32TABLE 31 .sup.13C Spectrum Resonans frequency: 100 MHz .delta.
(ppm) Assignment 26.4 to 34.3 hexyl --CH.sub.2, --CH .fwdarw. e to
j 40.1 --CH.sub.2 .fwdarw. d 41.9 --CH.sub.2 .fwdarw. b 69.3 --CH
.fwdarw. c 77.1 to 77.7 Solvent (CDCl.sub.3) 169.8 Carbonyl --C
.dbd.O .fwdarw. a
[0630]
33 TABLE 32 Cell (Dry weight) (mg/l) 1295 Polymer (Dry weight)
(mg/l) 350 Number average molecular weight (Mn) .times. 10.sup.4
3.9 Weight average molecular weight (Mw) .times. 10.sup.4 8.1
3-hydroxy-5-phenoxyvaleic acid (%) 60.0
3-hydroxy-7-phenoxyheptanoic acid (%) 40.0
[0631]
34 TABLE 33 Cell (Dry weight) (mg/l) 1070 Polymer (Dry weight)
(mg/l) 235 Number average molecular weight (Mn) .times. 10.sup.4
2.9 Weight average molecular weight (Mw) .times. 10.sup.4 5.7
3-hydroxy-5-phenoxyvaleic acid (%) 27.9
3-hydroxy-7-phenoxyheptanoic acid (%) 72.1
[0632]
35 TABLE 34 Cell (Dry weight) (mg/l) 1315 Polymer (Dry weight)
(mg/l) 415 Number average molecular weight (Mn) .times. 10.sup.4
2.5 Weight average molecular weight (Mw) .times. 10.sup.4 5.5
3-hydroxy-4-phenoxybutyric acid (%) 2.2 3-hydroxy-6-phenoxyhexanoic
acid (%) 68.7 3-hydroxy-8-phenoxyoctanoic acid (%) 29.1
[0633]
36 TABLE 35 Cell (Dry weight) (mg/l) 990 Polymer (Dry weight)
(mg/l) 225 Number average molecular weight (Mn) .times. 10.sup.4
1.8 Weight average molecular weight (Mw) .times. 10.sup.4 4.3
3-hydroxy-4-phenoxybutyric acid (%) 2.4 3-hydroxy-6-phenoxyhexanoic
acid (%) 73.2 3-hydroxy-8-phenoxyoctanoic acid (%) 24.4
[0634]
37 TABLE 36 Cell (Dry weight) (mg/l) 1510 Polymer (Dry weight)
(mg/l) 385 Number average molecular weight (Mn) .times. 10.sup.4
1.8 Weight average molecular weight (Mw) .times. 10.sup.4 3.8
3-hydroxy-5-phenoxyvaleric acid (%) 32.0
3-hydroxy-7-phenoxyheptanoic acid (%) 65.6
3-hydroxy-9-phenoxynonanoic acid (%) 2.4
[0635]
38 TABLE 37 Cell (Dry weight) (mg/l) 1015 Polymer (Dry weight)
(mg/l) 120 Number average molecular weight (Mn) .times. 10.sup.4
2.2 Weight average molecular weight (Mw) .times. 10.sup.4 4.5
3-hydroxy-5-phenoxyvaleric acid (%) 45.8
3-hydroxy-7-phenoxyheptanoic acid (%) 47.8
3-hydroxy-9-phenoxynonanoic acid (%) 6.4
[0636]
39 TABLE 38 Cell (Dry weight) (mg/l) 1095 Polymer (Dry weight)
(mg/l) 90 Number average molecular weight (Mn) .times. 10.sup.4 6.8
Weight average molecular weight (Mw) .times. 10.sup.4 17.9
3-hydroxy-6-phenylhexanoic acid (%) 100.0
[0637]
40 TABLE 39 Cell (Dry weight) (mg/l) 935 Polymer (Dry weight)
(mg/l) 90 Number average molecular weight (Mn) .times. 10.sup.4 6.9
Weight average molecular weight (Mw) .times. 10.sup.4 15.5
3-hydroxy-4-phenylbutyric acid (%) 1.7 3-hydroxy-6-phenylhexanoic
acid (%) 98.7
[0638]
41 TABLE 40 Cell (Dry weight) (mg/l) 1300 Polymer (Dry weight)
(mg/l) 330 Number average molecular weight (Mn) .times. 10.sup.4
5.0 Weight average molecular weight (Mw) .times. 10.sup.4 10.8
3-hydroxy-5-phenylvaleric acid (%) 62.3 3-hydroxy-5-phenoxyvaleric
acid (%) 37.7
[0639]
42 TABLE 41 Cell (Dry weight) (mg/l) 1050 Polymer (Dry weight)
(mg/l) 165 Number average molecular weight (Mn) .times. 10.sup.4
3.6 Weight average molecular weight (Mw) .times. 10.sup.4 7.7
3-hydroxy-5-phenylvaleric acid (%) 76.4 3-hydroxy-5-phenoxyvaleric
acid (%) 23.6
[0640]
Sequence CWU 1
1
1 1 1501 DNA Pseudomonas jessenii P161 ; FERM P-17445 1 tgaacgctgg
cggcaggcct aacacatgca agtcgagcgg 40 atgacgggag cttgctcctg
aattcagcgg cggacgggtg 80 agtaatgcct aggaatctgc ctggtagtgg
gggacaacgt 120 ctcgaaaggg acgctaatac cgcatacgtc ctacgggaga 160
aagcagggga ccttcgggcc ttgcgctatc agatgagcct 200 aggtcggatt
agctagttgg tgaggtaatg gctcaccaag 240 gcgacgatcc gtaactggtc
tgagaggatg atcagtcaca 280 ctggaactga gacacggtcc agactcctac
gggaggcagc 320 agtggggaat attggacaat gggcgaaagc ctgatccagc 360
catgccgcgt gtgtgaagaa ggtcttcgga ttgtaaagca 400 ctttaagttg
ggaggaaggg cattaaccta atacgttagt 440 gttttgacgt taccgacaga
ataagcaccg gctaactctg 480 tgccagcagc cgcggtaata cagagggtgc
aagcgttaat 520 cggaattact gggcgtaaag cgcgcgtagg tggtttgtta 560
agttggatgt gaaagccccg ggctcaacct gggaactgca 600 ttcaaaactg
acaagctaga gtatggtaga gggtggtgga 640 atttcctgtg tagcggtgaa
atgcgtagat ataggaagga 680 acaccagtgg cgaaggcgac cacctggact
gatactgaca 720 ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 760
cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg 800 ggagccttga
gctcttagtg gcgcagctaa cgcattaagt 840 tgaccgcctg gggagtacgg
ccgcaaggtt aaaactcaaa 880 tgaattgacg ggggcccgca caagcggtgg
agcatgtggt 920 ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac 960
atccaatgaa ctttccagag atggatgggt gccttcggga 1000 acattgagac
aggtgctgca tggctgtcgt cagctcgtgt 1040 cgtgagatgt tgggttaagt
cccgtaacga gcgcaaccct 1080 tgtccttagt taccagcacg taatggtggg
cactctaagg 1120 agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1160
caagtcatca tggcccttac ggcctgggct acacacgtgc 1200 tacaatggtc
ggtacagagg gttgccaagc cgcgaggtgg 1240 agctaatccc acaaaaccga
tcgtagtccg gatcgcagtc 1280 tgcaactcga ctgcgtgaag tcggaatcgc
tagtaatcgc 1320 gaatcagaat gtcgcggtga atacgttccc gggccttgta 1360
cacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1400 gtagctagtc
taaccttcgg gaggacggtt accacggtgt 1440 gattcatgac tggggtgaag
tcgtaccaag gtagccgtag 1480 gggaacctgc ggctggatca c 1501
* * * * *