Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acid esters
Reexamination Certificate
2001-09-14
2003-10-21
Killos, Paul J. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acid esters
C560S051000
Reexamination Certificate
active
06635782
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel polyhydroxyalkanoate (hereinafter, sometimes abbreviated as PHA) and also to a method for manufacturing PHA very efficiently using a microorganism having capability to produce the PHA and accumulate it in bacterial bodies.
In addition, the present invention relates to a method for producing PHA using a substituted alkane derivative as a raw material.
2. Related Background Art
It has been reported so for that a variety of microorganisms produce poly-3-hydroxybutyric acid (hereinafter, sometimes abbreviated as PHB) and other PHAs and accumulate them in bacterial bodies. (“Handbook of Biodegradable Plastic”, edited by Research Association of Biodegradable Plastic, NTS Co., Ltd., pp.178-197). These polymers as well as conventional plastics can be utilized for production of various products by melt processing or the like. Further, the polymers are biodegradable, and therefore they have an advantage of being completely degraded by microorganisms in nature, and causing no pollution by being left in natural environment unlike many conventional synthetic polymer compounds. Further, they are also excellent in biocompatibility and expected to be applied to a medical soft member or the like.
It has been known that such PHA produced by a microorganism may have a variety of compositions and structures depending on types of microorganism, culture medium composition, culture conditions and the like, and mainly from the viewpoint of improving physical properties of PHA, the study has been performed so far for controlling such composition and structure.
<<1>>First, the biosynthesis of PHAs which are obtained by polymerizing monomer units with a relatively simple structure such as 3-hydroxybutyric acid (hereinafter, sometimes abbreviated as 3HB) includes the followings.
For example, it is reported that
Alcaligenes eutropus
H16 strain (ATCC No. 17699) and its variants produce copolymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid in various composition ratios with a carbon source varied in their culturing (U.S. Pat. Nos. 4,393,167 and 4,876,331).
In U.S. Pat. No. 5,200,332, a method for producing copolymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by making a microorganism of Methylobacterium sp., Paracoccus sp., Alcaligenes sp., or Pseudomonas sp. contact a primary alcohol having 3 to 7 carbons is disclosed.
In U.S. Pat. No. 5,292,860 and Japanese Patent Application Laid-Open No. 7-265065, it is disclosed that a two-component copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is produced by culturing
Aeromonas caviae
using oleic acid and olive oil as carbon sources.
In Japanese Patent Application Laid-Open No. 9-191893, it is disclosed that a polyester having monomer units of 3-hydroxybutyric acid and 4-hydroxybutyric acid is produced by culturing
Comamonas acidovorans
IFO 13852 strain using gluconic acid and 1,4-butanediol as carbon sources.
Recently, studies on a PHA comprising a 3-hydroxyalkanoic acid having medium-chain-length (abbreviated as mcl) wherein the number of carbons is up to about 12 have been carried out energetically. The synthetic route of such PHAs can be roughly classified into two parts, specifically examples of which will be shown in the following (1) and (2).
(1) Synthesis Using &bgr;-Oxidation:
In U.S. Pat. No. 5,135,859, it is disclosed that a PHA having a monomer unit of 3-hydroxyalkanoic acid having 6 to 12 carbons is produced by supplying an acyclic aliphatic hydrocarbon as a carbon source to
Pseudomonas oleovorans
ATCC 29347 strain. In Appl. Environ Microbiol, 58 (2), 746 (1992), it is reported that
Pseudomonas resinovorans
produces a polyester with monomer units of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (the amount ratio: 1:15:75:9) using octanoic acid as a sole carbon source, and further a polyester with monomer units of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (the amount ratio: 8:62:23:7) using hexanoic acid as a sole carbon source. Herein, it is considered that a monomer unit of a 3-hydroxyalkanoic acid having longer chain than that of a fatty acid as a material passes through the synthetic route of the fatty acid described in (2).
(2) Synthesis Using Fatty Acid De Novo Biosynthesis
In Int. J. Biol. Macromol., 16 (3), 119 (1994), it is reported that Pseudomonas sp. 61-3 strain produces a polyester with monomer units of 3-hydroxyalkanoic acids such as 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid, and of 3-hydroxyalkenic acids such as 3-hydroxy-5-cis-decenic acid and 3-hydroxy-5-cis-dodecenic acid using sodium gluconate as a sole carbon source.
By the way, the biosynthesis of PHA is usually performed by PHA synthase using “D-3-hydroxyacyl-CoA” as a substrate which is generated as an intermediate of various metabolic pathways in the cells.
Herein, “CoA” means “coenzyme A”. As described in the prior art of the above (1), when using a fatty acid such as octanoic acid, nonanoic acid or the like as a carbon source, it is said that the biosynthesis of PHA is carried out using “D-3-hydroxyacyl-CoA” as a starting material which is generated during the “&bgr;-oxidation pathway”.
The reactions until PHA is biosynthesized through “&bgr;-oxidation pathway” are shown below.
On the other hand, as described in the prior art of the above described (2), when PHA is biosynthesized using saccharides such as glucose or the like, it is said that the biosynthesis is carried out using “D-3-hydroxyacyl-CoA” as a starting material converted from “D-3-hydroxyacyl-ACP” which is generated in the “fatty acid de novo biosynthesis”
Herein, “ACP” means “acyl carrier protein”.
By the way, any of PHAs synthesized in the above (1) and (2) as described above is PHA which comprises monomer units having an alkyl group in the side chain, i.e. “usual PHA”.
<<2>>However, when considering application of such PHAs produced by a microorganism to a wider range, e.g. as a functional polymer, PHAs (“unusual PHAs”) In which substituents other then an alkyl group are introduced into the side chain are expected to be extremely useful. Examples of the substituent include those containing an aromatic ring (such as a phenyl group, a phenoxy group, a bennzoyl group or the like), an unsaturated hydrocarbon, an ester group, an aryl group, a cyano group, a halogenated hydrocarbon, an epoxide or the like. Of them, PHAs having an aromatic ring have been studied extensively.
(a) Those Containing a Phenyl Group or its Partially Substituted Form
In Makromol. Chem., 191, 1957-1965 (1990) and Macromolecules, 24, 5256-5260 (1991), it is reported that
Pseudomonas oleovorans
produces PHA containing 3-hydroxy-5-phenylvaleric acid as a unit using 5-phenylvaleric acid as a substrate.
Specifically, it is reported that
Pseudomonas oleovorans
produces 160 mg (31.64 of the dry weight to the bacterial body) per liter of a culture solution of a PHA comprising 3-hydroxyvaleric acid, 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoic acid and 3-hydroxy-5-phenylvaleric acid in a ratio of 0.6:16.0:41.1:1.7:40.6 as monomer units using 5-phenylvaleric acid and nonanoic acid as substrates (molar ratio of 2:1, total concentration of 10 mmol/L), and also this produces 200 mg (39.2% of the dry weight to the bacterial body) per liter of a culture solution of PHA containing 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxy-5-phenylvaleric acid in a ratio of 7.3:64.5:3.9:24.3 as monomer units using 5-phenylvaleric acid and octanoic acid as substrates (molar ratio of 1:1, total concentration of 10 mmol/L). It is considered that the PHAs in this report is synthesized mainly through the &bgr;-oxidation pathway because nonanoic acid and octanoic acid are used.
The relating description is in Chirality, 3, 492-494 (1991) besides the above where change in th
Honma Tsutomu
Imamura Takeshi
Kenmoku Takashi
Sugawa Etsuko
Yano Tetsuya
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