Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2003-04-11
2003-11-18
Acquah, Samuel A. (Department: 1711)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C528S361000, C528S363000, C528S364000, C524S704000, C524S732000
Reexamination Certificate
active
06649381
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel polyhydroxyalkanoate (PHA), a method for production of such PHA and microorganisms for use in the same.
2. Related Background Art
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. On the other hand, polyhydroxyalkanoates (PHAs) produced by microorganisms (hereinafter referred to as “microbial polyester”) represented by poly-3-hydroxy butyric acid (PHB) can be used as the conventional plastics to make various kinds of products with melting processes etd., and can be decomposed by organisms unlike oil-derived synthetic polymers. Therefore, the microbial polyester is bio-decomposed and thus incorporated in the natural material cycle when discarded, and would not remain in the natural environment to cause pollution unlike many conventional synthetic polymer compounds. Furthermore, since the microbial polyesters do not require incineration processes, they are also effective in terms of prevention of air pollution and global warming. Thus, they can be used as a plastic enabling environmental integrity. In addition, the application of the microbial polyesters to medical soft members is under consideration (Japanese Patent Application Laid-Open No. 5-159, Japanese Patent Application Laid-Open No. 6-169980, Japanese Patent Application Laid-Open No. 6-169988, Japanese Patent Application Laid-Open No. 6-225921 and the like).
Heretofore, various bacteria have been reported to produce and accumulate PHB or copolymers of other hydroxyalkanoic acids in the cells (“Biodegradable Plastics Handbook”, edited by Biodegradable Plastics Society, issued by NTS Co. Ltd., P178-197, (1995)). It is known that such microbial PHAs may have a variety of compositions and structures depending on types of the producing microorganisms, the composition of culture media, culture conditions and the like, and up to now, studies regarding the control of these compositions and structures have been carried out to improve the properties of PHA.
For example,
Alcaligenes eutropus
H16 (ATCC No. 17699) and its mutant strains reportedly produce copolymers of 3-hydroxy butyric acid (3HB) and 3-hydroxy valeric acid (3HV) at a variety of composition ratios according to the carbon source in culture (Japanese Patent Publication No. 6-15604, Japanese Patent Publication No. 7-14352, Japanese Patent Publication No. 8-19227 and the like).
Japanese Patent Application Laid-Open No. 5-74492 discloses a method in which the copolymer of 3HB and 3HV is produced by bringing Methylobacterium sp., Paracoccus sp., Alcalugenes sp. or Pseudomonas sp. into contact primary alcohol having 3 to 7 carbons.
Japanese Patent Application Laid-Open No. 5-93049 and Japanese Patent Application Laid-Open No. 7-265065 disclose that two-component copolymers of 3HB and 3-hydroxy hexanoic acid (3HHx) are produced by culturing
Aeromonas caviae
using oleic acid or olive oil as a carbon source.
Japanese Patent Application Laid-Open No. 9-191893 discloses that
Comamonas acidovorans
IFO 13852 produces polyester having 3HB and 4-hydroxy butyric acid as monomer units in culture with gluconic acid and 1,4-butandiol as a carbon source.
Also, in recent years, active researches about PHA composed of 3-hydroxyalkanoate (3HA) of medium-chain-length (abbreviated to mcl) having up to about 12 carbons. Synthetic routes can be classified broadly into two types, and their specific examples will be shown in (1) and (2) below.
(1) Synthesis Using &bgr;-oxidation
Japanese Patent No. 2642937 discloses that PHA having monomer units of 3-hydroxyalkanoate having 6 to 12 carbons is produced by providing as a carbon source aliphatic hydrocarbon to
Pseudomonas oleovorans
ATCC 29347. Furthermore, it is reported in Appl. Environ. Microbiol, 58(2), 746 (1992) that
Pseudomonas resinovorans
produces polyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid at a ratio of 1:15:75:9 as monomer units, using octanoic acid as a single carbon source, and also produces polyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (quantitative ratio of 8:62:23:7) as units, using hexanoic acid as a single carbon source. Herein, it is assumed that 3HA monomer units having longer chain length than that of the starting fatty acid are made by way of fatty acid synthetic route that will be described next in (2).
(2) Synthesis Using Fatty Acid Synthetic Route
It is reported in Int. J. Biol. Macromol., 16(3), 119 (1994) that Pseudomonas sp. 61-3 strain produces polyester made of 3-hydroxyalkanoic acids such as 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid and 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-decenoic acid, 3-hydroxy-5-cis-dodecenoic acid, using sodium gluconate as a single carbon source.
By the way, the biosynthesis of PHA is usually carried out by a PHA synthase using as a substrate “D-3-hydroxyacyl-CoA” occurring as an intermediate of a variety of metabolic pathways in the cell.
Herein, “CoA” means a “coenzyme A”. And, as described in the prior art of the above (1), the biosynthesis of PHA is carried out with “D-3-hydroxyacyl-CoA” occurring in the “&bgr; oxidation cycle” being a starting substance in the case where fatty acids such as octanoic acid and nonanoic acid are used as carbon sources.
Reactions through which PHA is synthesized by way of the “&bgr; oxidation cycle” will be shown below.
On the other hand, as described in the prior art of the above (2), in the case where the PHA is biosynthesized using saccharides such as glucose and the like, the biosynthesis is carried out with “D-3-hydroxyacyl-CoA” converted from “D-3-hydroxyacyl-ACP” occurring in the “fatty acid synthesis pathway” being a starting substance. Herein, “ACP” means a “acyl carrier protein”.
By the way, as described previously, the PHA synthesized in both (1) and (2) described above is PHA constituted by monomer units having alkyl groups in side chains. However, if a wider range of application of the microbial PHA like this, for example an application as a functional polymer is considered, it is expected that PHA having various substituents (for example phenyl groups) introduced in the side chain is significantly useful. With respect to the synthesis of such PHA, for the synthesis using P oxidation, a report regarding PHA having the aryl group and the like in the side chain can be found in, for example, Macromolecules, 24, p5256-5260 (1991). Specifically, it is reported that
Pseudomonas oleovorans
produces polyester having 3-hydroxy valeric acid, 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoic acid and 3-hydroxy-5-phenyl valeric acid (quantitative ratio of 0.6:16.0:41.1:1.7:40.6) as units in the amount of 160 mg for 1 L of culture solution (ratio in dry weight to the cell mass is 31.6%), using 5-phenylvaleric acid and nonanoic acid (mole ratio of 2:1, total concentration of 10 mmol/L) as a medium, and also produces polyester having 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxy-5-phenyl valeric acid (quantitative ratio of 7.3:64.5:3.9:24.3) as units in the amount of 200 mg for 1 L of culture solution (ratio in dry weight to the cell mass is 39.2%), using 5-phenyl valeric acid and octanoic acid (mole ratio of 1:1, total concentration of 10 mmol/L). It is conceivable that the PHA in this report is principally synthesized
Honma Tsutomu
Imamura Takeshi
Kenmoku Takashi
Kobayashi Shin
Kobayashi Toyoko
Acquah Samuel A.
Canon Kabushiki Kaisha
Fitzpatrick ,Cella, Harper & Scinto
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