Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2002-03-26
2004-10-26
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C528S272000, C528S274000, C528S354000, C528S358000, C528S361000
Reexamination Certificate
active
06808907
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing polyhydroxyalkanoate (which is also referred to as “poly-3-hydroxyalkanoic acid”, and hereinafter abbreviated as “PHA” at times), using a microorganism having an ability to produce and accumulate PHA in a cell thereof, or a higher organism such as a plant cell, in which PHA production has enabled by introduction of a PHA synthesis gene.
More specifically, the present invention relates to a method for producing PHA, which comprises a step of removing cell components other than PHA by treating cells containing PHA with an oxidizing agent, which can efficiently remove cell components other than PHA with a few steps, at a low cost, and obtain highly purified PHA with high yield. Further, the present invention relates to a method for producing PHA, wherein PHA containing a reduced amount of residual chlorine or no residual chlorine can be obtained.
Furthermore, the present invention relates to an apparatus, with which the above-described methods for producing PHA can be carried out.
2. Related Background Art
Up till now, it has been reported that a large number of microorganisms produce and accumulate poly-3-hydroxybutyric acid (hereinafter abbreviated as “PHB” at times) or other PHAs in cells thereof (Biodegradable Plastic Handbook, ed. by A Study Group of Biodegradable Plastics, NTS, Inc., p. 178-197). As the conventional plastics, these polymers can also be used for production of various products by melt processing, etc. Moreover, these polymers have an advantage of biodegradability to be completely decomposed by microorganisms in the nature. Therefore, unlike many synthetic high polymers previously used, these polymers do not remain in the natural environment and cause no pollution, and they require no incineration treatment, so they can be useful materials in terms of prevention of air pollution or global warming. Further, these polymers are excellent in biocompatibility, and so it is expected that they will be applied as medical soft materials, etc. It is known that the PHA produced by microorganisms can have various compositions or structures depending on the type of microorganisms used for the production, the composition of medium, culture conditions, etc., and researches on the control of the composition or structure have been made mainly from the viewpoint of improvement of the physical properties of PHA.
For example, it has been reported that
Alcaligenes eutrophus
H16 (ATCC No. 17699) and mutants thereof produce a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid in various composition ratios by varying carbon sources when they are cultured (Japanese Patent Publication Nos. 6-15604, 7-14352, 8-19227, etc.). Japanese Patent Application Laid-Open No. 5-74492 discloses a method for allowing microorganisms of
Methylobacterium
sp.,
Paracoccus
sp.,
Alcaligenes
sp.,
Pseudomonas
sp. to produce a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid by contacting them with primary alcohol containing 3 to 7 carbon atoms. Japanese Patent Application Laid-Open No. 9-191893 discloses that
Comamonas acidovorans
IF013852 produces a polyester having 3-hydroxybutyric acid unit and 4-hydroxybutyric acid unit by performing culture using gluconic acid and 1,4-butanediol as carbon sources.
Japanese Patent No. 2642937 discloses that
Pseudomonas oleovorans
ATCC 29347 produces PHA having a 3-hydroxyalkanoic acid unit containing 6 to 12 carbon atoms, where noncyclic aliphatic hydrocarbon is provided thereto as a carbon source. Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065 disclose that Aeromonas caviae produces a copolymer of two components, 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, by performing culture using oleic acid or olive oil as a carbon source.
The above described PHAs are all PHAs consisting of monomer units having an alkyl group on a side chain, which are synthesized by &bgr;-oxidation of carbohydrate etc. or fatty acid synthesis from a sugar by a microorganism and each of these PHAs is what is called “usual PHA”.
It has been reported that some types of microorganisms produce, PHA having various substituents other than alkyl groups introduced into side chains thereof, that is what is called “unusual PHA”, and using such means, an attempt to improve the physical properties of the PHA produced by microorganisms has been started. Moreover, where a broader application of the PHA produced by microorganism, e.g. application as a functional polymer is considered, the “unusual PHA” is extremely useful. Examples of substituents include a substituent comprising an aromatic ring (a phenyl group, a phenoxy group, a benzoyl group, etc.), an unsaturated hydrocarbon, an ester group, an allyl group, a cyano group, a halogenated hydrocarbon, an epoxide, etc. Of these, particularly, intensive studies on PHA having an aromatic ring have been progressing.
(a) Substituents comprising a phenyl group or a partially substituted phenyl group
Makromol. Chem., 191, 1957-1965 (1990) and Macromolecules, 24, 5256-5260 (1991) report that
Pseudomonas oleovorans
produces PHA comprising 3-hydroxy-5-phenylvaleric acid as a unit from 5-phenylvaleric acid as a substrate. Macromolecules, 29, 1762-1766 (1996) reports that
Pseudomonas oleovorans
produces PHA comprising 3-hydroxy-5-(4′-tolyl)valeric acid as a unit from 5-(4′-tolyl)valeric acid as a substrate. Macromolecules, 32, 2889-2895 (1999) reports that
Pseudomonas oleovorans
produces PHA comprising 3-hydroxy-5-(2′,4′-dinitrophenyl)valeric acid and 3-hydroxy-5-(4′-nitrophenyl)valeric acid as units from 5-(2′,4′-dinitrophenyl)valeric acid as a substrate.
(b) Substituents comprising a phenoxy group or a partially substituted phenoxy group
Macromol. Chem. Phys., 195, 1665-1672 (1994) reports that
Pseudomonas oleovorans
produces a PHA copolymer of a 3-hydroxy-5-phenoxyvaleric acid unit and a 3-hydroxy-9-phenoxynonanoic acid unit, from 11-phenoxyundecanoic acid as a substrate. Japanese Patent No. 2989175 discloses inventions regarding: a homopolymer consisting of a 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MFP)P) unit or a 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DFP)P) unit; a copolymer comprising at least a 3H5(MFP)P unit or a 3H5(DFP)P unit; and a method for producing the above-described polymers, using
Pseudomonas putida
of
Pseudomonas
sp. that synthesizes these polymers. The publication states that the effect of the inventions is to provide stereoregularity and water repellency while retaining a high melting point and good workability. Moreover, Japanese Patent Application Laid-Open No. 2000-72865 reports that
Pseudomonas putida
27N01 produces PHA comprising various types of 3-hydroxyfluorophenoxyvaleric acid units.
In addition to such fluorine-substituted forms, studies on cyano- or nitro-substituted forms have also been progressed. Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996) report the production of PHA comprising 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit from octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as substrates, using
Pseudomonas oleovorans
ATCC 29347 and
Pseudomonas putida
KT 2442.
These reports are useful to obtain polymers having physical properties derived from PHA having an aromatic ring on a side chain thereof, unlike a usual PHA having an alkyl group on a side chain.
As a new category, not only regarding change of physical properties, but also a study for producing PHA having a suitable functional group on a side chain thereof to create a new function using the functional group, is progressing.
For example, Macromolecules, 31, 1480-1486 (1996), Journal of Polymer Science: Part A: Polymer Chemistry, 36, 2381-2387 (1998), etc. report that PHA comprising a highly reactive epoxy group at the terminus of a side chain thereof was synthesized by synthesizing PHA comprising a unit having a vinyl group at
Honma Tsutomu
Imamura Takeshi
Kenmoku Takashi
Nomoto Tsuyoshi
Sugawa Etsuko
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