Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2002-09-18
2004-11-02
Achutamurthy, P. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S006120, C435S071100, C435S252300, C435S320100, C435S440000, C536S023200
Reexamination Certificate
active
06812013
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a polyhydroxyalkanoate (hereinafter, referred to as a “PHA”) synthase, a gene encoding the synthase, a recombinant vector containing the gene, a transformant transformed by the vector, a process for producing the PHA synthase utilizing the transformant, and a process for preparing the PHA utilizing the transformant.
2. Related Background Art
There have been reported a number of microorganisms producing poly-3-hydroxybutyric acid (PHB) or another PHA and storing it therein (“Biodegradable Plastic Handbook”, edited by Biodegradable Plastic Research Society, NTS Co. Ltd., p. 178-197 1995). These polymers may be, as conventional plastics, used for producing a variety of products by, for example, melt processing. Since they are biodegradable, they have an advantage that they can be completely degraded by microorganisms in the natural environment, and they do not cause pollution due to remaining in the natural environment like many conventional polymer compounds. Furthermore, they are excellently biocompatible, and thus are expected to be used in applications such as a medical soft member.
It is known that a composition and a structure of such a PHA produced by a microorganism may considerably vary depending on the type of a microorganism used for the production, a culture-medium composition and culturing conditions. Investigations have been, therefore, mainly focused on controlling such a composition or structure for the purpose of improving physical properties of a PHA.
For example, Japanese Patent Application Nos. 7-14352 and 8-19227 and Japanese Examined Publication No. 6-15604 describe that
Alcaligenes eutropus
H16 (ATCC No. 17699) and its variants may produce 3-hydroxybutyric acid (3HB) and its copolymer with 3-hydroxyvaleric acid (3HV) with various composition ratios by changing a carbon source during culturing.
Japanese Patent Publication No. 2642937 has disclosed that PHA in which a monomer unit is 3-hydroxyalkanoate with 6 to 12 carbon atoms may be produced by supplying a non-cyclic aliphatic hydrocarbon as a carbon source to
Pseudomonas oleovorans
(ATCC No. 29347).
Japanese Patent Application Laid-Open No. 5-7492 discloses methods in which
Methylobacterium
sp.,
Paracoccus
sp.,
Alcaligenes
sp., and
Pseudomonas
sp. are contacted with a primary alcohol with 3 to 7 carbon atoms to produce a copolymer of 3HB and 3HV.
Japanese Patent Application Laid-Open No. 5-93049 and No. 7-265065 have disclosed that
Aeromonas caviae
is cultured using oleic acid or olive oil as a carbon source to produce a two-component copolymer of 3HB and 3-hydroxyhexanoic acid (3HHx).
Japanese Patent Application Laid-Open No. 9-191893 has disclosed that
Comamonas acidovorans
IF013852 is cultured using gluconic acid and 1,4-butanediol as carbon sources to produce a polyester having 3HB and 4-hydroxybutyric acid as monomer units.
Furthermore, it has been reported that certain microorganisms produce PHAs having a variety of substituents such as groups derived from an unsaturated hydrocarbon, ester, allyl, cyano, groups derived from a halogenated hydrocarbon and epoxide. Recently, there have been attempts for improving physical properties of a PHA produced by a microorganism using such a procedure.
As an example of such a polymer, a PHA having a phenyl group in its side chain has been developed. For example, Makromol Chem., 191, 1957-1965 (1990); Macromolecules, 24, 5256-5260 (1991); and Chirality, 3, 492-494 (1991) have described production of a PHA comprising 3-hydroxy-5-phenylvaleric acid (3HPV) as a monomer unit by
Pseudomonas oleovorans
, where there has been observed variation in polymer physical properties probably due to the presence of 3HPV.
As described above, microorganism-produced PHAs with various combinations of composition and structure have been obtained by varying factors such as the type of a microorganism used, a culture medium composition and culturing conditions. However, each microorganism or PHA synthase has significantly different substrate specificity. Therefore, it has been difficult to produce PHAs comprising different monomer units extensively suitable to a variety of applications using known microorganisms or PHA synthases alone.
SUMMARY OF THE INVENTION
A PHA having a substituent in its side chain as described above may be expected to be a “functional polymer” having significantly useful functions and properties owing to the properties of the introduced substituent. It is, therefore, extremely useful and important to prepare a gene encoding a PHA synthase from a microorganism which can produce and store a very useful polymer having both such functionality and biodegradability; prepare a recombinant vector comprising the gene, a transformant transformed by the vector; and develop a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant.
In view of usefulness of such a PHA synthase useful in PHA production, an object of the present invention is to provide a PHA synthase, a gene encoding the enzyme, a recombinant vector comprising the gene, a transformant transformed by the vector, a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant.
For developing a PHA having a novel side-chain structure useful as, for example, a device material or a medical material, the inventors have searched a novel microorganism capable of producing and storing the desired PHA. Additionally, the inventors have intensely investigated for preparing a gene encoding a PHA synthase from such a microorganism, a recombinant vector containing the gene, a transformant transformed by the vector and developing a process for producing a PHA synthase utilizing the transformant and a process for preparing a PHA utilizing the transformant.
The inventors have finally found a novel microorganism capable of producing and storing a novel PHA comprising 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) represented by formula (2) as a monomer unit from synthetic 5-(4-fluorophenyl)valeric acid (PPVA) represented by formula (1) as a starting material, and designate it as P91 strain.
The inventors have also found that P91 strain in capable of producing and storing a PHA comprising 3-hydroxy-4-phenoxy-n-butyric acid (3HPxB) represented by formula (4) as a monomer unit from 4-phenoxy-n-butyric acid (PxBA) represented by formula (3) as a starting material.
An example of a microorganism capable of producing and storing a PHA comprising 3HPxB as a monomer unit is
Pseudomonas oleovorans
involved in a process described in Macromolecules. 29, 3432-3435, 1996. This process is considerably different from the process using PxBA as a substrate in P91 strain in that 8-phenoxyoctanoic acid (PxOA) is used as a substrate. In addition, for a PHA produced, the above reported process provides a copolymer consisting of three monomer units, i.e., 3-hydroxy-8-phenoxyoctanoic acid derived from the substrate PxOA, 3-hydroxy-6-phenoxyhexanoic acid as a byproduct derived from a metabolite and 3HPxB. On the other hand, P91 strain can produce a PHA comprising 3HPxB derived from PxBA as a sole phenoxy-containing monomer unit. In this respect, P91 strain is basically different from the above reported strain.
There are no reports describing microbial production of a PHA comprising 3HPxD as a monomer unit using PxBA as a substrate or 3HPxB as a sole phenoxy-containing monomer unit.
Microbiological properties of P91 strain according to this invention are as follows.
<Microbiological Properties of P91 Strain>
(Morphologic Properties)
Cell shape and size: Bacilliform, 0.6 &mgr;m×1.5 &mgr;m
Cell polymorphism: No
Motility: Yes
Sporulation: No
Gram stainability: Negative
Colonization: Circular, smooth in the overallperiphery, low convex, smooth surface, gloss, cream color
(Physiological Properties)
Catalase: Positive
Oxidase: Positive
O/F test: oxidized form
Reduction of a nitrate: Negative
Indole formation: Negative
Honma Tsutomu
Imamura Takeshi
Suda Sakae
Yano Tetsuya
Achutamurthy P.
Canon Kabushiki Kaisha
Fitzpatrick ,Cella, Harper & Scinto
Pak Yong D.
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