JP2006320256A - Polyhydroxybutyrate-producing microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism capable of producing PHB (polyhydroxybutyrate) having a high average molecular weight, for the purpose of enhancing a performance of the PHB as a biodegradable plastic. <P>SOLUTION: This polyhydroxybutyrate-producing microorganism contains a sequence expressed by sequence number 1 in its 16S rRNA gene (16S rDNA), can accumulate the PHB having an average molecular weight of ≥3,000,000, and belongs to the genus Methylocystis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel polyhydroxybutyrate-producing bacterium.

In general, plastic is a compound that is produced from petroleum, a fossil resource, and is chemically very stable. Therefore, as long as the original use is performed, it is considered that there is almost no adverse effect on the environment by decomposition or the like.
On the other hand, the plastic is difficult to be rapidly decomposed by microorganisms because of its chemical stability. For this reason, if the plastic product is used and discarded in the environment as it is, it will cause harmful effects such as soiling the landscape and damaging wild animals. In addition, when plastic products are incinerated in a garbage incinerator, there are various problems such as generation of toxic gas, or generation of high temperature and damage of the incinerator.

  The biodegradable plastic can be used in the same manner as the plastic during use, and can be further decomposed into inorganic substances such as water and carbon dioxide by natural microorganisms after use. Therefore, it is expected that the above-mentioned problems in disposal of plastic can be solved.

  For this reason, research for the practical application of biodegradable plastics is underway. Biodegradable plastics are roughly classified into those using natural products such as starch as raw materials, those based on microbial synthesis, and those based on chemical synthesis.

Aliphatic polyester biodegradable plastics are produced by various microorganisms and can be obtained by chemical synthesis. In addition, they have physical properties similar to those of general-purpose resins (the plastics mentioned above), so they can be applied in various fields. Is considered.
One of the aliphatic polyester-based biodegradable plastics is polyhydroxybutyrate (hereinafter referred to as “PHB”), which is produced by microorganisms.

  As microorganisms that produce PHB, methane-utilizing bacteria, methanol-utilizing bacteria, and bacteria belonging to the genus Alcaligenes are known. Each of these bacteria accumulates PHB in the microbial cells using methane, methanol, and sugar as carbon sources.

  In the case of methanol-utilizing bacteria, PHB having an average molecular weight of about 200,000 can be produced, and it is put into practical use as a biodegradable plastic. However, PHB having such a molecular weight has a problem that its elongation is low and brittle, so that its application is limited (for example, see Non-Patent Document 1).

  In general, since the elongation percentage of plastic tends to increase as the average molecular weight increases, a microorganism capable of further accumulating high molecular PHB is desired. It is known that Methylocystis (methylocystis) sp. GB 25 DSMZ 7674, a kind of methane-utilizing bacterium, accumulates PHB having a molecular weight of 2.5 million in the body (for example, see Non-Patent Document 2).

Supervised by Yoshio Inoue, “Green Plastics Latest Technology”, CMC Publishing, June 2002, p271-290

KD Wendlandt, M. Jechorek, J. Helm, and U. Stottmeister, “High Molecular Weight Poly-3 from Methane -Production of hydroxybutyrate "(" Producing poly-3-hydroxybutyrate with a high molecular mass from methane. "), Journal of Biotechnology 86 (2001) p127-p133

  In view of the above problems, an object of the present invention is to provide a microorganism that produces PHB having a high average molecular weight in order to further improve the performance as a biodegradable plastic.

  In order to achieve this object, the characteristic constitution of the PHB-producing bacterium of the present invention includes, as described in claim 1, a 16S rRNA gene (16S rDNA) comprising the sequence of SEQ ID NO: 1, and an average molecular weight of 300 It is a bacterium belonging to the genus Methylocystis that can accumulate more than 10,000 PHBs.

  In the above characteristic configuration, as described in claim 2, the PHB-producing bacterium is Methylocystis sp. It is preferable that it is I431-029II-4A strain (FERM AP-20425).

  The inventors screened hot spring water in Japan to solve the above problems. As a result, the present inventors have succeeded in separating a novel microorganism that accumulates PHB having a higher average molecular weight than conventionally known, and completed the present invention.

That is, the present invention relates to a PHB-producing bacterium that is a methane-utilizing bacterium capable of accumulating PHB having an average molecular weight of 3 million or more. Specifically, the PHB-producing bacterium is a bacterium belonging to the genus Metylocystis, and a 16S rRNA gene (16S rDNA) containing the sequence described in SEQ ID NO: 1 is used as a methyrocystis sp. It is I431-029II-4A strain (FERM AP-20425).
The methylocystis sp. Taking the I431-029II-4A strain as an example, the method for obtaining PHB-producing bacteria and the properties thereof according to the present invention are shown below.

  We searched for microorganisms capable of producing high molecular weight PHB from soil and hot spring water in various parts of Japan. Specifically, enrichment culture was performed using an inorganic salt liquid medium (see Table 1) in which the soil and hot spring water were used as samples and the gas phase was methane: air = 1: 1. An agar medium prepared by adding agar to the inorganic salt liquid medium to 1% (by weight) is prepared, inoculated with an enriched culture solution in which growth has been observed, and the gas phase is methane: air = 1: Solid culture was performed as 1. In addition, both enrichment culture and solid culture were performed at 30 ° C.

  For the strain (10 strains) isolated on the solid medium above, the bacterial species is identified by examining the base sequence of DNA corresponding to 16S rRNA, and the optimum growth temperature, growth rate, PHB accumulation rate The average molecular weight of accumulated PHB was measured.

  Among the strains obtained by the above screening procedure, the mycological properties of the I431-029II-4A strain having the highest average molecular weight of accumulated PHB are shown below. In addition, the base sequence of 16S rDNA of this strain was determined and compared with the base sequence of 16S rDNA of known microorganisms. The results are as follows.

(A) Morphological properties Cell shape: Cocci (inorganic salt liquid medium), Aspergillus (inorganic salt liquid medium)
2. Cell size: 0.2-0.4 μm × 0.6-0.8 μm
3. 3. Cell polymorphism: Yes 4. Mobility: None Sporulation: None

(B) Culture properties Medium used: NMS-VCR medium (see Table 2)
2. Culture conditions: 25 ° C., air: methane = 4: 1 (volume), day 25 Diameter: 1mm
4). Color: Light pink Shape: Circular shape 6. 6. Raised shape: half-lens shape Perimeter: All edges 8. Surface shape, etc .: Smooth Transparency: opaque 10 Viscosity: Butter

(C) Physiological properties Gram stainability: negative Growth optimal temperature: 20-37 ° C
3. Growth optimum pH: 6.9
4). Attitude toward oxygen: aerobic

(D) Chemical properties 1.16S rDNA base sequence: as shown in SEQ ID NO: 1. The closest base strain of the strain: the partial base sequence of 16S rDNA of the I431-029II-4A strain was determined. A DNA database (DDBJ) was accessed, and a homology search was performed based on the determined base sequence of 16S rDNA using the BLAST program. As a result, the homology between Methylocystis echinoides and 16S rDNA of this strain was 98.9%, and the homology between Methylocystis parvus and 16S rDNA of this strain was 97.6%. On the other hand, the homology between 16S rDNA of microorganisms classified into the genus Methylosinus, which is closely related to the genus Methylosistis, and 16S rDNA of this strain was 96.4% at the maximum. Therefore, this strain was determined to be a methane-utilizing bacterium classified into the genus Methylocystis.

  Furthermore, reference was made to “Bergey's Manual of Systematic Bacteriology 1st Edition (1984)”, etc., but it is a known methylocystis bacterium with an average molecular weight of 300. A strain that accumulates more than 10,000 PHBs is not publicly known, and it has been clarified that this strain is a new strain classified into the genus methylocystis. Methylocystis sp. This strain, designated I431-029II-4A, was received on February 22, 2005 by the National Institute of Advanced Industrial Science and Technology as a receipt number FERM AP-20425.

  In addition, regarding the bacteria of the genus Methylostis that accumulate PHB disclosed in Non-Patent Document 2, publications in which mycological properties and the base sequence of 16S rDNA are disclosed cannot be found, and the present invention relates to Taxonomic comparison with other bacteria including PHB producing bacteria was not possible. However, the molecular weight of PHB to be produced is 2.5 million in the upper limit in the bacteria of the genus Methylostis disclosed in Non-Patent Document 2, whereas the PHB producing bacterium according to the present invention produces PHB having an average molecular weight of 3 million or more. They differed in that they were possible and were judged to be at least another strain.

  Here, according to JIS K7127, the elastic modulus of PHB having an average molecular weight of 3 million and the elastic modulus of PHB having an average molecular weight of 2.5 million were compared. The former had an elastic modulus of 3790 MPa, whereas the latter had 3030 MPa. there were. Thus, the elastic modulus of the PHB produced by the microorganism according to the present invention is improved by about 25% compared to the known microorganism-produced PHB. It is important to improve the elastic modulus in order to improve the durability of PHB and expand its applications. The PHB-producing bacterium according to the present invention supplies a very excellent PHB as a biodegradable plastic. Can do.

  Another PHB-producing bacterium belonging to the genus Methylocystis, which contains the sequence described in SEQ ID NO: 1 in the 16S rRNA gene (16S rDNA) according to the present invention and can accumulate PHB having an average molecular weight of 3 million or more, is the above-mentioned methylocystis sp. It can be obtained by the same screening method as the I431-029II-4A strain. In addition, mutants of the genus Metylocystis that are obtained by subjecting the above microorganisms to a known mutation treatment method (for example, ultraviolet irradiation) and can accumulate PHB having an average molecular weight of 3 million or more are also microorganisms according to the present invention. include.

  Hereinafter, embodiments of the present invention will be described with reference to methylocystis sp. The I431-029II-4A strain will be described as an example.

  Methylocystis sp. Isolated by the screening method described above. The I431-029II-4A strain can be cultured in the same manner as known methane-utilizing bacteria. That is, it is a normal aqueous medium containing a carbon source, a nitrogen source, and inorganic salts, and requires methane as a carbon source. The NMS-VCR medium is preferred as the medium for the strain, but is not limited thereto. For example, casein hydrolyzate, yeast extract, yeast autolysate, yeast hydrolyzate, soy flour, corn steep liquor, meat extract and the like can be used as a nitrogen source. Examples of inorganic salts include, but are not limited to, sodium, potassium, magnesium, ammonium, calcium, manganese, zinc, iron, cobalt, phosphate, sulfate, nitrate, chloride, carbonate, etc. The salt can be used. Preferably, the culture is shaken or aerated at pH 6.5 to 7.5, 20 to 37 ° C. (34 ° C. is optimal).

  The medium atmosphere contains methane as an essential component, for example, about 1 to 50% by volume, preferably about 10 to 50% by volume. In addition, since the methane-utilizing bacterium is an obligate aerobic bacterium, oxygen must be contained in the medium atmosphere at about 1 to 50% by volume, preferably about 10 to 50% by volume. The medium atmosphere allows other gases such as nitrogen and carbon dioxide to be contained within the range not impeding the growth of the PHB-producing bacteria of the present invention in addition to the above-mentioned methane and oxygen. For example, the PHB-producing bacterium according to the present invention can be cultured in an atmosphere of air: methane = 4: 1 (methane about 20% by volume, oxygen about 16% by volume).

  The methylocystis sp. In order to measure the average molecular weight of PHB accumulated in the cells of the I431-029II-4A strain, the following experiment was conducted. Examples of the present invention will be described below.

[Production of PHB]
In 200 mL of the inorganic salt liquid medium, the methylocystis sp. I431-029II-4A strain was inoculated and cultured for 72 hours (shaking or standing) while supplying methane: air = 1: 4 at a rate of 400 mL / day. Subsequently, the culture medium was centrifuged, and only the cells were collected and lyophilized. After 5 g of this freeze-dried microbial cell was well ground in a mortar, 300 mL of chloroform was added and heated at 60 ° C. for 5 hours. After removing the cell residue through a glass filter, chloroform was removed from the liquid fraction using an evaporator.
Thereafter, the obtained film-like substance was washed with methanol three times to remove impurities such as fat, and then residual methanol was removed using an evaporator.

[Average molecular weight of PHB]
The obtained film-like substance was dissolved in chloroform, and the average molecular weight was measured under the conditions shown in Table 3 using gel permeation chromatography (hereinafter referred to as GPC).

When the average molecular weight of PHB was calculated based on the data obtained as a result of GPC using polystyrene as the molecular weight standard, it was found to be at least 3 × 10 ⁶ .

  The PHB-producing bacterium according to the present invention can produce a very high molecular weight PHB having an average molecular weight of 3 million or more. Since the PHB obtained from the PHB-producing bacterium has a high elongation rate, a plastic excellent in strength and durability can be provided.

Claims (2)

  A polyhydroxybutyrate-producing bacterium belonging to the genus Metylocystis, which can accumulate polyhydroxybutyrate having an average molecular weight of 3 million or more, comprising the sequence of SEQ ID NO: 1 in a 16S rRNA gene (16S rDNA).   Metylocystis sp. The polyhydroxybutyrate-producing bacterium according to claim 1, which is strain I431-029II-4A (FERM AP-20425).