JPH07155180A - New phb degrading enzyme, its production and microorganism capable of producing the same enzyme - Google Patents
New phb degrading enzyme, its production and microorganism capable of producing the same enzymeInfo
- Publication number
- JPH07155180A JPH07155180A JP5339196A JP33919693A JPH07155180A JP H07155180 A JPH07155180 A JP H07155180A JP 5339196 A JP5339196 A JP 5339196A JP 33919693 A JP33919693 A JP 33919693A JP H07155180 A JPH07155180 A JP H07155180A
- Authority
- JP
- Japan
- Prior art keywords
- phb
- enzyme
- degrading enzyme
- pseudomonas
- degrading
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 51
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 51
- 230000000593 degrading effect Effects 0.000 title claims abstract description 21
- 244000005700 microbiome Species 0.000 title claims description 18
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 229920000331 Polyhydroxybutyrate Polymers 0.000 claims abstract description 38
- 239000005015 poly(hydroxybutyrate) Substances 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 238000002523 gelfiltration Methods 0.000 claims abstract description 3
- 241000589516 Pseudomonas Species 0.000 claims description 22
- 229920000704 biodegradable plastic Polymers 0.000 abstract description 10
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 241000589774 Pseudomonas sp. Species 0.000 abstract description 2
- 239000006227 byproduct Substances 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 abstract 1
- 230000001473 noxious effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 39
- 230000000694 effects Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 239000013587 production medium Substances 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101100243764 Caenorhabditis elegans phb-1 gene Proteins 0.000 description 2
- 229910002570 KH2PO4-Na2HPO4 Inorganic materials 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000011256 inorganic filler Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 239000012766 organic filler Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013502 plastic waste Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 102220201851 rs143406017 Human genes 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、新規なポリヒドロキシ
ブチレート分解酵素（ポリヒドロキシブチレートデポリ
メラーゼ）に関する。また、本発明は、シュードモナス
属に属する微生物から新規なポリヒドロキシブチレート
分解酵素を製造する方法に関する。さらに、本発明は、
新規なポリヒドロキシブチレート分解酵素を産生するシ
ュードモナス属に属する微生物に関する。本発明では、
このポリヒドロキシブチレート分解酵素、あるいは、こ
のポリヒドロキシブチレート分解酵素を産生するシュー
ドモナス属に属する微生物を用いることにより、ポリヒ
ドロキシブチレートを成分とする生分解性プラスティッ
クを効率的に分解することが可能である。TECHNICAL FIELD The present invention relates to a novel polyhydroxybutyrate degrading enzyme (polyhydroxybutyrate depolymerase). The present invention also relates to a method for producing a novel polyhydroxybutyrate-degrading enzyme from a microorganism belonging to the genus Pseudomonas. Further, the present invention provides
The present invention relates to a microorganism belonging to the genus Pseudomonas that produces a novel polyhydroxybutyrate degrading enzyme. In the present invention,
By using this polyhydroxybutyrate-degrading enzyme, or a microorganism belonging to the genus Pseudomonas that produces this polyhydroxybutyrate-degrading enzyme, it is possible to efficiently decompose a biodegradable plastic containing polyhydroxybutyrate as a component. It is possible.
【０００２】[0002]
【従来の技術】近年、生分解性プラスティック製品は、
土中に投棄した後に埋め立てまたは海中投棄などの処理
方法によって自然界の微生物に分解され得る素材とし
て、他のプラスティック素材製品よりも高価であるにも
かかわらず、これらの素材に代わる物として使用が増加
してきている。しかし、現在のプラスティックごみの発
生量を考慮すると、仮に全てのプラスティック製品が生
分解性プラスティックになったとしても、これを処理す
るのに自然界の微生物の働きによる分解を待っているだ
けでは処理しきれなくなることが予想される。そこで、
微生物による分解を促進するために生分解性プラスティ
ック素材に微生物培地成分を配合する方法（特開平4-16
8150号公報）、生分解性プラスティック素材にリパー
ゼ、アミラーゼ、セルラーゼ、乳酸脱水素酵素などの加
水分解酵素を添加する方法（特開平4-168149号公報）、
生分解性プラスティック素材成形品を製造する際に配合
する無機及び／または有機フィラーの配合量と平均体積
を特定範囲に調節し、成形品使用中の生分解及び崩壊を
抑制する方法（特開平４−146953号公報）などが提案さ
れている。しかしながら、ポリヒドロキシブチレート
（以下、ＰＨＢと略記する）を主な素材とする生分解性
プラスティックを特異的に分解するＰＨＢ分解酵素、あ
るいはそのＰＨＢ分解酵素を菌体外に産生する微生物に
ついては、実用化されていない現状にある。2. Description of the Related Art In recent years, biodegradable plastic products have been
Although it is more expensive than other plastic material products as a material that can be decomposed into natural microorganisms by disposal methods such as landfill or sea dumping, it is increasingly used as an alternative to these materials. I'm doing it. However, considering the current amount of generated plastic waste, even if all plastic products become biodegradable plastics, it is not possible to process them by simply waiting for the decomposition by the action of natural microorganisms. It is expected that they will not be able to run out. Therefore,
A method of incorporating a microbial medium component into a biodegradable plastic material in order to promote microbial degradation (Japanese Patent Laid-Open No. 4-16
8150), a method of adding a hydrolytic enzyme such as lipase, amylase, cellulase, lactate dehydrogenase to a biodegradable plastic material (JP-A-4-168149),
A method of suppressing the biodegradation and disintegration during use of a molded product by adjusting the compounding amount and the average volume of the inorganic and / or organic filler to be mixed when manufacturing the molded product of the biodegradable plastic material (Japanese Patent Application Laid-Open No. Hei 4) -146953) has been proposed. However, regarding a PHB-degrading enzyme that specifically decomposes a biodegradable plastic whose main material is polyhydroxybutyrate (hereinafter abbreviated as PHB), or a microorganism that produces the PHB-degrading enzyme outside the cells, The situation is that it has not been put to practical use.
【０００３】[0003]
【発明が解決しようとする課題】本発明者らは、このよ
うな状況を鑑み鋭意研究を行った結果、東京都新宿区の
土壌より分離したシュードモナス（Pseudomonas)属に属
する微生物が、ＰＨＢを特異的に分解するＰＨＢ分解酵
素を産生することを見出した。さらに、こうして得られ
た菌株からこのＰＨＢ分解酵素を分離、精製して本発明
を完成するに至った。したがって、本発明は、生分解性
プラスティックのＰＨＢを分解するＰＨＢ分解酵素を提
供することを課題とする。また、本発明は、シュードモ
ナス(Peudomonas)属に属する微生物を培養してＰＨＢ分
解酵素を採取する方法を提供することを課題とする。さ
らに、本発明は、ＰＨＢ分解酵素を産生するシュードモ
ナス(Pseudomonas) 属に属する微生物を提供することを
課題とする。SUMMARY OF THE INVENTION It is an object of the present invention have found that, as a result of this situation intense research in view of the, the microorganism belonging to Pseudomonas (Pseudomonas) genus were isolated from the soil of Shinjuku-ku, Tokyo, specifically the PHB It was found that it produces a PHB-degrading enzyme that decomposes selectively. Furthermore, the PHB-degrading enzyme was separated and purified from the strain thus obtained, and the present invention was completed. Therefore, an object of the present invention is to provide a PHB degrading enzyme that degrades PHB of biodegradable plastic. Another object of the present invention is to provide a method for culturing a microorganism belonging to the genus Peudomonas and collecting a PHB degrading enzyme. Furthermore, the present invention aims to provide a microorganism belonging to Pseudomonas (Pseudomonas) genus that produce PHB enzymes.
【０００４】[0004]
【課題を解決する手段】本発明は、上記課題を解決する
ためになされたものである。本発明のＰＨＢを特異的に
分解する酵素を産生する微生物は、前記のように東京都
新宿区の土壌から分離された。この微生物の分離方法に
ついては後に記述する。ここで本発明のＰＨＢ分解酵素
を産生する微生物の分類学的性状及び生理学的性状を以
下に示す。 （１）菌形 桿菌 （２）サイズ 1.0 ×1.5 μｍ （３）連鎖 − （４）運動性 ＋ （５）グラム染色性 ＋ （６）好気性 ＋ （７）オキシダーゼテスト ＋ （８）カタラーゼテスト ＋ （９）ＤＮａｓｅ − （１０）ＤＮＡ中のＧＣ含量 63.9 ％ ＋はありを、−はなしを示す。この結果、Bergey's Man
ual of Systematic Bacteriology(1986)に基づいて、こ
の微生物菌体をシュードモナス・スピーシーズと同定し
た。そして、この菌体、シュードモナス（Pseudomonas)
sp.328 株はSBT-3444株として工業技術院生命工学工業
技術研究所に寄託されている（受託番号 FERM P-1395
4)。The present invention has been made to solve the above problems. The microorganism that produces the enzyme that specifically decomposes PHB of the present invention was isolated from the soil in Shinjuku Ward, Tokyo, as described above. The method for separating this microorganism will be described later. Here, the taxonomic and physiological properties of the PHB-degrading enzyme-producing microorganism of the present invention are shown below. (1) Bacterial bacillus (2) Size 1.0 × 1.5 μm (3) Chain − (4) Motility + (5) Gram stainability + (6) Aerobic + (7) Oxidase test + (8) Catalase test + (9) DNase- (10) GC content in DNA 63.9% + means yes, -means none. As a result, Bergey's Man
Based on ual of Systematic Bacteriology (1986), this microbial cell was identified as Pseudomonas species. Then, the bacteria, Pseudomonas (Pseudomonas)
The sp. 328 strain has been deposited as SBT-3444 strain at the Institute of Biotechnology, Institute of Industrial Science and Technology (accession number FERM P-1395.
Four).
【０００５】次に、本発明のＰＨＢ分解酵素を製造する
方法について説明する。シュードモナス・スピーシーズ
をＰＨＢ添加培地、例えば、Succinate 培地[J.Bacteri
ol.119, 152-161(1974)]のクエン酸の代わりにＰＨＢを
１％添加した培地に添加し、30℃前後で15−20時間培養
した後、菌体を遠心分離で回収する。次に、その菌体を
ＰＨＢ分解酵素生産培地に添加し、30℃前後で菌数が 5
×109 cells/ml程度になるまで培養してＰＨＢ分解酵素
を産生させる。そして、その培養上清を回収して70％硫
安分画を行った後、この沈殿画分にＰＨＢを添加して、
ＰＨＢと本酵素の親和性を利用してＰＨＢ−酵素複合体
を形成させ、遠心分離によってＰＨＢ分解酵素蛋白を回
収する。さらに、このＰＨＢ分解酵素蛋白を種々のクロ
マトグラフィーにより精製し、SDS-PAGEで単一なＰＨＢ
分解酵素を得ることができる。Next, a method for producing the PHB degrading enzyme of the present invention will be described. Pseudomonas species were added to a medium containing PHB, such as Succinate medium [J. Bacteri
119 , 152-161 (1974)] to the medium containing 1% of PHB instead of citric acid, and culturing at about 30 ° C. for 15 to 20 hours, and then collecting the cells by centrifugation. Next, the cells were added to a PHB-degrading enzyme production medium, and the number of cells was increased to 5 at around 30 ° C.
The cells are cultured to about 10 9 cells / ml to produce PHB degrading enzyme. Then, after collecting the culture supernatant and performing 70% ammonium sulfate fractionation, PHB was added to the precipitate fraction,
A PHB-enzyme complex is formed by utilizing the affinity between PHB and this enzyme, and the PHB-degrading enzyme protein is recovered by centrifugation. Furthermore, this PHB degrading enzyme protein was purified by various chromatographies, and a single PHB was obtained by SDS-PAGE.
A degrading enzyme can be obtained.
【０００６】このようにして得られるＰＨＢ分解酵素の
性質は、以下の通りである。 （１）至適ｐＨ pH 約 4.3 （２）等電点 pI 約 8.4 （３）最適温度 約 40℃ （４）分子量 約 513,000 ダルトン（ゲル
濾過） 約 42,700ダルトン(SDS-PAGE)The properties of the PHB-degrading enzyme thus obtained are as follows. (1) Optimum pH pH approx. 4.3 (2) Isoelectric point pI approx. 8.4 (3) Optimum temperature approx. 40 ℃ (4) Molecular weight approx. 513,000 Dalton (gel filtration) approx. 42,700 Dalton (SDS-PAGE)
【０００７】次に実施例を挙げて、本発明をさらに詳し
く説明する。Next, the present invention will be described in more detail with reference to examples.
【実施例１】ＰＨＢ分解酵素の土壌からの分離方法につ
いて説明する。すなわち、東京都新宿区から採取した土
壌を生理食塩水で希釈し、この希釈液を遠心分離した上
清を段階的に希釈して、表２に示したＰＨＢ分解酵素生
産培地に寒天を 1.5％添加して作成したプレートに塗抹
する。ＰＨＢ分解酵素を生産する菌株は白濁したプレー
ト上に透明なハローを形成することにより分離すること
ができる。さらに、こうして得られた菌株をＰＨＢ分解
酵素生産培地より培養し、ＰＨＢ分解酵素を生産するこ
とを確認した。得られた菌株は、ＰＨＢ添加培地に寒天
を 1.5％加えて作成したスラントで植え継ぎを行った。
このようにして得られた菌株はＰＨＢ分解活性が著しく
強力で、この菌株を同定したところ、前述のような菌学
的性質を示し、シュードモナス(Pseudomonas)sp.328 株
と命名し、生命工学工業技術研究所に寄託した（受託番
号 FERM P-13954)。Example 1 A method for separating PHB degrading enzyme from soil will be described. That is, soil collected from Shinjuku-ku, Tokyo was diluted with physiological saline, and the supernatant obtained by centrifuging the diluted solution was diluted stepwise, and 1.5% of agar was added to the PHB-degrading enzyme production medium shown in Table 2. Smear onto the plate created by addition. The PHB-degrading enzyme-producing strain can be isolated by forming a transparent halo on a cloudy plate. Furthermore, it was confirmed that the strain thus obtained was cultured in a PHB-degrading enzyme production medium to produce a PHB-degrading enzyme. The obtained strain was subcultured with a slant prepared by adding 1.5% agar to a PHB-containing medium.
The strain thus obtained has a remarkably strong PHB-degrading activity, and when this strain was identified, it showed the above-mentioned mycological properties and was named Pseudomonas sp. 328 strain. Deposited at Technical Research Institute (accession number FERM P-13954).
【０００８】[0008]
【実施例２】実施例１で得られたシュードモナス（Pseu
domonas)sp. 328 株(FERM P-13954)を表１に示すＰＨＢ
添加培地に添加し、30℃で17時間培養した後、遠心分離
で回収した菌体を表２に示すＰＨＢ分解酵素生産培地に
添加し、30℃で菌数が５×109cells/ml となるまで培養
した。次に、遠心分離で培養上清を回収した後、70％硫
安分画を行って、ＰＨＢ分解酵素活性のある沈殿画分を
回収した。そして、このＰＨＢ分解酵素画分にＰＨＢを
添加してＰＨＢ−酵素複合体を形成させ、10,000rpm 15
分間の遠心分離により沈澱したＰＨＢ−酵素複合体を回
収した。この操作を３回繰り返した。さらに、このＰＨ
Ｂ分解酵素蛋白をDEAE-TOYOPEARL (商品名) 650Mに10mM
トリス＋５％ グリセロール(pH8.8）緩衝液を用いて
吸着させ、0.08M NaClで洗浄後、0.18M NaClで溶出させ
た。以上の操作により、SDS-PAGEで単一なＰＨＢ分解酵
素を得た。Example 2 Pseudomonas ( Pseu) obtained in Example 1
domonas) sp. 328 strain (FERM P-13954) shown in Table 1
After adding to the addition medium and culturing at 30 ° C for 17 hours, the cells collected by centrifugation were added to the PHB-degrading enzyme production medium shown in Table 2, and the number of cells was 5 × 10 9 cells / ml at 30 ° C. It culture | cultivated until it became. Next, after collecting the culture supernatant by centrifugation, 70% ammonium sulfate fractionation was carried out to collect a precipitate fraction having PHB degrading enzyme activity. Then, PHB was added to this PHB-degrading enzyme fraction to form a PHB-enzyme complex,
The PHB-enzyme complex precipitated was recovered by centrifugation for minutes. This operation was repeated 3 times. Furthermore, this PH
10 mM of B-degrading enzyme protein in DEAE-TOYOPEARL (trade name) 650M
It was adsorbed using Tris + 5% glycerol (pH 8.8) buffer, washed with 0.08M NaCl, and then eluted with 0.18M NaCl. By the above operation, a single PHB degrading enzyme was obtained by SDS-PAGE.
【０００９】以上の操作における精製度、および回収率
を以下に示した。 ────────────────────────────────── 操 作 活 性 蛋白量 比活性 回収率 ────────────────────────────────── 培養上清 84.8 17.2 4.9 100 硫安分画 72.6 5.8 12.4 85.6 アフィニティ─＊ 50.3 0.99 50.8 59.3 イオン交換クロマト 15.5 0.18 87.6 18.3 ────────────────────────────────── ＊ＰＨＢ−酵素複合体を作成させ、これを遠心分離によ
り回収する方法を示した。単位はそれぞれ以下に示し
た。 活性 U/ml（1Uは、１分間に吸収波長650nm の吸光度
が0.001 減少する量） 蛋白量 mg/ml 比活性 U/mg 回収率 ％The degree of purification and the recovery rate in the above operation are shown below. ────────────────────────────────── Operation activity Protein amount Specific activity recovery rate ────── ──────────────────────────── Culture supernatant 84.8 17.2 4.9 100 Ammonium sulfate fraction 72.6 5.8 12.4 85.6 Affinity ─ * 50.3 0.99 50.8 59.3 Ion Exchange Chromatography 15.5 0.18 87.6 18.3 ────────────────────────────────── * Let the PHB-enzyme complex form, A method of collecting this by centrifugation was shown. The units are shown below. Activity U / ml (1U is the amount by which the absorbance at the absorption wavelength of 650 nm decreases 0.001 per minute) Protein amount mg / ml Specific activity U / mg Recovery rate%
【００１０】[0010]
【表１】 ＰＨＢ添加培地 ──────────────────────── 成 分 濃 度 ──────────────────────── ＰＨＢ 1 ％ KH2PO4-Na2HPO4(pH=6.8) 33 mM 塩化アンモニウム 18 mM 硫酸マグネシウム・７水塩 2 mM 塩化鉄（III)・６水塩 0.037mM 塩化カルシウム 0.045mM ────────────────────────[Table 1] PHB-added medium ──────────────────────── Component concentration ──────────────── ───────── PHB 1% KH 2 PO 4 -Na 2 HPO 4 (pH = 6.8) 33 mM ammonium chloride 18 mM magnesium sulfate heptahydrate 2 mM iron (III) chloride hexahydrate 0.037 mM calcium chloride 0.045mM ─────────────────────────
【００１１】[0011]
【表２】 ＰＨＢ分解酵素生産培地 ─────────────────────── 成 分 濃 度 ─────────────────────── ＰＨＢ 1 ％ KH2PO4-Na2HPO4(pH=6.8) 33 mM 塩化アンモニウム 18 mM 硫酸マグネシウム・７水塩 1 mM ───────────────────────[Table 2] PHB-degrading enzyme production medium ─────────────────────── Component concentration ─────────────── ───────── PHB 1% KH 2 PO 4 -Na 2 HPO 4 (pH = 6.8) 33 mM ammonium chloride 18 mM magnesium sulfate heptahydrate 1 mM ────────── ─────────────
【００１２】[0012]
【試験例１】実施例２で得られたＰＨＢ分解酵素につい
て、ＰＨＢ分解活性の有無を試験した。精製水 0.8ml、
0.1M 酢酸緩衝液 1ml、10mg/ml濃度のＰＨＢ溶液40μｌ
から成る反応液1.84mlに、実施例２で得られたＰＨＢ分
解酵素液 200μl を添加し、30℃で20分間保持したとこ
ろ、ＰＨＢに特異的な吸収波長 650nmの吸光度が1.0 か
ら0.283 に減少した。Test Example 1 The PHB degrading enzyme obtained in Example 2 was tested for the presence or absence of PHB degrading activity. 0.8 ml of purified water,
0.1M acetate buffer 1ml, 10mg / ml PHB solution 40μl
To 1.84 ml of the reaction solution consisting of 200 μl of the PHB-degrading enzyme solution obtained in Example 2 and kept at 30 ° C. for 20 minutes, the absorbance at the absorption wavelength of 650 nm specific to PHB decreased from 1.0 to 0.283. .
【００１３】[0013]
【発明の効果】本発明のＰＨＢ分解酵素、あるいは、本
発明のＰＨＢ分解酵素を産生するシュードモナス属に属
する微生物を用いることにより、有害な副産物を伴うこ
と無く生分解性プラスティックのＰＨＢを短時間で効率
的に処理することができ、環境保護に有用である。By using the PHB-degrading enzyme of the present invention or the microorganism belonging to the genus Pseudomonas producing the PHB-degrading enzyme of the present invention, PHB of biodegradable plastic can be produced in a short time without causing harmful by-products. It can be processed efficiently and is useful for environmental protection.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:38） ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Office reference number FI technical display location C12R 1:38)
Claims (6)
ト分解酵素。 （１）作用：ポリヒドロキシブチレートを分解する。 （２）至適 pH ：約 4.3 （３）等電点：約 8.4 （４）最適温度：約 40 ℃ （５）分子量：約513,000 ダルトン（ゲル濾過） 約42,700ダルトン(SDS-PAGE)1. A polyhydroxybutyrate degrading enzyme having the following properties. (1) Action: Decomposes polyhydroxybutyrate. (2) Optimum pH: approx. 4.3 (3) Isoelectric point: approx. 8.4 (4) Optimum temperature: approx. 40 ° C (5) Molecular weight: approx. 513,000 daltons (gel filtration) approx. 42,700 daltons (SDS-PAGE)
る微生物に由来する請求項１記載の酵素。2. The enzyme according to claim 1, which is derived from a microorganism belonging to the genus Pseudomonas .
p. 328株(FERM P-13954)である請求項２記載の酵素。According to claim 3 wherein the microorganism is Pseudomonas (Pseudomonas) s
The enzyme according to claim 2, which is p. 328 strain (FERM P-13954).
し、請求項１記載のポリヒドロキシブチレート分解酵素
産生能を有する微生物を培養し、培地または菌体に該酵
素を産生せしめ、これを採取することを特徴とするポリ
ヒドロキシブチレート分解酵素の製造法。4. A method of culturing a microorganism belonging to the genus Pseudomonas and capable of producing a polyhydroxybutyrate-degrading enzyme according to claim 1, allowing the medium or cells to produce the enzyme, and collecting the enzyme. A method for producing a characteristic polyhydroxybutyrate degrading enzyme.
p.328 株(FERM P-13954)である請求項４記載の製造法。5. A microorganism Pseudomonas (Pseudomonas) s
The method according to claim 4, which is strain p.328 (FERM P-13954).
(FERM P-13954)。6. Pseudomonas (Pseudomonas) sp.328 shares
(FERM P-13954).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5339196A JPH07155180A (en) | 1993-12-06 | 1993-12-06 | New phb degrading enzyme, its production and microorganism capable of producing the same enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5339196A JPH07155180A (en) | 1993-12-06 | 1993-12-06 | New phb degrading enzyme, its production and microorganism capable of producing the same enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07155180A true JPH07155180A (en) | 1995-06-20 |
Family
ID=18325152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5339196A Pending JPH07155180A (en) | 1993-12-06 | 1993-12-06 | New phb degrading enzyme, its production and microorganism capable of producing the same enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07155180A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005045017A1 (en) * | 2003-11-05 | 2005-05-19 | National Institute Of Advanced Industrial Science And Technology | Method of decomposing polyhydroxyalkanoate resin |
| CN115942963A (en) * | 2020-07-31 | 2023-04-07 | 金伯利-克拉克环球有限公司 | Absorbent article with automatic biodegradation |
-
1993
- 1993-12-06 JP JP5339196A patent/JPH07155180A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005045017A1 (en) * | 2003-11-05 | 2005-05-19 | National Institute Of Advanced Industrial Science And Technology | Method of decomposing polyhydroxyalkanoate resin |
| JPWO2005045017A1 (en) * | 2003-11-05 | 2007-05-24 | 独立行政法人産業技術総合研究所 | Method for decomposing polyhydroxyalkanoate resin |
| JP4649593B2 (en) * | 2003-11-05 | 2011-03-09 | 独立行政法人産業技術総合研究所 | Method for decomposing polyhydroxyalkanoate resin |
| CN115942963A (en) * | 2020-07-31 | 2023-04-07 | 金伯利-克拉克环球有限公司 | Absorbent article with automatic biodegradation |
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