JPH09191893A - Production of hydroxyalkanoic acid copolymer - Google Patents

Production of hydroxyalkanoic acid copolymer

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Publication number
JPH09191893A
JPH09191893A JP857796A JP857796A JPH09191893A JP H09191893 A JPH09191893 A JP H09191893A JP 857796 A JP857796 A JP 857796A JP 857796 A JP857796 A JP 857796A JP H09191893 A JPH09191893 A JP H09191893A
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Prior art keywords
acid copolymer
hydroxyalkanoic acid
4hb
3hb
cells
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JP857796A
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Japanese (ja)
Inventor
Osamu Hiruta
Yuji Saito
Hideaki Takebe
Takashi Tomosawa
孝 友沢
祐二 斎藤
英日 武部
修 蛭田
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Meiji Seika Kaisha Ltd
Taisei Corp
大成建設株式会社
明治製菓株式会社
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Abstract

PROBLEM TO BE SOLVED: To effectively obtain the subject polymer having biodegradability by extracting the wet cells of a microorganism producing a copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units with acetone containing a surfactant under heating.
SOLUTION: This method for producing a hydroxyalkanoic acid copolymer comprises culturing a microorganism (e.g. Comamonans acidovorans IFO 13852) having an ability to produce the hydroxyalkanoic acid copolymer comprising 3-hydroxybutyrate units of formula I and 4-hydroxybutyrate units of formula II, centrifuging the cultured solution, and subsequently mixing the recovered wet cells with acetone containing a surfactant under heating to extract the hydroxyalkanoic acid copolymer accumulated in the recovered cells. Thus, the objective hydroxyalkanoic acid copolymer provided with a larger dynamic strength than those of general purpose polymers such as polyethylene or nylon and with excellent biodegradability is obtained.
COPYRIGHT: (C)1997,JPO

Description

【発明の詳細な説明】 DETAILED DESCRIPTION OF THE INVENTION

【0001】 [0001]

【発明の属する技術分野】本発明は、生分解性の3−ヒドロキシブチレート単位(以下、3HB成分という。) The present invention relates to the biodegradable 3-hydroxybutyrate units (hereinafter, referred to as 3HB component.)
と4−ヒドロキシブチレート単位(以下、4HB成分という。)とからなるヒドロキシアルカン酸共重合体(以下、P(3HB-co-4HB) という。)を、微生物を用いて効率よく製造する方法に関する。 When 4-hydroxybutyrate units (hereinafter, referred to as 4HB component.) And hydroxyalkanoic acid copolymer consisting (hereinafter, P (3HB-co-4HB) as.), And relates to a process for producing efficiently using microorganisms .

【0002】 [0002]

【従来の技術】多くの微生物は、3−ヒドロキシ酪酸(3HB)のホモポリエステルをエネルギー貯蔵物質として蓄積する。 BACKGROUND OF THE INVENTION Many microorganisms accumulates homopolyester 3-hydroxybutyric acid (3HB) as an energy storage material. さらに近年では、用いる微生物や炭素源の種類に応じて、3−ヒドロキシ酪酸と、3−ヒドロキシプロピオン酸(3HP)や4−ヒドロキシ酪酸(4H In more recent years, it used depending on the type of microorganisms and carbon sources, 3-hydroxy butyric acid, 3-hydroxypropionic acid (3HP) and 4-hydroxybutyrate (4H
B)などの他のヒドロキシアルカン酸とがランダムに共重合したヒドロキシアルカン酸共重合体の発酵合成も確認されている。 B) and other hydroxy acids, such as has been confirmed fermentation synthesis of hydroxyalkanoic acid copolymer obtained by copolymerizing randomly. これらの共重合体は、その共重合組成に応じて多様な性質を示すことから、微生物によって分解可能である、生分解性プラスチック材料として大いに注目されている。 These copolymers, because they exhibit a variety of properties depending on the copolymer composition, can be decomposed by microorganisms, it has attracted much attention as a biodegradable plastic material. 特に、4HB成分含量の高いP(3HB In particular, high 4HB ingredient content P (3HB
-co-4HB) は、ポリエチレンやナイロンなどの汎用ポリマー以上の力学的強度と、優れた生分解性を兼ね備えていることから、環境に調和したプラスチック素材として期待されている。 -co-4HB), since it has both the mechanical strength of the above generic polymers, such as polyethylene and nylon, the excellent biodegradability, it is expected as a plastic material in harmony with the environment.

【0003】ところで、微生物を用いてヒドロキシアルカン酸重合体又は共重合体を製造する場合、これらの重合体又は共重合体はエネルギー貯蔵物質として微生物体内に蓄積されるため、菌体から分離・精製するための工程が必要となる。 Meanwhile, when manufacturing hydroxyalkanoic acid polymer or copolymer using a microorganism, since these polymers or copolymers are accumulated in the microbial body as an energy storage material, separation and purification from the cells process for is required.

【0004】菌体内からの代表的な分離・精製方法としては、例えば、ヒドロキシアルカン酸重合体及び/又は共重合体が蓄積された微生物の菌体を乾燥し、乾燥菌体からクロロホルムや塩化メチレンなどのハロゲン系有機溶剤を用いて前記重合体及び/又は共重合体を抽出した後、抽出液をメタノールやヘキサンなどの貧溶媒と混合することによって前記重合体及び/又は共重合体を析出させて回収する方法(第1の方法)、ヒドロキシアルカン酸重合体及び/又は共重合体が蓄積された微生物の細胞質をプロテアーゼで溶解させ、界面活性剤を用いて当該重合体及び/又は共重合体を精製する方法(第2の方法)が挙げられる。 [0004] As a typical separation and purification method from the microbial cells, for example, hydroxy alkanoic acid polymers and / or copolymers microbial cells accumulated was dried, chloroform dried cells and methylene chloride after extracting the polymer and / or copolymers with a halogenated organic solvent such as, the extract to precipitate the polymer and / or copolymer by mixing with a poor solvent such as methanol or hexane method for recovering Te (first method), the cytoplasm of a microorganism hydroxyalkanoic acid polymer and / or copolymer is accumulated dissolved protease, the polymer with a surfactant and / or copolymer method of purifying (second method) and the like.

【0005】しかしながら、上記第1の方法は、高純度かつ高収率でヒドロキシアルカン酸重合体及び/又は共重合体の分離・精製が可能であるが、菌体を乾燥させる工程が必要であるため効率が悪く、さらに環境規制に関わるハロゲン系有機溶剤を使用することなどの問題がある。 However, the first method is susceptible to separation and purification of high purity and hydroxyalkanoic acid polymer in high yield and / or copolymers, it is necessary drying the cells because efficiency is low, there are problems such as the use of halogenated organic solvents further related to environmental regulations. また、第2の方法では、抽出方法として細胞質分解酵素であるプロテアーゼを用いるために高価であり、実用化には問題がある。 In the second method, as an extraction method is expensive to use a protease, a cytoplasmic enzyme, the practical use is problematic.

【0006】一方、用いる微生物の種類や培養条件によっては、3HB成分含量が高いP(3HB-co-4HB) [0006] On the other hand, depending on the microbial type and culture conditions used, 3HB ingredient content is high P (3HB-co-4HB)
と、4HB成分の含量が高いP(3HB-co-4HB) とを同時に蓄積することがある。 If, sometimes content of 4HB components accumulate high P (3HB-co-4HB) and at the same time. このような場合、従来の方法では、3HB成分の含量が高いP(3HB-co-4H In this case, in the conventional method, a high content of 3HB component P (3HB-co-4H
B) と4HB成分の含量が高いP(3HB-co-4HB) B) the content of 4HB component high P (3HB-co-4HB)
とが混合した状態で一緒に抽出されるので、どちらか一方を得るためにはそれぞれを分離するための精製工程が必要となるという問題もある。 Because it is extracted together in a state in which bets are mixed, there is a problem that a purification step for in order to obtain either the separating each is required.

【0007】 [0007]

【発明が解決しようとする課題】したがって、本発明の課題は、環境規制に関わるハロゲン系有機溶剤を用いずに、効率よく経済的に3HB成分と4HB成分とからなるヒドロキシアルカン酸共重合体(以下、P(3HB-c OBJECTS OF THE INVENTION It is therefore an object of the present invention, without using a halogen-based organic solvent on environmental regulations, efficient economically 3HB component and 4HB hydroxyalkanoic acid copolymer comprising a component ( below, P (3HB-c
o-4HB) という。 o-4HB) called. )を微生物を用いて製造する方法を提供することにある。 ) To to provide a method of manufacturing using a microorganism. 特に、本発明は、微生物菌体内に3HB成分含量が高いP(3HB-co-4HB) と、4H In particular, the present invention includes a 3HB ingredient content in microbial cells within a high P (3HB-co-4HB), 4H
B成分含量が高いP(3HB-co-4HB)が蓄積される場合に、4HB成分含量が高いP(3HB-co-4HB) B component content is higher P if (3HB-co-4HB) is accumulated, 4HB ingredient content is high P (3HB-co-4HB)
(通常、4HB成分含量が60モル%以上のもの)を該菌体から選択的に抽出することが可能なヒドロキシアルカン酸共重合体の製造方法を提供することを課題とする。 (Usually, 4HB component content of 60 mol% or more) and to provide a method for manufacturing a can a selectively extracted from microbial cells such hydroxyalkanoic acid copolymer.

【0008】 [0008]

【課題を解決するための手段】 In order to solve the problems]

本発明は、下記式(1): −OCH(CH 3 )CH 2 CO− (1) で表される3−ヒドロキシブチレート単位(3HB成分)と、下記式(2): −OCH 2 CH 2 CH 2 CO− (2) で表される4−ヒドロキシブチレート単位(4HB成分)とからなるヒドロキシアルカン酸共重合体(P(3 The present invention has the following formula (1): -OCH (CH 3 ) and CH 2 CO- (1) represented by 3-hydroxybutyrate units (3HB component), the following equation (2): -OCH 2 CH 2 CH 2 CO- (2) represented by 4-hydroxybutyrate units (4HB component) and consisting of hydroxyalkanoic acid copolymer (P (3
HB-co-4HB) )生産能を有する微生物の菌体内に蓄積された前記ヒドロキシアルカン酸共重合体を抽出・分離する工程を含む前記ヒドロキシアルカン酸共重合体の製造方法において、前記ヒドロキシアルカン酸共重合体の抽出を、前記菌体の湿菌体に界面活性剤含有アセトンを混合し、加熱することにより行うことを特徴とする、 HB-co-4HB)) in the production method of the hydroxyalkanoic acid copolymer comprising the step of extracting and separating the hydroxyalkanoic acid copolymer accumulated in the cells of microorganism having the ability to produce, the hydroxyalkanoic acid the extraction of the copolymer, by mixing the surfactant-containing acetone wet cells of said cell, characterized in that by heating,
ヒドロキシアルカン酸共重合体の製造方法を提供するものである。 There is provided a method of manufacturing a hydroxyalkanoic acid copolymer.

【0009】本発明においては、微生物菌体内に蓄積された上記P(3HB-co-4HB) の抽出を、界面活性剤を含有するアセトンと混合して加熱することにより行う。 [0009] In the present invention, it carried out by extraction of the accumulated in the microbial cells within P (3HB-co-4HB), heated and mixed with acetone containing a surfactant. この場合、湿菌体をそのまま界面活性剤含有アセトンに混合すればよいので菌体を乾燥する必要がなく、生産性、経済性に優れる。 In this case, it is not necessary to dry the cells because it may be mixed into the surfactant containing acetone wet cells, productivity, excellent in economical efficiency. また、湿菌体と界面活性剤含有アセトンとを混合して加熱する際の加熱温度はアセトンの沸点程度又はそれ以上であれば問題はなく、好ましくは50〜60℃である。 The heating temperature in heating and mixing the wet cells and a surfactant-containing acetone is not a problem as long as the boiling point of about or more acetone, preferably 50-60 ° C.. 加熱温度が低すぎると抽出効率及び抽出速度が低下する。 And the heating temperature is too low extraction efficiency and rate of extraction is decreased. 更に、抽出時間は、通常3〜10時間程度である。 Further, the extraction time is usually from about 3 to 10 hours.

【0010】界面活性剤の種類は特に限定されず、具体的には、N−アシルアミノ酢酸塩、アルキルスルホン酸塩、アルキルベンゼンスルホン酸塩、アルケンスルホン酸塩、脂肪酸塩、硫酸エステル塩、硫酸アルキルフェニルポリオキシエチレン塩、硫酸アルキルポリオキシエチレン塩、リン酸アルキルポリオキシエチレン塩等の陰イオン界面活性剤;アルキルフェニルポリオキシエチレンエーテル、アルキルポリオキシエチレンエーテル、脂肪酸ポリオキシエチレンエステル、Tween 系界面活性剤(ポリオキシエチレンソルビタン脂肪酸エステル)、ポリオキシエチレンポリオキシプロピレン、N−ヒドロキシエチルアルカンアミド等の非イオン界面活性剤;1-(2 [0010] type of surfactant is not particularly limited, specifically, N- acylamino acid salts, alkyl sulfonates, alkyl benzene sulfonates, alkene sulfonates, fatty acid salts, sulfuric acid ester salts, alkyl phenyl sulfate polyoxyethylene salts, alkyl sulfates, polyoxyethylene salts, anionic surfactants such as alkyl phosphates, polyoxyethylene salts, alkyl polyoxyethylene ether, alkyl polyoxyethylene ethers, fatty acid polyoxyethylene esters, Tween series surfactants (polyoxyethylene sorbitan fatty acid esters), polyoxyethylene polyoxypropylene, nonionic surfactants such as N- hydroxyethyl alkane amide; 1- (2
-アシルアミノエチル)-1-メチル-2-アルキルイミダゾリニウム塩、アルキルトリメチルアンモニウム塩、アルキルピリジニウム塩、アルキルベンジルジメチルアンモニウム塩、アルキルメチルジポリエトキシアンモニウム塩等の陽イオン界面活性剤;N−アルキルアミノ酸、N− - acyl aminoethyl) -1-methyl-2-alkyl imidazolinium salts, alkyl trimethyl ammonium salts, alkyl pyridinium salts, alkyl benzyl dimethyl ammonium salts, cationic surfactants such as alkyl methyl di polyethoxy ammonium salt; N- alkyl amino acids, N-
アルキルジメチルアミノ酸、アルキルジメチルアミンオキシド等の両性界面活性剤が例示され、これらの中で好ましいのは陰イオン界面活性剤及び非イオン界面活性剤であり、更に好ましいのはTween80 及びドデシル硫酸ナトリウムである。 Alkyl dimethyl amino acids, is exemplified amphoteric surfactants such as alkyl dimethyl amine oxides, those Preferred among are anionic surfactants and nonionic surfactants, more preferred are Tween80 and sodium dodecyl sulfate .

【0011】また、アセトン中の界面活性剤の配合量は、0.05〜0.5 重量%の範囲が好ましい。 [0011] The amount of surfactant in acetone, preferably in the range of 0.05 to 0.5 wt%. 界面活性剤の配合量が高すぎると抽出したP(3HB-co-4HB) に界面活性剤が混在するとともにP(3HB-co-4HB) P and the extracted amount of the surfactant is too high (3HB-co-4HB) with a surfactant are mixed in the P (3HB-co-4HB)
の分子量の低下を招き、界面活性剤の配合量が低すぎると抽出効率が低下する。 Cause a decrease in the molecular weight, the extraction efficiency decreases with the amount of the surfactant is too low.

【0012】菌体から上記P(3HB-co-4HB) を抽出した後、P(3HB-co-4HB)を回収するには、従来公知の方法で行うことができる。 [0012] After extracting the P (3HB-co-4HB) from the cells, to the recovery of P (3HB-co-4HB) can be carried out by a conventionally known method. 具体的には、上記菌体とアセトンの混合液から菌体残渣を濾過又は遠心分離により除去し、次いで残ったアセトン溶液を貧溶媒と混合してP(3HB-co-4HB) を析出させることによってP(3HB-co-4HB) を回収することができる。 Specifically, the cell debris was removed by filtration or centrifugation from a mixture of the bacteria and acetone, and then the remaining to acetone solution is mixed with a poor solvent to precipitate P (3HB-co-4HB) it can be recovered P (3HB-co-4HB) by. 貧溶媒の種類は特に限定されず、具体的にはメタノール、 Type of poor solvent is not particularly limited, specifically methanol,
ヘキサン、ペンタン、水等が例示され、好ましいのはメタノール及びヘキサンである。 Hexane, pentane, water, etc. are exemplified, preferred are methanol and hexane.

【0013】本発明で用いる微生物は、P(3HB-co- [0013] The microorganism used in the present invention, P (3HB-co-
4HB) 生産能を有する微生物であればいずれのものでもよい。 4HB) may be any one as long as a microorganism having an ability to produce. 例えば、コマモナス(comamonas) 属、アルカリゲネス(Alcaligenes) 属、ロドコッカス(Rhodococcus) For example, Comamonas (Comamonas) genus Alcaligenes (Alcaligenes) genus Rhodococcus (Rhodococcus)
属等に属するものであって、P(3HB-co-4HB) 生産能を有する微生物が挙げられる。 Be those belonging to the genus or the like, and a microorganism having a P (3HB-co-4HB) producing ability. 具体的には、コマモナス アシドボランズ(Comamonas acidovorans)、アルカリゲネス ユートロファス(Alcaligenes eutrophu Specifically, Comamonas Ashidoboranzu (Comamonas acidovorans), Alcaligenes eutrophus (Alcaligenes eutrophu
s)、アルカリゲネス ラタス(Alcaligenes latus)等がある。 s), there is Alcaligenes Ratasu (Alcaligenes latus) and the like. 入手容易な菌株としては、コマモナス アシドボランズ IFO13852、アルカリゲネス ユートロファスATC Available as easy strains, Comamonas Ashidoboranzu IFO13852, Alcaligenes eutrophus ATC
C 17699、アルカリゲネス ユートロファスATCC 1159 C 17699, Alcaligenes eutrophus ATCC 1159
9、アルカリゲネス ラタスATCC 29713、ロドコッカスs 9, Alcaligenes Ratasu ATCC 29713, Rhodococcus s
p. NCIMB 40126 、ロドコッカスsp. ATCC 19070等がある。 p. NCIMB 40126, there is a Rhodococcus sp. ATCC 19070, or the like.

【0014】上記のような微生物の菌体内にP(3HB [0014] P (3HB in the cells of microorganisms such as described above
-co-4HB) を蓄積させるには、微生物をその微生物の種類に応じた適当な培地に接種して、常法にしたがって培養して増殖させればよい。 To -co-4HB) to accumulate, the microorganism was inoculated into a suitable medium in accordance with the type of the microorganism, it is sufficient to proliferate in culture in a conventional manner. 培地としては、公知のものをいずれも使用できるが、コマモナス属に属する微生物を用いる場合、炭素源としては、3−ヒドロキシ酪酸及び4−ヒドロキシ酪酸を使用する。 As the medium, although any may be used known ones, in the case of using a microorganism belonging to the Comamonas genus, as the carbon source, using 3-hydroxybutyric acid and 4-hydroxybutyric acid. その他の炭素源として、炭素原子数が偶数のアルカンジオール、γ−ブチロラクトン、4-アミノ酪酸等が例示される。 Other carbon sources, the number of carbon atoms is even alkanediol, .gamma.-butyrolactone, 4-aminobutyric acid, and the like. その他、培地のpH、培養温度、培養時間等の培養条件も微生物の種類により適宜設定する。 Other, pH of the medium, the culture temperature is appropriately set according to the type of culture conditions also microorganisms such as incubation time.

【0015】 [0015]

【実施例】以下、本発明を実施例によりさらに具体的に説明する。 EXAMPLES The following more specifically describes the invention based on examples. 尚、本発明は、これらの実施例に限定されるものではない。 The present invention is not limited to these examples. 〔実施例1〜5〕各実施例において、以下のようにしてP(3HB-co-4HB) を製造した。 In Example 1-5] Each example was prepared with P (3HB-co-4HB) as follows. コマモナス アシドボランズ(Comamonas acidovorans) IFO13852 を、肉エキス5g/L、ポリペプトン10g/L、及び塩化ナトリウム5g/Lを含む天然培地で24時間好気的に培養し増殖させて、次いで、菌体を遠心分離で回収した。 The Comamonas Ashidoboranzu (Comamonas acidovorans) IFO13852, meat extract 5 g / L, and polypeptone 10 g / L, and for 24 hours aerobically cultured grown in natural medium containing sodium chloride 5 g / L, then centrifuged the cells in were recovered. 続いて、下記の組成の窒素制限のミネラル培地に、炭素源として3-ヒドロキシ酪酸(3HB)及び4-ヒドロキシ酪酸(4HB)を表1に示す割合でそれぞれ配合し、回収した菌体を懸濁して48時間培養した。 Subsequently, the mineral medium nitrogen limitation of the following composition, as carbon sources 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4HB) were respectively compounded in the proportions shown in Table 1, it was suspended the recovered cells They were cultured for 48 hours Te.

【0016】 窒素制限のミネラル培地組成 MgSO 4・7H 2 O 0.6 g/L Na 2 HPO 4・12H 2 O 7.16 g/L KH 2 PO 4 2.65 g/L 微量元素溶液(1) 1 ml/L (1) 微量元素溶液組成(1N−HCl中) FeSO 4・7H 2 O 2.78 g/L MnCl 2・4H 2 O 1.98 g/L CoSO 4・7H 2 O 2.81 g/L CaCl 2・2H 2 O 1.67 g/L CuCl 2・2H 2 O 0.17 g/L ZnSO 4・7H 2 O 0.29 g/L [0016] Mineral Medium composition of nitrogen limitation MgSO 4 · 7H 2 O 0.6 g / L Na 2 HPO 4 · 12H 2 O 7.16 g / L KH 2 PO 4 2.65 g / L trace element solution (1) 1 ml / L ( 1) trace element solution composition (in 1N-HCl) FeSO 4 · 7H 2 O 2.78 g / L MnCl 2 · 4H 2 O 1.98 g / L CoSO 4 · 7H 2 O 2.81 g / L CaCl 2 · 2H 2 O 1.67 g / L CuCl 2 · 2H 2 O 0.17 g / L ZnSO 4 · 7H 2 O 0.29 g / L

【0017】培養終了後、各培地から得た菌体を凍結乾燥した。 [0017] After completion of the culture, it was lyophilized cells from each culture. 培地1L当たりの乾燥菌体重量(g/L)を表1に示す。 Cell dry weight per medium 1L and (g / L) are shown in Table 1. 次いで、乾燥菌体を熱クロロホルムと混合して該菌体からポリマーを抽出した後、ヘキサンを添加した。 The dried cells after extracting the polymer from microbial cells is mixed with hot chloroform, hexane was added. 析出したポリマーの乾燥菌体重量中の含量(重量%)を表1に示す。 The content of the dried cells of the weight of the precipitated polymer (% by weight) shown in Table 1. また、各ポリマーをメチルエステル化してガスクロマトグラフィーにて分析した。 It was also analyzed by gas chromatography each polymer and methyl-esterified. 各ポリマーの3HB成分含量と4HB成分含量を表1に示す。 The 3HB ingredient content and 4HB ingredient content of each polymer shown in Table 1. その結果、実施例1〜4では、表1に示すようにそれぞれ4HB含量が56モル%、73モル%、80モル%及び83モル%のP(3HB-co-4HB) が得られ、実施例5では、 As a result, in Examples 1-4, respectively, as shown in Table 1 4HB content of 56 mol%, 73 mol%, 80 mol% and 83 mol% of P (3HB-co-4HB) can be obtained, Example In 5,
ポリ-4-ヒドロキシブチレートが得られたことが確認された。 The poly-4-hydroxybutyrate was obtained was confirmed.

【0018】 [0018]

【表1】 [Table 1]

【0019】〔実施例6〜9〕上記実施例1〜4で得られた4種類のP(3HB-co-4HB) をそれぞれアセトンに混合し、55℃で3時間加熱した後、遠心分離を行うことによって熱アセトンに可溶なポリマーと不溶なポリマーとに分別した。 [0019] [Examples 6-9] above obtained in Examples 1 to 4 Four P a (3HB-co-4HB) respectively were mixed in acetone, heated 3 hours at 55 ° C., centrifuged It was fractionated into a soluble polymer and insoluble polymer in hot acetone by performing. 熱アセトン混合前のポリマーに対する熱アセトン可溶ポリマーと、熱アセトン不溶ポリマーの割合(重量%)を表2に示す。 And hot acetone soluble polymer to the hot acetone before mixing of the polymer, the proportion of hot acetone-insoluble polymer (wt%) shown in Table 2. 各実施例とも、P(3 In each Example, P (3
HB-co-4HB) は熱アセトンに可溶なポリマーと不溶なポリマーとに分けられ、可溶ポリマーと不溶ポリマーの共重合組成をそれぞれメチルエステル化してガスクロマトグラフィーにて分析した。 HB-co-4HB) is divided into a soluble polymer and insoluble polymer in hot acetone and analyzed by gas chromatography the copolymerization composition of soluble polymer and insoluble polymer to each methyl esterification. 各ポリマーの3HB成分含量(モル%)と4HB成分含量(モル%)を表2に示す。 3HB ingredient content (mol%) and 4HB ingredient content of the polymer (mol%) shown in Table 2. この結果から、可溶ポリマーはすべて4HB成分含量が高いP(3HB-co-4HB) であり、不溶ポリマーは3HB成分含量が高いP(3HB-co-4HB) であることが確認された。 This result soluble polymers are all 4HB ingredient content is high P (3HB-co-4HB), insoluble polymer was confirmed 3HB ingredient content is high P (3HB-co-4HB).

【0020】 [0020]

【表2】 [Table 2]

【0021】これらの実施例から、微生物によるP(3 [0021] From these examples, P by microorganisms (3
HB-co-4HB) の合成では、3HB成分含量が高いものと、4HB成分含量が高いものの2種類の共重合体が混合して得られる可能性があることがわかった。 The HB-co-4HB) synthesis, it was found that as 3HB ingredient content is high, may be obtained by mixing 4HB component but a high content of two of the copolymer. また、 Also,
熱アセトンを用いることによって、4HB成分含量の高いP(3HB-co-4HB) を選択的に分離できることがわかった。 By using hot acetone, it was able to be selectively separate high P (3HB-co-4HB) of 4HB ingredient content.

【0022】〔実施例10〜13〕実施例6〜9で得られた熱アセトン可溶ポリマーを13 C−NMRで解析した。 [0022] were analyzed hot acetone soluble polymer obtained in Example 10 to 13] Examples 6-9 at 13 C-NMR. 40 40
0MHz 13 C−NMRにおけるカルボニル連鎖の相対ピーク面積から決定したダイアド連鎖のモル分率F 33 0 MHz 13 C-NMR dyad chain determined from the relative peak areas of the carbonyl linkage in the mole fraction of F 33,
34 、F 43 、及びF 44を表3に示す。 F 34, F 43, and F 44 shown in Table 3. また、熱アセトン可溶ポリマー中の3HB成分と4HB成分のダイアド連鎖のモル分率から、モノマー反応比の積であるD値を下記式により算出した結果も表3に示す。 Further, the mole fraction of dyad chains of 3HB component and 4HB component of hot acetone soluble polymer, the results of the D value which is the product of monomer reaction ratios were calculated by the following equation are shown in Table 3. D=(F 33 ×F 44 )/(F 34 ×F 43 D = (F 33 × F 44 ) / (F 34 × F 43)

【0023】 [0023]

【表3】 [Table 3]

【0024】これらの結果から、各ポリマーともD値が1に極めて近いことが確認された。 [0024] From these results, D value in each polymer was confirmed very close to 1. これは、統計的に3 This is, statistically 3
HB成分と4HB成分とがランダムに共重合していることを示しており(例えば、Yuji Saito and Yoshiharu D And HB component and 4HB component indicates that it is randomly copolymerized (e.g., Yuji Saito and Yoshiharu D
oi, Int. J. Biol. Macromol., 16, 99-104 (1994)参照)、熱アセトン可溶ポリマーはP(3HB-co-4H oi, Int. J. Biol. Macromol., 16, see 99-104 (1994)), hot acetone soluble polymer P (3HB-co-4H
B) ランダム共重合体であることが確認された。 B) a random copolymer was confirmed.

【0025】〔実施例14〜22、比較例1〜4〕実施例1 [0025] Example 14-22, Comparative Examples 1-4] Example 1
にしたがって、4HB成分含量が84モル%のP(3HB According, 4HB component content of 84 mol% of P (3HB
-co-4HB)を乾燥菌体重量当たり21重量%の含量で蓄積した菌体を得た。 -co-4HB) to give the accumulated cells at a content of dry cell weight per 21 wt%. 各実施例及び比較例において、表4 In the Examples and Comparative Examples, Table 4
に示した界面活性剤、即ちTween 80(非イオン系界面活性剤)、SDS(ドデシル硫酸ナトリウム、陰イオン系界面活性剤)、又はCTAB(臭化セチルトリメチルアンモニウム、陽イオン系界面活性剤)をそれぞれ 0.1重量%配合した 300mlの抽出溶媒(表4参照)に、前記菌体(培養液 300mlから遠心分離で得た湿菌体)を懸濁させた。 Surfactants shown in, i.e. Tween 80 (nonionic surfactant), SDS (sodium dodecyl sulfate, an anionic surface active agent), or CTAB (cetyltrimethylammonium bromide, cationic surfactant) the extraction solvent 300ml blended respectively 0.1 wt% (see Table 4) were suspended the cells (wet cells obtained by centrifuging the culture broth 300ml). 得られた菌体懸濁液を、60℃に調整したウォーターバス内に浸してマグネチックスターラーで撹拌した。 The resulting cell suspension was stirred with a magnetic stirrer and placed in a water bath adjusted to 60 ° C..
5時間後、菌体懸濁液をPTFE製のメンブランフィルターで吸引濾過し、得られた各アセトン溶液の 100ml After 5 hours, the cell suspension was suction filtered through a PTFE membrane filter, each acetone solution obtained 100ml
を、それぞれ表4に示した析出溶媒 100mlに混合し、ポリマーを析出させた。 The respective mixed precipitation solvent 100ml shown in Table 4, to precipitate a polymer.

【0026】抽出前に菌体に含まれていたポリマーに対する抽出ポリマーの回収率(重量%)を表4に示す。 The recovery of the extracted polymer to polymer contained in cells prior to extraction (% by weight) shown in Table 4. また、得られたポリマーの純度、組成(3HB成分含量(モル%)及び4HB成分含量(モル%))、数平均分子量、並びに多分散度を表4に示す。 Further, the purity of the obtained polymer, the composition (3HB ingredient content (mol%) and 4HB ingredient content (mol%)), indicating the number-average molecular weight, and the polydispersity in Table 4. 尚、表中の比較例1は乾燥菌体から熱クロロホルムで抽出した結果を示し、比較例2から4は湿菌体から界面活性剤を含まないアセトンを用いて抽出した結果を示している。 In Comparative Example 1 in the table shows the results of extraction with hot chloroform from dried cells, from 4 to Comparative Example 2 shows the result of extracting with acetone containing no surfactant from wet cells.

【0027】 [0027]

【表4】 [Table 4]

【0028】共重合体の回収率は、アセトンに配合する界面活性剤と析出に用いる貧溶媒の種類によって異なった。 The recovery rate of the copolymer differed depending on the type of the poor solvent used for the precipitation and surfactant to be blended in acetone. ただし、回収した共重合体の純度をみると、界面活性剤としてSDS又はTween 80を用いた条件で抽出し、 However, looking at the purity of the recovered copolymer was extracted with conditions using SDS or Tween 80 as the surfactant,
メタノール又はヘキサンに析出させた場合に高純度の共重合体が得られる結果となった。 High purity of the copolymer resulted obtained when precipitated in methanol or hexane. また、各条件で抽出した共重合体の分子量をみると、すべての条件とも析出させる貧溶媒として、メタノール>ヘキサン>蒸留水の序列で高くなることがわかった。 Looking at the molecular weight of the copolymer was extracted with each condition, as a poor solvent to deposit with all conditions was found to be higher in the order of methanol> hexane> distilled water. さらに、多分散度をみると、メタノールに析出させた共重合体は、他の条件よりも狭くなることがわかった。 Further, looking at the polydispersity, the copolymer was precipitated in methanol, was found to be narrower than the other conditions.

【0029】 [0029]

【発明の効果】本発明のP(3HB-co-4HB) の製造方法によれば、湿菌体からP(3HB-co-4HB) を抽出することができるため菌体の乾燥を行う必要がなく、 According to the production method of the Effects of the Invention] P of the present invention (3HB-co-4HB), it is necessary to dry the cells since it is possible to extract the P (3HB-co-4HB) from wet cells without,
しかも抽出時間も短縮することができるので、製造工程の効率化を図ることができる。 Moreover it is possible to shortened extraction time, it is possible to improve the efficiency of the manufacturing process. 更に、このような製造工程の効率化によりエネルギー消費量を低減することができ、経済性に優れる。 Furthermore, it is possible to reduce the energy consumption by more efficient such a manufacturing process with high economic efficiency. また、菌体からのP(3HB-co- In addition, P from the bacterial cells (3HB-co-
4HB) の抽出溶媒に用いるアセトンは回収・再利用が可能であり、抽出温度も通常50〜60℃程度であることから、省エネルギーで安全に抽出することができる。 Acetone used for the extraction solvent of 4HB) is possible to recover and reuse, since the extraction temperature is generally about 50-60 ° C., it can be safely extracted with energy saving. 更に、本発明によれば、菌体に3HB成分含量の高いP Further, according to the present invention, a high 3HB ingredient content in cells P
(3HB-co-4HB)と4HB成分の含量の高いP(3 (3HB-co-4HB) with high content of 4HB component P (3
HB-co-4HB) (通常、4HB成分含量が60モル%以上のもの)とが蓄積されている場合に、4HB成分含量の高いP(3HB-co-4HB) を容易に精度よく選択的に分離・精製することができる。 HB-co-4HB) (usually, when 4HB ingredient content is 60 mol% or more) and is stored, high P (3HB-co-4HB) readily accurately selectively the 4HB ingredient content it can be separated and purified.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 武部 英日 神奈川県小田原市栢山788 明治製菓株式 会社薬品技術研究所内 (72)発明者 蛭田 修 神奈川県小田原市栢山788 明治製菓株式 会社薬品技術研究所内 ────────────────────────────────────────────────── ─── of the front page continued (72) inventor Takebe English-Japanese Odawara, Kanagawa Prefecture Kiama 788 Meiji Seika Kaisha, Ltd. medicine intra-technology Research Institute (72) inventor Osamu Hiruta Odawara, Kanagawa Prefecture Kiama 788 Meiji Seika Kaisha, Ltd. chemical technology within the Institute

Claims (6)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】 下記式(1): −OCH(CH 3 )CH 2 CO− (1) で表される3−ヒドロキシブチレート単位と、下記式(2): −OCH 2 CH 2 CH 2 CO− (2) で表される4−ヒドロキシブチレート単位とからなるヒドロキシアルカン酸共重合体生産能を有する微生物の菌体内に蓄積された前記ヒドロキシアルカン酸共重合体を抽出・分離する工程を含む前記ヒドロキシアルカン酸共重合体の製造方法において、 前記ヒドロキシアルカン酸共重合体の抽出を、前記菌体の湿菌体に界面活性剤含有アセトンを混合し、加熱することにより行うことを特徴とする、ヒドロキシアルカン酸共重合体の製造方法。 1. A formula (1): -OCH (CH 3 ) CH 2 CO- (1) and 3-hydroxybutyrate units represented by the following formula (2): -OCH 2 CH 2 CH 2 CO - comprising the step of extracting and separating the hydroxyalkanoic acid copolymer accumulated in the cells of a microorganism having a hydroxyalkanoic acid copolymer-producing ability consisting of 4-hydroxybutyrate units represented by (2) in the manufacturing method of the hydroxyalkanoic acid copolymer, the extraction of the hydroxyalkanoic acid copolymer, a mixture of surfactant-containing acetone wet cells of said cell, characterized in that by heating the method of hydroxyalkanoic acid copolymer.
  2. 【請求項2】 微生物がコマモナス(Comamonas) 属に属する微生物である、請求項1に記載のヒドロキシアルカン酸共重合体の製造方法。 2. is a microorganism belonging to the microorganism Comamonas (Comamonas) genus, method of manufacturing a hydroxyalkanoic acid copolymer according to claim 1.
  3. 【請求項3】 界面活性剤が陰イオン又は非イオン界面活性剤である、請求項1又は2に記載のヒドロキシアルカン酸共重合体の製造方法。 3. A surfactant is anionic or non-ionic surface active agent, method for producing a hydroxyalkanoic acid copolymer according to claim 1 or 2.
  4. 【請求項4】 湿菌体に界面活性剤含有アセトンを混合し、加熱した後、菌体残渣を除去し、次いで残ったアセトン溶液を貧溶媒と混合してヒドロキシアルカン酸共重合体を析出させて分離することを特徴とする、請求項1 4. A mixture of surfactant-containing acetone wet cells, after heating to remove the cell residues, then the remaining acetone solution to precipitate by mixing with a poor solvent hydroxyalkanoic acid copolymer and separating Te, claim 1
    〜3のいずれか1項に記載のヒドロキシアルカン酸共重合体の製造方法。 Method for producing a hydroxyalkanoic acid copolymer according to any one of to 3.
  5. 【請求項5】 貧溶媒が、メタノール又はヘキサンであることを特徴とする、請求項4に記載のヒドロキシアルカン酸共重合体の製造方法。 5. A poor solvent, characterized in that methanol or hexane, a manufacturing method of the hydroxyalkanoic acid copolymer of claim 4.
  6. 【請求項6】 菌体内に蓄積される前記ヒドロキシアルカン酸共重合体が、3−ヒドロキシブチレート単位含量が高い共重合体と4−ヒドロキシブチレート単位含量が高い共重合体とを含む場合に、4−ヒドロキシブチレート単位含量が高い共重合体を選択的に抽出する、請求項1〜5のいずれか1項に記載のヒドロキシアルカン酸共重合体の製造方法。 Wherein said hydroxyalkanoic acid copolymer is accumulated in the cells it is, if the 3-hydroxybutyrate unit content is high copolymer and 4-hydroxybutyrate units content and a high copolymer , 4-hydroxybutyrate units content selectively extracting higher copolymer, method for producing a hydroxyalkanoic acid copolymer according to any one of claims 1 to 5.
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