JP4104932B2 - Biodegradable polymer and novel microorganism producing the same, method for producing biodegradable polymer, biodegradable random copolymer and method for isolating the same - Google Patents
Biodegradable polymer and novel microorganism producing the same, method for producing biodegradable polymer, biodegradable random copolymer and method for isolating the same Download PDFInfo
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- JP4104932B2 JP4104932B2 JP2002221440A JP2002221440A JP4104932B2 JP 4104932 B2 JP4104932 B2 JP 4104932B2 JP 2002221440 A JP2002221440 A JP 2002221440A JP 2002221440 A JP2002221440 A JP 2002221440A JP 4104932 B2 JP4104932 B2 JP 4104932B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、医薬品、食品、衛生用品、建設用品、工業用品、農園芸品、包装材料など広範な分野に応用可能な新規生分解性重合体および生分解性ランダムコポリマー、それらの製造方法、ならびに該生分解性重合体の産生に供し得る新規微生物に関する。
【０００２】
【従来の技術】
長年にわたって、石油由来の合成高分子がプラスチックなどとして利用されてきたが、石油資源の枯渇や上記合成高分子が分解されにくく自然界に残留することによる産業廃棄物の蓄積などが大きな社会問題となってきている。上記の合成高分子は、焼却処理を行うと二酸化炭素排出量の増加となり、またある場合には、ダイオキシンや環境ホルモンなどの有害物質の発生原因ともなることも問題視されている。
【０００３】
近年、上記の石油由来の合成高分子に換わり、自然界の微生物によって分解される生分解性高分子が注目されてきている。生分解性高分子の中でも、天然生物資源、微生物を利用して生産するポリヒドロキシアルカノエート（ＰＨＡ）は、上記問題の解決が可能であり、現在までに様々な研究開発、実用化が行われてきている。
【０００４】
【発明が解決しようとする課題】
しかしながら、微生物が生成する生分解性高分子は、その理化学的性質が多岐にわたっており、それぞれの使用目的に適合する生分解性高分子の探索が要求されている段階であり、従来なかったような有用な特性を有する生分解性高分子の開発が進められている。
本発明の目的は、有用な新規生分解性高分子およびその製造方法を提供することである。
【０００５】
【課題を解決するための手段】
本発明者らは、上記課題を解決するため鋭意研究を行った結果、本発明を完成するに至った。即ち、本発明は以下のとおりである。
（１）６０モル％〜９５モル％の下記式
【０００６】
【化１６】
で表される３−ヒドロキシブチレート単位と、
５モル％〜４０モル％の下記式
【０００８】
【化１７】
で表される３−ヒドロキシヘキサノエート単位、下記式
【００１０】
【化１８】
で表される３−ヒドロキシオクタノエート単位、下記式
【００１２】
【化１９】
で表される３−ヒドロキシデカノエート単位、および下記式
【００１４】
【化２０】
で表される３−ヒドロキシドデカノエート単位から選ばれる少なくともいずれかとからなる生分解性重合体。
（２）６０モル％〜９５モル％の３−ヒドロキシブチレート単位と、０モル％〜１０モル％の３−ヒドロキシヘキサノエート単位と、１モル％〜３０モル％の３−ヒドロキシオクタノエート単位と、３モル％〜３０モル％の３−ヒドロキシデカノエート単位と、０モル％〜１５モル％の３−ヒドロキシドデカノエート単位とからなる、上記（１）に記載の生分解性重合体。
（３）６０モル％〜７０モル％の３−ヒドロキシブチレート単位と、０モル％〜５モル％の３−ヒドロキシヘキサノエート単位と、５モル％〜２５モル％の３−ヒドロキシオクタノエート単位と、５モル％〜２５モル％の３−ヒドロキシデカノエート単位と、０モル％〜５モル％の３−ヒドロキシドデカノエート単位とからなる、上記（１）または（２）に記載の生分解性重合体。
（４）３−ヒドロキシブチレート単位、３−ヒドロキシヘキサノエート単位、３−ヒドロキシオクタノエート単位、３−ヒドロキシデカノエート単位および３−ヒドロキシドデカノエート単位のうちから選ばれる少なくとも２種以上からなる生分解性ランダムコポリマーと、３−ヒドロキシブチレート単位のみからなるホモポリマーとの混合物であって、上記ランダムコポリマーを５重量％〜５０重量％含有するものである、上記（１）〜（３）のいずれかに記載の生分解性重合体。
（５）上記ランダムコポリマーが、１モル％〜１５モル％の３−ヒドロキシブチレート単位と、０モル％〜１５モル％の３−ヒドロキシヘキサノエート単位と、１５モル％〜５５モル％の３−ヒドロキシオクタノエート単位と、１５モル％〜５５モル％の３−ヒドロキシデカノエート単位と、１モル％〜１５モル％の３−ヒドロキシドデカノエート単位からなるものである上記（４）に記載の生分解性重合体。
（６）シュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＲＦＬ−０１０株（ＦＥＲＭ Ｐ−１８８９３）を、炭素源を含有する培地で培養して得られたものである、上記（１）〜（５）のいずれかに記載の生分解性重合体。
（７）炭素源を含有する培地でシュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＲＦＬ−０１０株（ＦＥＲＭ Ｐ−１８８９３）を培養する工程を含有することを特徴とする生分解性重合体の製造方法。
（８）上記炭素源がグルコース、糖蜜、デンプンおよびマルトースから選ばれる少なくともいずれかである、上記（７）に記載の生分解性重合体の製造方法。
（９）１モル％〜１５モル％の下記式
【００１６】
【化２１】
で表される３−ヒドロキシブチレート単位と、
０モル％〜１５モル％の下記式
【００１８】
【化２２】
で表される３−ヒドロキシヘキサノエート単位と、
１５モル％〜５５モル％の下記式
【００２０】
【化２３】
で表される３−ヒドロキシオクタノエート単位と、
１５モル％〜５５モル％の下記式
【００２２】
【化２４】
で表される３−ヒドロキシデカノエート単位と、
１モル％〜１５モル％の下記式
【００２４】
【化２５】
で表される３−ヒドロキシドデカノエート単位とからなる生分解性ランダムコポリマー。
（１０）炭素源を含有する培地でシュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＲＦＬ−０１０株（ＦＥＲＭ Ｐ−１８８９３）を培養して生分解性重合体を産生させる工程と、
得られた生分解性重合体から、１モル％〜１５モル％の下記式
【００２６】
【化２６】
で表される３−ヒドロキシブチレート単位と、
０モル％〜１５モル％の下記式
【００２８】
【化２７】
で表される３−ヒドロキシヘキサノエート単位と、
１５モル％〜５５モル％の下記式
【００３０】
【化２８】
で表される３−ヒドロキシオクタノエート単位と、
１５モル％〜５５モル％の下記式
【００３２】
【化２９】
で表される３−ヒドロキシデカノエート単位と、
１モル％〜１５モル％の下記式
【００３４】
【化３０】
で表される３−ヒドロキシドデカノエート単位とからなるランダムコポリマーを単離する工程とを含有する、生分解性ランダムコポリマーの単離方法。
（１１）シュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＲＦＬ−０１０株（ＦＥＲＭ Ｐ−１８８９３）。
【００３６】
【発明の実施の形態】
以下、本発明を詳細に説明する。
本発明の生分解性重合体は、下記式
【００３７】
【化３１】
で表される３−ヒドロキシブチレート（３ＨＢ）単位（以下、「Ｃ４単位」と呼ぶことがある。）を６０モル％〜９５モル％含有するポリヒドロキシアルカノエート（ＰＨＡ）である。Ｃ４単位が６０モル％未満であると、生分解性重合体の結晶化度が落ち、ゴム状物または粘稠な油状物となり、成形品への加工が困難となってしまう。またＣ４単位が９５モル％を超えると、生分解性重合体の結晶化度が高くなり過ぎて柔軟性がなくなり、加工性に劣る。
【００３９】
また本発明のＰＨＡは、上記Ｃ４単位以外の残余の５モル％〜４０モル％を占める成分が、下記式
【００４０】
【化３２】
で表される３−ヒドロキシヘキサノエート（３ＨＨｘ）単位（以下、「Ｃ６単位」と呼ぶことがある。）、下記式
【００４２】
【化３３】
で表される３−ヒドロキシオクタノエート（３ＨＯ）単位（以下、「Ｃ８単位」と呼ぶことがある。）、下記式
【００４４】
【化３４】
で表される３−ヒドロキシデカノエート（３ＨＤ）単位（以下、「Ｃ１０単位」と呼ぶことがある。）、および下記式
【００４６】
【化３５】
で表される３−ヒドロキシドデカノエート（３ＨＤＤ）単位（以下、「Ｃ１２単位」と呼ぶことがある。）から選ばれる少なくともいずれかである（以下、これらＣ６単位、Ｃ８単位、Ｃ１０単位およびＣ１２単位から選ばれる少なくともいずれかを総称して「長鎖単位」ということがある。）。長鎖単位が５モル％未満であると、可塑化効果が落ち、生分解性重合体が結晶性の高い硬いものとなってしまう。また長鎖単位が４０モル％を超えると、可塑化効果が大きくなり過ぎて、生分解性重合体がゴム状物または粘稠な油状物となり、成形品への加工が困難となってしまう。
【００４８】
上述したモノマー組成を特徴とする本発明の生分解性重合体（ポリヒドロキシアルカノエート（ＰＨＡ））は新規であり、生分解性を有するものである。なお生分解性を有することは、ＪＩＳ Ｋ ６９５０（活性汚泥による好気的生分解度試験方法）の規定に準拠して、対象とするプラスチックを混合した活性汚泥を攪拌培養し、培養液の生物化学的酸素消費量及びプラスチックの残存量から生分解度を求めることで確認することができる。本発明のＰＨＡは、かくして求められた生分解度が６０％〜１００％である。
このような生分解性重合体は、従来のプラスチックと同様、溶融加工などにより各種製品の生産に利用することができるとともに、石油由来の合成高分子とは異なり、生物により分解され得るという特性を有する。したがって、廃棄した際、微生物産生ポリエステルは生分解されることにより自然界の物質循環に取り込まれるので、従来利用されていた多くの合成高分子化合物のように自然環境に残留して汚染を引き起こすことがない。また生分解処理を行うことで、燃焼処理を行う必要もないため、大気汚染や地球温暖化を防止するという点で有効な材料であり、環境保全を可能とするプラスチックとして利用することができる。
【００４９】
本発明の生分解性重合体は、引張強度が６ＭＰａ〜４０ＭＰａ、ヤング率（引張弾性率）が３００ＭＰａ〜１１００ＭＰａ、さらに伸び率が１０％〜１５０％であり、プラスチックとして良好な機械的特性を有するものである。
なお上記引張強度は、ＪＩＳ Ｋ ７１２７（プラスチック及びシートの引張試験方法）の規定に準拠して、たとえば、生分解性重合体にてキャストフィルム（１０ｍｍ×５ｍｍ×０．０２ｍｍ）を作製し、これを精密万能試験機（島津製作所製）を用いて２０ｍｍ／ｍｉｎの速度で引張試験を行い、フィルムが破断するまでにかけられた最大の引張応力として測定できる。また上記ヤング率は、ＪＩＳ Ｋ ７１２７の規定に準拠して、上記引張試験を同様に行って、弾性限界内での（直線上の任意の２点の引張応力差）／（直線上の任意の２点間の伸びの差）として測定できる。上記伸び率は、ＪＩＳ Ｋ ７１２７の規定に準拠して、上記引張試験を行い、フィルムが破断したときの伸び率として測定できる。
【００５０】
また本発明の生分解性重合体は、ガラス転移点（Ｔｇ）が−１０℃〜−３℃、結晶化点（Ｔｃ）が５３℃〜５８℃、さらに融点（Ｔｍ）が１５５℃〜１７５℃であり、プラスチックとして良好な熱的性質を有するものである。
なお上記ガラス転移点、結晶化点および融点は、いずれもＤＳＣ（示差走査熱量計）を用いて測定することができる。
【００５１】
上述のように良好な機械的強度および熱的性質を有する本発明の生分解性重合体は、容器、袋、フィルムなどの包装材料、魚網、釣糸などの水産用材料、植生シート、育苗ポットなどの農園芸材料、縫合糸などの医療材料などの分野への適用が期待できる。
【００５２】
本発明における長鎖単位は、生分解性重合体中、Ｃ６単位が０モル％〜１０モル％、Ｃ８単位が１モル％〜３０モル％、Ｃ１０単位が３モル％〜３０モル％、Ｃ１２単位が０モル％〜１５モル％の範囲内からそれぞれ選ばれて、上述のように生分解性重合体中５モル％〜４０モル％を占めるのが好ましい。
【００５３】
さらに本発明においては、Ｃ４単位が６０モル％〜７０モル％、Ｃ６単位が０モル％〜５モル％、Ｃ８単位が５モル％〜２５モル％、Ｃ１０単位が５モル％〜２５モル％、かつＣ１２単位が０モル％〜５モル％であると、伸び率が６０％〜１５０％という延伸性に特に優れた生分解性重合体を実現することができ、特に好ましい。このような優れた伸び率を有する生分解性重合体は、加工性に優れ、延伸によりフィルムを作製することができる、または容易に成形品に加工できるという点で特に有用であり、上記中でも容器、袋、食品包装材料などの包装材料分野などへの適用が期待できる。
【００５４】
本発明の生分解性重合体は、具体的には、Ｃ４単位、Ｃ６単位、Ｃ８単位、Ｃ１０単位およびＣ１２単位のうちから選ばれる少なくとも２種以上からなる生分解性ランダムコポリマーと、Ｃ４単位のみからなるホモポリマーとの混合物である。該生分解性重合体は、上記ランダムコポリマーを５重量％〜５０重量％含有するのが好ましい。ランダムコポリマーの含有率が５重量％未満であると、生分解性重合体が柔軟性に乏しいものとなってしまう傾向にあるためであり、またランダムコポリマーの含有率が５０重量％を超えると、生分解性重合体が柔軟性が高すぎて成形品への適用が困難となってしまう傾向にあるためである。生分解性重合体において、上記生分解性ランダムコポリマーは、Ｃ４単位のみからなるホモポリマーの可塑剤として働いていると考えられる。すなわち、本発明の生分解性重合体においては、可塑剤として働く上記生分解性ランダムコポリマーが含有されているため、Ｃ４単位のみからなるホモポリマーと比較して柔軟性があり、上述したような機械的特性（ヤング率、伸び率など）、熱的性質（ガラス転移点など）に優位性が現れるものと考えられる。
【００５５】
本発明では、上述してきた生分解性重合体に加え、この生分解性重合体に含有される生分解性ランダムコポリマーをも提供するものである。
本発明の生分解性ランダムコポリマーは、具体的には、１モル％〜１５モル％のＣ４単位、０モル％〜１５モル％のＣ６単位、１５モル％〜５５モル％のＣ８単位、１５モル％〜５５モル％のＣ１０単位、および１モル％〜１５モル％のＣ１２単位からなるものであり、生分解性を有する。このような本発明の生分解性ランダムコポリマーは、長鎖単位のモノマーの組成比が高く、ガラス転移点（Ｔｇ）が−４０℃前後、明確な結晶化点および融点が認められず、粘稠な油状物であり、柔軟性に特に優れたものであり、たとえば他の生分解性プラスチックへの可塑剤としての利用が期待できる。
【００５６】
なお本発明の生分解性重合体および生分解性ランダムコポリマーにおける各モノマー単位の含有率は、たとえばガスクロマトグラフィを用い、生分解性重合体または生分解性ランダムコポリマーにアルコール、酸、クロロホルムを加え、加熱して反応させ、各モノマー単位のエステルとして定量することができる。
【００５７】
本発明の生分解性重合体は、上述したものであるならばその製造方法に特に制限はないが、石油資源を原料とした化学合成による製造方法では達成できない高分子量化、高純度化が達成できるという理由から、以下の本発明者らが新規に見出した微生物を利用する本発明の製造方法によって製造されたものであることが好ましい。すなわち、本発明は、以下の菌学的性質を有する微生物を、炭素源を含有する培地で培養して、上記生分解性重合体を製造する方法をも提供するものである。
本発明の製造方法においては、以下の菌学的性質を有する微生物を用いる。
【００５８】
〔１〕形態学的性質
寒天(Nutrient Agar)培地、３０℃、一日培養で０．８μｍ×１．５μｍ〜２．０μｍの直状桿菌である。鞭毛染色で極毛が観察され、運動性が認められる。細胞の多形性および胞子は認められない。
【００５９】
〔２〕各種培地における生育状態
（１）肉汁寒天(Nutrient Agar)平板培地（３０℃）
コロニーは平滑で周縁はなめらかであり、クリーム色を呈している。光沢を有し、拡散性色素の産生は認められない。
（２）肉汁液体培地（３０℃）
培地表面での生育及び被膜の形成は認められないが、培地の混濁は認められた。
（３）肉汁ゼラチン穿刺培養（３０℃）
生育は認められず、液化も認められない。
（４）リトマスミルク
酸、アルカリの産生は認められず、凝固および液化も認められない。
【００６０】
〔３〕生理学的性質
（１）グラム染色性：陰性
（２）硝酸塩の還元：陽性
（３）脱窒反応：陽性
（４）ＭＲテスト：陰性
（５）ＶＰテスト：陰性
（６）インドールの生成：陰性
（７）硫化水素の生成：陰性
（８）デンプンの加水分解：陰性
（９）クエン酸の利用
・Ｋｏｓｅｒの培地：陽性
・Ｃｈｒｉｓｔｅｎｓｅｎの培地：陽性
（１０）無機窒素源の利用
・硝酸塩：陽性
・アンモニウム塩：陽性
（１１）色素の生成：陰性
（１２）ウレアーゼ：陰性
（１３）オキシダーゼ：陽性
（１４）カタラーゼ：陽性
（１５）生育の範囲
１．ｐＨ
・ｐＨ６．０：陽性
・ｐＨ７．０：陽性
・ｐＨ８．０：陽性
・ｐＨ９．０：陽性
２．温度
・１５℃：陰性
・２０℃：微弱
・３０℃：陽性
・３５℃：陰性
（１６）酸素に対する態度：好気性
（１７）Ｏ−Ｆテスト（Hugh Leifson法）：陰性
（１８）糖類からの酸およびガスの生成
１．Ｌ−アラビノース
・酸の生成：陽性
・ガスの生成：陰性
２．Ｄ−キシロース
・酸の生成：陽性
・ガスの生成：陰性
３．Ｄ−グルコース
・酸の生成：陽性
・ガスの生成：陰性
４．Ｄ−マンノース
・酸の生成：陽性
・ガスの生成：陰性
５．Ｄ−フラクトース
・酸の生成：陽性
・ガスの生成：陰性
６．Ｄ−ガラクトース
・酸の生成：陽性
・ガスの生成：陰性
７．マルトース
・酸の生成：陰性
・ガスの生成：陰性
８．シュークロース
・酸の生成：陽性
・ガスの生成：陰性
９．ラクトース
・酸の生成：陰性
・ガスの生成：陰性
１０．トレハロース
・酸の生成：陽性
・ガスの生成：陰性
１１．Ｄ−ソルビトール
・酸の生成：陰性
・ガスの生成：陰性
１２．Ｄ−マンニトール
・酸の生成：陽性
・ガスの生成：陰性
１３．イノシトール
・酸の生成：陽性
・ガスの生成：陰性
１４．グリセリン
・酸の生成：陽性
・ガスの生成：陰性
１５．デンプン
・酸の生成：陰性
・ガスの生成：陰性
（１９）その他の特徴
１．β−ガラクトシダーゼ活性：陰性
２．アルギニンジヒドロラーゼ：陽性
３．リジンデカルボキシラーゼ活性：陰性
４．トリプトファンデアミナーゼ活性：陰性
５．アセトイン産生：陰性
６．ゼラチナーゼ活性：陰性
【００６１】
以上の菌学的性質から、この微生物はシュードモナス属に属する菌であり、さらに公知の菌株と比較しても同じものが存在しないため新規の菌株と判断し、シュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＲＦＬ−０１０株と命名して、独立行政法人産業技術総合研究所特許生物寄託センターにＦＥＲＭ
Ｐ−１８８９３（受託番号）として寄託した。
該新規微生物は、各地の森林、田畑や河川などの土壌、および活性汚泥その他を分離源として、グルコースを単一炭素源に使用した培地を用いて鋭意スクリーニングを行った結果、平成１３年５月３０日に福井県坂井郡金津町の土壌より得られたものである。
本発明の微生物は、自然界においてまたは人工的な操作（たとえば、紫外線照射、Ｘ線照射、化学薬品処理など）により、変異を起こす。したがって、後述する本発明の生分解性重合体の製造方法には、これらの変異株を用いることもできる。
【００６２】
本発明の製造方法は、上述した新規微生物シュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＲＦＬ−０１０株を、炭素源を含有する培地で培養することで、上記本発明の生分解性重合体を製造することを特徴とするものである。
本発明において使用される培地に含有される炭素源としては、特に制限はないが、たとえば、グルコース、フラクトース、シュークロース、マルトースやデンプンなどの糖類、メタノールやエタノールなどのアルコール類、グルコン酸、オクタン酸や吉草酸などの有機酸とその塩類、糖蜜、酵母エキス、肉エキス、大豆油、ナタネ油や魚油などの複合炭素源などが挙げられる。中でも、本発明の微生物は、比較的安価で入手が容易であり、微生物により代謝され易いという利点を有するグルコースを炭素源として用いることができるという格別の利点がある。
【００６３】
上記培地における炭素源の含有量に、特に制限はないが、効率よく菌体の生育と生分解性重合体の生産を行うことができることから、０．１ｇ／Ｌ〜１００ｇ／Ｌであるのが好ましく、１ｇ／Ｌ〜３０ｇ／Ｌであるのがより好ましい。炭素源が０．１ｇ／Ｌ未満であると、充分な菌体生育が行われず、生分解性重合体の生産性が著しく低下する傾向にあるためであり、また炭素源が１００ｇ／Ｌを超えると、培養液の浸透圧が高くなり過ぎ、菌体生育を阻害する傾向にあるためである。
【００６４】
また本発明において使用される培地に含有される窒素源としては、硫酸アンモニウム、塩化アンモニウムなどのアンモニウム塩、硝酸ナトリウム、硝酸カリウムなどの硝酸塩をはじめとする無機窒素源や、酵母エキス、肉エキス、ペプトン、トリプトン、尿素、大豆粉、油粕、コーンスティープリカーなどの有機窒素源が挙げられる。中でも、生産される生分解性重合体の菌体からの分離・精製の効率化の面から、無機窒素源を用いることが好ましい。
【００６５】
上記窒素源を含有する培地を用いる場合、その含有量に特に制限はないが、効率よく菌体の生育と生分解性重合体の生産を行えるという理由からは、０．１ｇ／Ｌ〜８ｇ／Ｌであるのが好ましく、０．２ｇ／Ｌ〜３ｇ／Ｌであるのがより好ましい。窒素源が０．１ｇ／Ｌ未満であると、菌体が充分に生育せず、生分解性重合体の絶対的な生産量が著しく低下する傾向にあるためであり、また窒素源が８ｇ／Ｌを超えると、菌体は充分に生育するが、生分解性重合体の含有率が低下し、生産性が著しく低下する傾向にあるためである。
【００６６】
上述した炭素源、および所望に応じて窒素源を含有するものであれば、本発明で用いる培地には特に制限はなく、当分野において従来より広く用いられてきている各種の培地を用いることができるが、上述したシュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．） ＲＬＦ−０１０株の菌学的性質より、たとえば、炭素源と窒素源の濃度をある一定の範囲に設定した培地を用いるのが好ましい。かかる培地を用いることで、効率よく菌体の生育と生分解性重合体の生産を行えるというような利点がある。
【００６７】
上記培地には、本発明の目的を阻害しない範囲で、必要に応じ、マグネシウム、ナトリウム、カリウム、カルシウム、マンガン、銅、コバルト、モリブデン、亜鉛、鉄などの金属塩、リン酸塩、硝酸塩、塩化物、炭酸塩など、クエン酸ナトリウムなどの有機酸塩、さらに必要に応じてビタミンなどの発育素が含有されたものであってもよい。
【００６８】
培養は、好気的条件下で行うのが好ましく、静置、振盪、通気攪拌培養のいずれも可能であるが、振盪あるいは通気攪拌培養が有利である。このとき、培養方式は回分培養、連続培養のいずれであってもよい。培養温度は、１５℃〜３４℃が好ましく、２０℃〜３２℃が特に好ましい。また、培地のｐＨは５．８〜９．５が適当であるが、６．５〜８．５が最適である。
【００６９】
本研究では窒素あるいはリンを制限した培地で菌体増殖培養と生分解性重合体生産培養を同時に行っているが、一般によく用いられているように菌体増殖培養と生分解性重合体生産培養を分けて行ってもよい。すなわち、肉汁培地などで菌体増殖培養を行った後、その培養液から菌体を濾過或いは遠心分離などの通常の手段により分離回収し、その菌体を窒素或いはリンを制限した培地に移行させて生分解性重合体生産培養を行ってもよい。
【００７０】
上記により培養された培養物中から、濾過あるいは遠心分離などの通常の手段によって菌体を分離回収し、この菌体を洗浄、乾燥して乾燥菌体を得る。菌体の収量は、通常、０．５ｇ／Ｌ〜１０ｇ／Ｌ程度である。
【００７１】
上記培養により、菌体は、生分解性ランダムコポリマーとＣ４ホモポリマーとを、通常、５０：５０〜５：９５（重量比）の割合で含有する混合物（生分解性重合体）を生産する。該混合物を菌体より単離する方法としては、常法にしたがって、たとえば、得られた乾燥菌体にクロロホルムを加え、還流させて化合物を抽出した後に濃縮し、これをメタノールに再沈殿させた後、上澄みのメタノールを取り除き、真空乾燥機で化合物を乾燥する。
このようにして、６０モル％〜９５モル％のＣ４単位と、５モル％〜４０モル％の長鎖単位（Ｃ６単位、Ｃ８単位、Ｃ１０単位およびＣ１２単位から選ばれる少なくともいずれか）とからなる本発明の生分解性重合体を製造することができる。
【００７２】
本発明の生分解性ランダムコポリマーは、上述のようにして得られた生分解性重合体から単離することができる。生分解性ランダムコポリマーの単離は、常法に従って、たとえば、得られた生分解性重合体（混合物）にアセトンを加え、還流させた後アセトン不溶部を取り除き、アセトン可溶部のアセトンを除去し、真空乾燥機で乾燥して生分解性ランダムコポリマーを得る、というような手順にて行う。このようにして、１モル％〜１５モル％のＣ４単位と、０モル％〜１５モル％のＣ６単位と、１５モル％〜５５モル％のＣ８単位と、１５モル％〜５５モル％のＣ１０単位と、１モル％〜１５モル％のＣ１２単位とからなる本発明の生分解性ランダムコポリマーを得ることができる。
【００７３】
【実施例】
以下に実施例を示し、本発明を具体的に説明するが、本発明は下記の実施例に制限されるものではない。
実施例１
〔前培養〕
表１に示す組成の培地２０ｍＬを入れた１００ｍＬ三角フラスコに下記培地組成のスラントに生育させたシュードモナス エスピー（Ｐｓｅｕｄｏｍｏｎａｓｓｐ．）ＲＦＬ−０１０（受託番号ＦＥＲＭ Ｐ−１８８９３）を一白金耳植菌し、３０℃で７２時間振盪（１５０ｒｐｍ）培養した。
【００７４】
【表１】
〔本培養〕
上記表１に示した培地組成のうち、硫酸アンモニウムの濃度を０．２ｇ／Ｌに変えた培地１００ｍＬを入れた５００ｍＬ三角フラスコに前培養液を１％植菌し、３０℃で７２時間振盪（１５０ｒｐｍ）培養した。
【００７６】
〔菌体分離〕
このようにして得られた培養液から遠心分離（８０００ｒｐｍ）によって菌体を回収し、純水で２回洗浄を行った。この菌体を１０５℃で２４時間乾燥して乾燥菌体０．９７ｇ／Ｌを得た。
【００７７】
〔混合物の分離・精製〕
得られた乾燥菌体にクロロホルムを加え、還流させて菌体内より化合物を抽出した。濾過により菌体を除去しクロロホルム溶液を濃縮して、メタノールに再沈殿させた後、上澄のメタノールを取り除き、真空乾燥機で化合物を乾燥して、菌体の生産物（本発明の生分解性重合体）（０．５１ｇ／Ｌ）を得た。
【００７８】
〔生分解性ランダムコポリマーの単離〕
得られた生産物（生分解性重合体）にアセトンを加え、還流させた後、濾過によりアセトン不溶部を取り除いた。アセトン不溶部を真空乾燥機で乾燥し、Ｃ４ホモポリマーを得た。また、アセトン可溶部を濃縮して乾燥し、生分解性ランダムコポリマーを得た。このようにして分別したところ、菌体の生産物（本発明の生分解性重合体）は生分解性ランダムコポリマーとＣ４ホモポリマーとが４１．６：５８．４で混合した混合物であった。
また、ガスクロマトグラフィ（ガスクロマトグラフＧＣ−１７Ａ、島津製作所製）によって、上記単離した生分解性ランダムコポリマーのモノマー組成を測定した結果、モノマー組成比は、Ｃ４単位：Ｃ６単位：Ｃ８単位：Ｃ１０単位：Ｃ１２単位＝６．５：８．２：５３．８：２７．０：４．５であった。
【００７９】
実施例２
本培養の際、窒素源として硫酸アンモニウムを０．４ｇ／Ｌ用いた以外は、実施例１と同様に行った。菌体の生産物（本発明の生分解性重合体）（０．８０ｇ／Ｌ）は、生分解性ランダムコポリマーとＣ４ホモポリマーとが２７．２：７２．８で混合した混合物であった。実施例１と同様に単離して得られた生分解性ランダムコポリマーのモノマー組成比は、Ｃ４単位：Ｃ６単位：Ｃ８単位：Ｃ１０単位：Ｃ１２単位＝８．７：４．５：４８．７：３１．２：６．９であった。
【００８０】
実施例３
本培養の際、炭素源としてグルコースを１０ｇ／Ｌ、窒素源として硫酸アンモニウムを０．８ｇ／Ｌ用いた以外は、実施例１と同様に行った。菌体の生産物（本発明の生分解性重合体）（１．７１ｇ／Ｌ）は、生分解性ランダムコポリマーとＣ４ホモポリマーとが１７．６：８２．４で混合した混合物であった。実施例１と同様に単離して得られた生分解性ランダムコポリマーのモノマー組成比は、Ｃ４単位：Ｃ６単位：Ｃ８単位：Ｃ１０単位：Ｃ１２単位＝１４．１：３．８：３６．３：３５．８：１０．０であった。
【００８１】
比較例１
ＰＨＢ（Ｐｏｌｙ（３−ｈｙｄｒｏｘｙｂｕｔｙｒｉｃ ａｃｉｄ）、ＡＬＤＲＩＣＨ社製）（Ｃ４ホモポリマー）を用いた。
【００８２】
比較例２
３ＨＢ−３ＨＶランダムコポリマー（Ｐｏｌｙ（３−ｈｙｄｒｏｘｙｂｕｔｙｌｉｃ ａｃｉｄ−ｃｏ−３−ｈｙｄｒｏｘｙｖａｌｅｒｉｃ ａｃｉｄ）、ＡＬＤＲＩＣＨ社製）（３ＨＶ：１２重量％）を用いた。
【００８３】
比較例３
ポリ乳酸（Ｐｏｌｙ（Ｌ−ｌａｃｔｉｄｅ）、ＳＩＧＭＡ社製）を用いた。
【００８４】
（評価試験）
実施例１〜３で得られた生分解性重合体および比較例１〜３で得られた重合体について、以下の物性試験を行った。
（１）引張強さ
上述したように、ＪＩＳ Ｋ ７１２７の規定に準拠して、精密万能試験機（オートグラフＡＧＳ−１００Ｇ、島津製作所製）を用いて測定した。
（２）ヤング率
上述したように、ＪＩＳ Ｋ ７１２７の規定に準拠して、上記精密万能試験機を用いて測定した。
（３）伸び率
上述したように、ＪＩＳ Ｋ ７１２７の規定に準拠して、上記精密万能試験機を用いて測定した。
（４）ガラス転移点
上述したようにＤＳＣ装置（ＤＳＣ−８２３０、リガク社製）を用いて測定した（−２０℃／ｍｉｎ）。
（５）結晶化点
上述したように上記ＤＳＣ装置を用いて測定した（１０℃／ｍｉｎ）。
（６）融点
上述したように上記ＤＳＣ装置を用いて測定した（１０℃／ｍｉｎ）。
結果を表２に示す。
【００８５】
【表２】
【発明の効果】
以上の説明で明らかなように、本発明によれば、有用な新規生分解性高分子およびその製造方法、ならびにかかる高分子を産生する新規微生物を提供することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to novel biodegradable polymers and biodegradable random copolymers that can be applied to a wide range of fields such as pharmaceuticals, foods, hygiene products, construction products, industrial products, agricultural and horticultural products, packaging materials, methods for producing the same, and The present invention relates to a novel microorganism that can be used for production of the biodegradable polymer.
[0002]
[Prior art]
For many years, synthetic polymers derived from petroleum have been used as plastics, but the depletion of petroleum resources and the accumulation of industrial waste due to the fact that the synthetic polymers are difficult to decompose and remain in nature have become major social problems. It is coming. The above-mentioned synthetic polymer increases the amount of carbon dioxide emission when incinerated, and in some cases, it causes a generation of harmful substances such as dioxins and environmental hormones.
[0003]
In recent years, biodegradable polymers that are degraded by natural microorganisms have attracted attention in place of the above-described synthetic polymers derived from petroleum. Among the biodegradable polymers, polyhydroxyalkanoate (PHA) produced using natural biological resources and microorganisms can solve the above problems, and various research and development and practical use have been conducted so far. It is coming.
[0004]
[Problems to be solved by the invention]
However, biodegradable polymers produced by microorganisms have a wide range of physicochemical properties, and there is a demand for the search for biodegradable polymers suitable for each purpose of use. Biodegradable polymers with useful properties are being developed.
An object of the present invention is to provide a useful novel biodegradable polymer and a method for producing the same.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, the present invention is as follows.
(1) 60 mol% to 95 mol% of the following formula
[0006]
Embedded image
3-hydroxybutyrate unit represented by:
The following formula of 5 mol% to 40 mol%
[0008]
Embedded image
3-hydroxyhexanoate unit represented by the following formula
[0010]
Embedded image
3-hydroxyoctanoate unit represented by the following formula
[0012]
Embedded image
A 3-hydroxydecanoate unit represented by the formula:
[0014]
Embedded image
A biodegradable polymer comprising at least one selected from 3-hydroxydodecanoate units represented by:
(2) 60 mol% to 95 mol% 3-hydroxybutyrate units, 0 mol% to 10 mol% 3-hydroxyhexanoate units, and 1 mol% to 30 mol% 3-hydroxyoctanoate The biodegradable weight according to the above (1), comprising a unit, 3 mol% to 30 mol% of 3-hydroxydecanoate unit, and 0 mol% to 15 mol% of 3-hydroxydodecanoate unit. Coalescence.
(3) 60 mol% to 70 mol% of 3-hydroxybutyrate units, 0 mol% to 5 mol% of 3-hydroxyhexanoate units, and 5 mol% to 25 mol% of 3-hydroxyoctanoate The unit according to (1) or (2), comprising a unit, 5 mol% to 25 mol% of 3-hydroxydecanoate unit, and 0 mol% to 5 mol% of 3-hydroxydodecanoate unit. Biodegradable polymer.
(4) At least two or more selected from 3-hydroxybutyrate units, 3-hydroxyhexanoate units, 3-hydroxyoctanoate units, 3-hydroxydecanoate units and 3-hydroxydodecanoate units A mixture of a biodegradable random copolymer comprising: and a homopolymer comprising only 3-hydroxybutyrate units, wherein the random copolymer is contained in an amount of 5 wt% to 50 wt%. The biodegradable polymer according to any one of 3).
(5) The random copolymer comprises 1 mol% to 15 mol% 3-hydroxybutyrate units, 0 mol% to 15 mol% 3-hydroxyhexanoate units, and 15 mol% to 55 mol% 3 (4), which comprises a hydroxyoctanoate unit, 15 mol% to 55 mol% of 3-hydroxydecanoate unit, and 1 mol% to 15 mol% of 3-hydroxydodecanoate unit. The biodegradable polymer as described.
(6) Any of (1) to (5) above, which is obtained by culturing Pseudomonas sp. RFL-010 strain (FERM P-18893) in a medium containing a carbon source. The biodegradable polymer described in 1.
(7) A method for producing a biodegradable polymer, comprising a step of culturing Pseudomonas sp. RFL-010 strain (FERM P-18893) in a medium containing a carbon source.
(8) The method for producing a biodegradable polymer according to (7), wherein the carbon source is at least one selected from glucose, molasses, starch, and maltose.
(9) 1 mol% to 15 mol% of the following formula
[0016]
Embedded image
3-hydroxybutyrate unit represented by:
The following formula of 0 mol% to 15 mol%
[0018]
Embedded image
A 3-hydroxyhexanoate unit represented by:
15 mol% to 55 mol% of the following formula
[0020]
Embedded image
A 3-hydroxyoctanoate unit represented by:
15 mol% to 55 mol% of the following formula
[0022]
Embedded image
A 3-hydroxydecanoate unit represented by:
1 mol% to 15 mol% of the following formula
[0024]
Embedded image
A biodegradable random copolymer consisting of 3-hydroxydodecanoate units represented by:
(10) a step of culturing Pseudomonas sp. RFL-010 strain (FERM P-18893) in a medium containing a carbon source to produce a biodegradable polymer;
From the obtained biodegradable polymer, 1 mol% to 15 mol% of the following formula
[0026]
Embedded image
3-hydroxybutyrate unit represented by:
The following formula of 0 mol% to 15 mol%
[0028]
Embedded image
A 3-hydroxyhexanoate unit represented by:
15 mol% to 55 mol% of the following formula
[0030]
Embedded image
A 3-hydroxyoctanoate unit represented by:
15 mol% to 55 mol% of the following formula
[0032]
Embedded image
A 3-hydroxydecanoate unit represented by:
1 mol% to 15 mol% of the following formula
[0034]
Embedded image
And isolating a random copolymer comprising 3-hydroxydodecanoate units represented by the formula:
(11) Pseudomonas sp. RFL-010 strain (FERM P-18893).
[0036]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The biodegradable polymer of the present invention has the following formula:
[0037]
Embedded image
A polyhydroxyalkanoate (PHA) containing 60 mol% to 95 mol% of a 3-hydroxybutyrate (3HB) unit represented by the formula (hereinafter sometimes referred to as “C4 unit”). If the C4 unit is less than 60 mol%, the crystallinity of the biodegradable polymer is lowered, resulting in a rubbery or viscous oil, making it difficult to process into a molded product. On the other hand, if the C4 unit exceeds 95 mol%, the crystallinity of the biodegradable polymer becomes too high, the flexibility is lost, and the processability is poor.
[0039]
In the PHA of the present invention, the component occupying the remaining 5 mol% to 40 mol% other than the C4 unit is represented by the following formula.
[0040]
Embedded image
3-hydroxyhexanoate (3HHx) unit represented by the following (hereinafter sometimes referred to as “C6 unit”), the following formula
[0042]
Embedded image
3-hydroxyoctanoate (3HO) unit represented by the following (hereinafter sometimes referred to as “C8 unit”), the following formula
[0044]
Embedded image
A 3-hydroxydecanoate (3HD) unit represented by the following (hereinafter sometimes referred to as “C10 unit”), and the following formula
[0046]
Embedded image
At least one selected from 3-hydroxydodecanoate (3HDD) units (hereinafter sometimes referred to as “C12 units”) (hereinafter referred to as C6 units, C8 units, C10 units, and C12). At least one selected from units may be collectively referred to as “long chain unit”). When the long chain unit is less than 5 mol%, the plasticizing effect is lowered, and the biodegradable polymer becomes hard and highly crystalline. On the other hand, if the long chain unit exceeds 40 mol%, the plasticizing effect becomes too great, and the biodegradable polymer becomes a rubbery or viscous oil, making it difficult to process into a molded product.
[0048]
The biodegradable polymer (polyhydroxyalkanoate (PHA)) of the present invention characterized by the monomer composition described above is novel and biodegradable. In order to have biodegradability, in accordance with the provisions of JIS K 6950 (aerobic biodegradation test method using activated sludge), activated sludge mixed with the target plastic is stirred and cultured, This can be confirmed by determining the degree of biodegradation from the chemical oxygen consumption and the residual amount of plastic. The PHA of the present invention has a biodegradability thus determined of 60% to 100%.
Such biodegradable polymers, like conventional plastics, can be used for the production of various products by melt processing and the like, and, unlike petroleum-derived synthetic polymers, can be decomposed by living organisms. Have. Therefore, when discarded, the microbially produced polyester is biodegraded and taken into the natural material circulation, so it can remain in the natural environment and cause pollution like many synthetic polymer compounds that have been used in the past. Absent. In addition, by performing biodegradation treatment, it is not necessary to perform combustion treatment. Therefore, it is an effective material in terms of preventing air pollution and global warming, and can be used as a plastic capable of environmental protection.
[0049]
The biodegradable polymer of the present invention has a tensile strength of 6 MPa to 40 MPa, a Young's modulus (tensile elastic modulus) of 300 MPa to 1100 MPa, and an elongation of 10% to 150%, and has good mechanical properties as a plastic. Is.
In addition, the said tensile strength produces cast film (10 mm x 5 mm x 0.02 mm) with a biodegradable polymer, for example based on the prescription | regulation of JISK7127 (Plastic and sheet tensile test method) Is measured at a speed of 20 mm / min using a precision universal testing machine (manufactured by Shimadzu Corporation) and measured as the maximum tensile stress applied until the film breaks. In addition, the Young's modulus was determined in the same manner as in the above tensile test in accordance with the provisions of JIS K 7127, and within the elastic limit (the difference in tensile stress between any two points on the straight line) / (any one on the straight line). Difference in elongation between two points). The said elongation rate can be measured as an elongation rate when the said tensile test is done based on the prescription | regulation of JISK7127, and a film fractures | ruptures.
[0050]
The biodegradable polymer of the present invention has a glass transition point (Tg) of -10 ° C to -3 ° C, a crystallization point (Tc) of 53 ° C to 58 ° C, and a melting point (Tm) of 155 ° C to 175 ° C. As a plastic, it has good thermal properties.
The glass transition point, crystallization point, and melting point can all be measured using a DSC (differential scanning calorimeter).
[0051]
As described above, the biodegradable polymer of the present invention having good mechanical strength and thermal properties includes packaging materials such as containers, bags and films, fishery materials such as fish nets and fishing lines, vegetation sheets, seedling pots and the like. Application to fields such as agricultural and horticultural materials and medical materials such as sutures can be expected.
[0052]
In the biodegradable polymer, the long chain unit in the present invention is C6 unit 0 mol% to 10 mol%, C8 unit 1 mol% to 30 mol%, C10 unit 3 mol% to 30 mol%, C12 unit. Are preferably selected from the range of 0 mol% to 15 mol%, and occupy 5 mol% to 40 mol% in the biodegradable polymer as described above.
[0053]
Furthermore, in the present invention, C4 units are 60 mol% to 70 mol%, C6 units are 0 mol% to 5 mol%, C8 units are 5 mol% to 25 mol%, C10 units are 5 mol% to 25 mol%, In addition, when the C12 unit is 0 mol% to 5 mol%, a biodegradable polymer having particularly excellent extensibility with an elongation of 60% to 150% can be realized, which is particularly preferable. A biodegradable polymer having such an excellent elongation is particularly useful in that it is excellent in processability and can be produced into a film by stretching, or can be easily processed into a molded product. Application to packaging materials such as bags and food packaging materials can be expected.
[0054]
The biodegradable polymer of the present invention specifically includes a biodegradable random copolymer composed of at least two or more selected from C4 units, C6 units, C8 units, C10 units and C12 units, and only C4 units. It is a mixture with a homopolymer consisting of The biodegradable polymer preferably contains 5% to 50% by weight of the random copolymer. If the content of the random copolymer is less than 5% by weight, the biodegradable polymer tends to be poor in flexibility, and if the content of the random copolymer exceeds 50% by weight, This is because the biodegradable polymer is too flexible and tends to be difficult to apply to a molded product. In the biodegradable polymer, the biodegradable random copolymer is considered to function as a homopolymer plasticizer composed of only C4 units. That is, in the biodegradable polymer of the present invention, the biodegradable random copolymer that acts as a plasticizer is contained, so that it is flexible as compared with a homopolymer consisting of only C4 units, as described above. It is thought that superiority appears in mechanical properties (such as Young's modulus and elongation) and thermal properties (such as glass transition point).
[0055]
In the present invention, in addition to the biodegradable polymer described above, a biodegradable random copolymer contained in the biodegradable polymer is also provided.
The biodegradable random copolymer of the present invention specifically includes 1 mol% to 15 mol% C4 units, 0 mol% to 15 mol% C6 units, 15 mol% to 55 mol% C8 units, 15 mols. It consists of C10 units of% to 55 mol% and C12 units of 1 mol% to 15 mol%, and has biodegradability. Such a biodegradable random copolymer of the present invention has a high composition ratio of monomers of a long chain unit, a glass transition point (Tg) of around −40 ° C., no clear crystallization point and no melting point, and no viscosity. The oily product is particularly excellent in flexibility. For example, it can be expected to be used as a plasticizer for other biodegradable plastics.
[0056]
The content of each monomer unit in the biodegradable polymer and biodegradable random copolymer of the present invention is, for example, using gas chromatography, adding alcohol, acid, chloroform to the biodegradable polymer or biodegradable random copolymer, The reaction can be carried out by heating, and quantified as an ester of each monomer unit.
[0057]
The production method of the biodegradable polymer of the present invention is not particularly limited as long as it is described above, but high molecular weight and high purity that cannot be achieved by a chemical synthesis method using petroleum resources as raw materials have been achieved. For the reason that it can be produced, it is preferably produced by the production method of the present invention using a microorganism newly found by the following inventors. That is, the present invention also provides a method for producing the biodegradable polymer by culturing a microorganism having the following mycological properties in a medium containing a carbon source.
In the production method of the present invention, a microorganism having the following mycological properties is used.
[0058]
[1] Morphological properties
It is a straight gonococcus of 0.8 μm × 1.5 μm to 2.0 μm in an agar (Nutrient Agar) medium at 30 ° C. for one day. Polar hairs are observed by flagella staining and motility is observed. Cell polymorphism and spores are not observed.
[0059]
[2] Growth state in various media
(1) Nutrient Agar plate medium (30 ° C)
The colony is smooth and the periphery is smooth and has a cream color. Glossy, no production of diffusible pigment is observed.
(2) Meat broth liquid medium (30 ° C)
Although no growth or film formation was observed on the surface of the medium, turbidity of the medium was observed.
(3) Broth gelatin puncture culture (30 ° C)
No growth or liquefaction is observed.
(4) Litmus milk
Production of acid and alkali is not observed, and coagulation and liquefaction are not observed.
[0060]
[3] Physiological properties
(1) Gram stainability: negative
(2) Reduction of nitrate: positive
(3) Denitrification reaction: positive
(4) MR test: negative
(5) VP test: negative
(6) Indole production: negative
(7) Production of hydrogen sulfide: negative
(8) Starch hydrolysis: negative
(9) Use of citric acid
-Koser medium: positive
・ Christensen's medium: positive
(10) Use of inorganic nitrogen source
・ Nitrate: Positive
・ Ammonium salt: Positive
(11) Dye generation: Negative
(12) Urease: Negative
(13) Oxidase: positive
(14) Catalase: positive
(15) Range of growth
1. pH
・ PH 6.0: Positive
・ PH 7.0: Positive
・ PH 8.0: Positive
・ PH 9.0: Positive
2. temperature
・ 15 ℃: Negative
・ 20 ℃: Weak
・ 30 ℃: Positive
・ 35 ℃: Negative
(16) Attitude toward oxygen: aerobic
(17) OF test (Hugh Leifson method): negative
(18) Generation of acid and gas from saccharides
1. L-arabinose
Acid generation: positive
・ Gas generation: Negative
2. D-xylose
Acid generation: positive
・ Gas generation: Negative
3. D-glucose
Acid generation: positive
・ Gas generation: Negative
4). D-Mannose
Acid generation: positive
・ Gas generation: Negative
5. D-fructose
Acid generation: positive
・ Gas generation: Negative
6). D-galactose
Acid generation: positive
・ Gas generation: Negative
7). Maltose
Acid generation: negative
・ Gas generation: Negative
8). Sucrose
Acid generation: positive
・ Gas generation: Negative
9. Lactose
Acid generation: negative
・ Gas generation: Negative
10. Trehalose
Acid generation: positive
・ Gas generation: Negative
11. D-sorbitol
Acid generation: negative
・ Gas generation: Negative
12 D-mannitol
Acid generation: positive
・ Gas generation: Negative
13. Inositol
Acid generation: positive
・ Gas generation: Negative
14 Glycerin
Acid generation: positive
・ Gas generation: Negative
15. Starch
Acid generation: negative
・ Gas generation: Negative
(19) Other features
1. β-galactosidase activity: negative
2. Arginine dihydrolase: positive
3. Lysine decarboxylase activity: negative
4). Tryptophan deaminase activity: negative
5. Acetoin production: negative
6). Gelatinase activity: negative
[0061]
From the above bacteriological properties, this microorganism belongs to the genus Pseudomonas, and since it does not exist in comparison with known strains, it is judged as a novel strain, and Pseudomonas sp. Named 010 shares, FERM in the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary
Deposited as P-18893 (Accession Number).
As a result of intensive screening using a culture medium in which glucose is used as a single carbon source from forests in various places, soils such as fields and rivers, and activated sludge as a separation source, the novel microorganisms were obtained in May 2001. It was obtained from the soil of Kanazu-cho, Sakai-gun, Fukui Prefecture on the 30th.
The microorganism of the present invention is mutated in nature or by artificial manipulation (for example, ultraviolet irradiation, X-ray irradiation, chemical treatment, etc.). Therefore, these mutant strains can also be used in the method for producing the biodegradable polymer of the present invention described later.
[0062]
The production method of the present invention comprises producing the above-described biodegradable polymer of the present invention by culturing the above-mentioned novel microorganism Pseudomonas sp. RFL-010 strain in a medium containing a carbon source. It is a feature.
The carbon source contained in the medium used in the present invention is not particularly limited, and examples thereof include glucose, fructose, sucrose, saccharides such as maltose and starch, alcohols such as methanol and ethanol, gluconic acid, and octane. Examples include organic acids such as acids and valeric acid and their salts, molasses, yeast extract, meat extract, complex carbon sources such as soybean oil, rapeseed oil and fish oil. Among them, the microorganism of the present invention has a particular advantage that glucose, which is relatively inexpensive and easily available, can be used as a carbon source, having the advantage of being easily metabolized by the microorganism.
[0063]
Although there is no restriction | limiting in particular in content of the carbon source in the said culture medium, since it can perform the growth of a microbial cell and production of a biodegradable polymer, it is 0.1g / L-100g / L. Preferably, it is more preferably 1 g / L to 30 g / L. This is because when the carbon source is less than 0.1 g / L, sufficient cell growth is not performed, and the productivity of the biodegradable polymer tends to be significantly reduced, and the carbon source exceeds 100 g / L. This is because the osmotic pressure of the culture solution becomes too high and tends to inhibit bacterial cell growth.
[0064]
The nitrogen source contained in the medium used in the present invention includes ammonium salts such as ammonium sulfate and ammonium chloride, inorganic nitrogen sources including nitrates such as sodium nitrate and potassium nitrate, yeast extract, meat extract, peptone, Organic nitrogen sources such as tryptone, urea, soybean powder, oil cake, corn steep liquor and the like can be mentioned. Among these, it is preferable to use an inorganic nitrogen source from the viewpoint of improving the efficiency of separation and purification of the biodegradable polymer produced from the cells.
[0065]
When the medium containing the nitrogen source is used, the content is not particularly limited. However, from the reason that the bacterial cells can be efficiently grown and the biodegradable polymer can be produced, 0.1 g / L to 8 g / L It is preferable that it is L, and it is more preferable that it is 0.2 g / L-3g / L. This is because if the nitrogen source is less than 0.1 g / L, the cells do not grow sufficiently and the absolute production amount of the biodegradable polymer tends to be remarkably reduced, and the nitrogen source is 8 g / L. When L is exceeded, the cells grow sufficiently, but the content of the biodegradable polymer is lowered, and the productivity tends to be remarkably lowered.
[0066]
The medium used in the present invention is not particularly limited as long as it contains the above-described carbon source and, if desired, a nitrogen source, and various media that have been widely used in the art can be used. However, it is preferable to use, for example, a medium in which the concentrations of the carbon source and the nitrogen source are set within a certain range because of the bacteriological properties of the above-mentioned Pseudomonas sp. RLF-010 strain. By using such a medium, there is an advantage that the bacterial cells can be efficiently grown and the biodegradable polymer can be produced.
[0067]
In the above medium, metal salts such as magnesium, sodium, potassium, calcium, manganese, copper, cobalt, molybdenum, zinc, iron, phosphates, nitrates, chlorides, and the like, as long as the purpose of the present invention is not impaired. Products, carbonates, organic acid salts such as sodium citrate, and growth factors such as vitamins as necessary.
[0068]
Cultivation is preferably performed under aerobic conditions, and any of stationary, shaking, and aeration-agitation culture is possible, but shaking or aeration-agitation culture is advantageous. At this time, the culture method may be either batch culture or continuous culture. The culture temperature is preferably 15 ° C to 34 ° C, particularly preferably 20 ° C to 32 ° C. The pH of the medium is suitably 5.8 to 9.5, but 6.5 to 8.5 is optimal.
[0069]
In this study, cell growth culture and biodegradable polymer production culture are carried out simultaneously in a medium restricted with nitrogen or phosphorus. As is generally used, cell growth culture and biodegradable polymer production culture are performed. May be performed separately. That is, after cell growth culture in a broth medium or the like, the cells are separated and recovered from the culture solution by a normal means such as filtration or centrifugation, and the cells are transferred to a medium restricted with nitrogen or phosphorus. Biodegradable polymer production culture may be performed.
[0070]
From the culture cultured as described above, microbial cells are separated and collected by ordinary means such as filtration or centrifugation, and the microbial cells are washed and dried to obtain dry microbial cells. The yield of microbial cells is usually about 0.5 g / L to 10 g / L.
[0071]
By the above culture, the cells produce a mixture (biodegradable polymer) containing the biodegradable random copolymer and the C4 homopolymer in a ratio of usually 50:50 to 5:95 (weight ratio). As a method for isolating the mixture from the cells, according to a conventional method, for example, chloroform was added to the obtained dry cells, refluxed to extract the compound, concentrated, and reprecipitated in methanol. Thereafter, the supernatant methanol is removed, and the compound is dried in a vacuum dryer.
Thus, it is composed of 60 mol% to 95 mol% C4 units and 5 mol% to 40 mol% long chain units (at least one selected from C6 units, C8 units, C10 units and C12 units). The biodegradable polymer of the present invention can be produced.
[0072]
The biodegradable random copolymer of the present invention can be isolated from the biodegradable polymer obtained as described above. Isolation of the biodegradable random copolymer is carried out according to a conventional method, for example, adding acetone to the obtained biodegradable polymer (mixture), refluxing, removing the acetone insoluble part, and removing the acetone soluble part of acetone. And drying in a vacuum dryer to obtain a biodegradable random copolymer. Thus, 1 mol% to 15 mol% C4 units, 0 mol% to 15 mol% C6 units, 15 mol% to 55 mol% C8 units, and 15 mol% to 55 mol% C10 units. The biodegradable random copolymer of the present invention comprising units and 1 mol% to 15 mol% C12 units can be obtained.
[0073]
【Example】
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.
Example 1
[Pre-culture]
Pseudomonas sp. RFL-010 (accession number FERM P-18893) grown in a slant having the following medium composition was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of the medium shown in Table 1 in a platinum ear, 30 Cultured with shaking (150 rpm) at 72 ° C. for 72 hours.
[0074]
[Table 1]
[Main culture]
Of the medium composition shown in Table 1 above, 1% of the preculture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of medium with the ammonium sulfate concentration changed to 0.2 g / L, and shaken at 150 ° C. for 72 hours (150 rpm ) Cultured.
[0076]
[Bacterial cell isolation]
Bacteria were collected from the culture solution thus obtained by centrifugation (8000 rpm) and washed twice with pure water. The cells were dried at 105 ° C. for 24 hours to obtain 0.97 g / L of dried cells.
[0077]
[Separation and purification of mixture]
Chloroform was added to the obtained dried cells and refluxed to extract compounds from the cells. After removing the cells by filtration and concentrating the chloroform solution and reprecipitating in methanol, the supernatant methanol is removed, the compound is dried in a vacuum dryer, and the product of the cells (the biodegradation of the present invention) Polymer (0.51 g / L) was obtained.
[0078]
[Isolation of biodegradable random copolymer]
Acetone was added to the obtained product (biodegradable polymer) and refluxed, and then the acetone insoluble part was removed by filtration. The acetone insoluble part was dried with a vacuum dryer to obtain a C4 homopolymer. Further, the acetone soluble part was concentrated and dried to obtain a biodegradable random copolymer. When fractionated in this way, the product of the bacterial cells (the biodegradable polymer of the present invention) was a mixture in which the biodegradable random copolymer and the C4 homopolymer were mixed at 41.6: 58.4.
Moreover, as a result of measuring the monomer composition of the isolated biodegradable random copolymer by gas chromatography (gas chromatograph GC-17A, manufactured by Shimadzu Corporation), the monomer composition ratio was C4 unit: C6 unit: C8 unit: C10 unit. : C12 unit = 6.5: 8.2: 53.8: 27.0: 4.5.
[0079]
Example 2
The main culture was performed in the same manner as in Example 1 except that 0.4 g / L of ammonium sulfate was used as a nitrogen source. The cell product (the biodegradable polymer of the present invention) (0.80 g / L) was a mixture of a biodegradable random copolymer and a C4 homopolymer mixed at 27.2: 72.8. The monomer composition ratio of the biodegradable random copolymer obtained by isolation in the same manner as in Example 1 was C4 unit: C6 unit: C8 unit: C10 unit: C12 unit = 8.7: 4.5: 48.7: 31.2: 6.9.
[0080]
Example 3
The main culture was performed in the same manner as in Example 1 except that 10 g / L of glucose was used as the carbon source and 0.8 g / L of ammonium sulfate was used as the nitrogen source. The cell product (the biodegradable polymer of the present invention) (1.71 g / L) was a mixture of a biodegradable random copolymer and a C4 homopolymer mixed at 17.6: 82.4. The monomer composition ratio of the biodegradable random copolymer obtained by isolation as in Example 1 was C4 unit: C6 unit: C8 unit: C10 unit: C12 unit = 14.1: 3.8: 36.3: It was 35.8: 10.0.
[0081]
Comparative Example 1
PHB (Poly (3-hydroxybutyric acid), manufactured by ALDRICH) (C4 homopolymer) was used.
[0082]
Comparative Example 2
3HB-3HV random copolymer (Poly (3-hydroxybutyric-co-3-hydroxyvaleric acid), ALDRICH) (3HV: 12% by weight) was used.
[0083]
Comparative Example 3
Polylactic acid (Poly (L-lactide), manufactured by SIGMA) was used.
[0084]
(Evaluation test)
The following physical property tests were conducted on the biodegradable polymers obtained in Examples 1 to 3 and the polymers obtained in Comparative Examples 1 to 3.
(1) Tensile strength
As described above, measurement was performed using a precision universal testing machine (Autograph AGS-100G, manufactured by Shimadzu Corporation) in accordance with the provisions of JIS K 7127.
(2) Young's modulus
As described above, the measurement was performed using the precision universal testing machine in accordance with JIS K 7127.
(3) Elongation rate
As described above, the measurement was performed using the precision universal testing machine in accordance with JIS K 7127.
(4) Glass transition point
As described above, measurement was performed using a DSC apparatus (DSC-8230, manufactured by Rigaku Corporation) (−20 ° C./min).
(5) Crystallization point
Measurement was performed using the DSC apparatus as described above (10 ° C./min).
(6) Melting point
Measurement was performed using the DSC apparatus as described above (10 ° C./min).
The results are shown in Table 2.
[0085]
[Table 2]
【The invention's effect】
As is apparent from the above description, according to the present invention, a useful novel biodegradable polymer, a method for producing the same, and a novel microorganism that produces such a polymer can be provided.
Claims (4)
上記生分解性重合体が、６０モル％〜９５モル％の下記式
５モル％〜４０モル％の下記式
当該生分解性重合体が、１モル％〜１５モル％の３−ヒドロキシブチレート単位と、０モル％〜１５モル％の３−ヒドロキシヘキサノエート単位と、１５モル％〜５５モル％の３−ヒドロキシオクタノエート単位と、１５モル％〜５５モル％の３−ヒドロキシデカノエート単位と、１モル％〜１５モル％の３−ヒドロキシドデカノエート単位からなる生分解性ランダムコポリマーと、３−ヒドロキシブチレート単位のみからなるホモポリマーとの混合物であって、当該ランダムコポリマーを５重量％〜５０重量％含有するものである、生分解性重合体の製造方法。 Pseudomonas sp in a medium containing a carbon source (Pseudomonas sp.) A RFL-010 strain (FERM P-18893) producing process of a biodegradable polymer you characterized by containing a step of culturing,
The biodegradable polymer is 60 mol% to 95 mol% of the following formula
The following formula of 5 mol% to 40 mol%
The biodegradable polymer comprises 1 mol% to 15 mol% 3-hydroxybutyrate units, 0 mol% to 15 mol% 3-hydroxyhexanoate units, and 15 mol% to 55 mol% 3 A biodegradable random copolymer comprising hydroxyoctanoate units, 15 mol% to 55 mol% 3-hydroxydecanoate units, and 1 mol% to 15 mol% 3-hydroxydodecanoate units; A method for producing a biodegradable polymer, which is a mixture with a homopolymer consisting only of hydroxybutyrate units and containing 5 to 50% by weight of the random copolymer.
得られた生分解性重合体から、１モル％〜１５モル％の下記式
０モル％〜１５モル％の下記式
１５モル％〜５５モル％の下記式
１５モル％〜５５モル％の下記式
１モル％〜１５モル％の下記式
From the obtained biodegradable polymer, 1 mol% to 15 mol% of the following formula
The following formula of 0 mol% to 15 mol%
15 mol% to 55 mol% of the following formula
15 mol% to 55 mol% of the following formula
1 mol% to 15 mol% of the following formula
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