JP2004250629A - Preparation process of polyhydroxyalkanoic acid - Google Patents
Preparation process of polyhydroxyalkanoic acid Download PDFInfo
- Publication number
- JP2004250629A JP2004250629A JP2003044135A JP2003044135A JP2004250629A JP 2004250629 A JP2004250629 A JP 2004250629A JP 2003044135 A JP2003044135 A JP 2003044135A JP 2003044135 A JP2003044135 A JP 2003044135A JP 2004250629 A JP2004250629 A JP 2004250629A
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- JP
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- Prior art keywords
- polyhydroxyalkanoate
- producing
- oil
- phbx
- genus
- 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.)
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- 238000002360 preparation method Methods 0.000 title 1
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 claims abstract description 49
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- 229920000331 Polyhydroxybutyrate Polymers 0.000 claims description 28
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- WHBMMWSBFZVSSR-UHFFFAOYSA-M 3-hydroxybutyrate Chemical compound CC(O)CC([O-])=O WHBMMWSBFZVSSR-UHFFFAOYSA-M 0.000 claims description 9
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、結晶固化速度の速いポリヒドロキシアルカノエートの製造方法に関する。
【０００２】
【従来の技術】
ポリヒドロキシアルカノエート（以後、ＰＨＢＸと略す）は、多くの微生物種の細胞内にエネルギー蓄積物質として生成、蓄積される熱可塑性ポリエステルである。微生物によって天然の有機酸や油脂を炭素源に生産されるＰＨＢＸは、土中や水中の微生物により完全に生分解されるため自然界の炭素循環プロセスに取り込まれることになる。従って、ＰＨＢＸは生態系への悪影響がほとんどない環境調和型のプラスチック材料と言える。近年、合成プラスチックが環境汚染、廃棄物処理、石油資源の観点から深刻な社会問題となるに至り、ＰＨＢＸが環境に優しいグリーンプラスチックとして注目され、その実用化が切望されている。
微生物中に最初に発見されたＰＨＢＸが３−ヒドロキシブチレートのホモポリマーであるポリ−３−ヒドロキシブチレート（以下、ＰＨＢと略す）である。ＰＨＢは、高結晶性で結晶化度が高いため硬くて脆く、しかも融点付近の温度（１８０℃）で速やかに熱分解するために、溶融加工性が低いといった物性上の欠点を抱えており、実用化が困難な素材といえる。
【０００３】
そこで、ＰＨＢの結晶化度を下げて脆性を改善するため、他の３−ヒドロキシアルカン酸（以下、３ＨＸと略す）をＰＨＢ骨格中に導入する試みがなされた。そのような３ＨＸとして３−ヒドロキシ吉草酸（以下、３ＨＶと略す）を使用することにより得られた共重合体Ｐ（３ＨＢ−ｃｏ−３ＨＶ）（以下、ＰＨＢＶと略す）（特許文献１〜３）は、広い３ＨＶ組成範囲にわたって高い結晶固化速度を維持したまま、ＰＨＢよりも融点はさがったため、高融点・易熱分解性のＰＨＢよりは溶融加工性の点で優れていた。この共重合体は実用性の高い高分子材料として注目され、一時Ｉｍｐｅｒｉａｌ Ｃｈｅｍｉｃａｌ Ｉｎｄｕｓｔｒｉｅｓからバイオポールの名前で販売されていた。しかしながら、ＰＨＢＶは結晶化度が５０％以下に下がることはなかったため、脆性を改善できなかった。
【０００４】
そこで、ＰＨＢＶの脆性をさらに改善することを目的とし、３ＨＶより大きな側鎖を持つヒドロキシ酸をＰＨＢ骨格に導入したＰＨＢＸの研究がされた。例えば、３−ヒドロキシプロピオン酸（以下、３ＨＰと略す）、３−ヒドロキシペンタン酸（以下、３ＨＣと略す）、３−ヒドロキシヘキサン酸（以下、３ＨＨと略す）、３−ヒドロキシオクタン酸（以下、３ＨＯと略す）、３−ヒドロキシノナン酸（以下、３ＨＮと略す）、３−ヒドロキシデカン酸（以下、３ＨＤと略す）、３−ヒドロキシドデカン酸（以下、３ＨＤＤと略す）などを３ＨＸとして含む共重合ポリエステルが精力的に研究された（非特許文献１）。これらは３ＨＶより長いペンチルやアルキル測鎖を持つため、これらが骨格に導入されたＰＨＢＸは実質的に非晶質となる。これらポリマーの特性は非常にしなやかで柔らかいため脆性が改善され、しかも、低融点（８０℃〜１５０℃）であるため溶融加工性も改善されることが期待された。しかし、これらのポリマーは結晶固化速度が非常に遅く、加熱溶融し冷却後、室温においても柔らかく長時間粘性を持つことや、粘着性があるため成型時にすぐに金型から取り出すことができず、実生産が連続的に行なえないという問題が新たに生じた。また、結晶固化速度の速い既存の汎用樹脂の加工に使用している加工機器が生分解性プラスチックの加工に利用できないなどの問題も明らかとなった。フィルムやシート、繊維、発泡体、成型品、不織布への加工では、生産工程中溶融加工したポリマーの冷却後の結晶固化速度が速いことが、生産工程の連続化、ひいては生産性の向上と低コストに繋がるため非常に重要である。
【０００５】
そこで、ＰＨＢＸの結晶固化速度を速める試みがされてきた。そのための一般的な方法として、結晶核剤を添加する方法が試みられている。例えば、特許文献４では、ＰＨＢＨに結晶核剤として窒化ホウ素を使用し結晶化促進効果が得られている。しかし、これは高価な材料であり、しかも生分解性を持たない。このため、より安価で生分解性のある結晶核剤が検討された。特許文献５及び６では、ＰＨＢＶあるいはＰＨＢＨに対してより高い融解温度を有し、しかも生分解性であるＰＨＢを結晶核剤として添加し、結晶固化速度を速くする技術が開示されている。これらの先行技術では、ＰＨＢＸとＰＨＢの混合法として、例えばＰＨＢＸとＰＨＢを熱クロロホルムに溶解・混合後クロロホルムを蒸発させることによるポリマーの析出やメタノールで晶析を行い混合ポリマーを回収する方法、あるいは２種類のポリマーをドライアイスで冷却しながら粉砕し混合する方法、ＰＨＢは溶融しないでＰＨＢＸのみ溶融させた状態での混合、乾燥ポリマー粉末のミキシングによる混合などが試みられている。しかしながら、溶媒へ溶解して混合する方法では、ＰＨＢＸの溶解や晶析のために非常に大量の溶媒が必要になり、コスト高となる。また、晶析時にポリマーと結晶核剤の溶解度の違いから、均一に分散した状態で晶析が行われないなどの可能性があり、実用的でない。ポリマーを粉砕後混合する方法や、乾燥粉末のミキシングではポリマーを均一に混合することは困難であり、結晶核剤の効果が減少する。特に、ＰＨＢＸ及び結晶核剤の粒子系は小さいほど良く混合し、また核形成部位の数が多くなるため高い効果が期待できるが、上記の混合法では微粒子での混合効果は期待できない。現在まで、このような微粒子を混合し結晶固化速度を調節する技術は今までに作られていない。
【０００６】
【特許文献１】
特開昭５７−１５０３９３号公報
【０００７】
【特許文献２】
特開昭５９−２２０１９２号公報
【０００８】
【特許文献３】
特表平１１−５００００８号公報
【０００９】
【特許文献４】
特開平６−１５７８７８号公報
【００１０】
【特許文献５】
特表平８−５１０４９８号公報
【００１１】
【特許文献６】
ＷＯ ０２／５０１５６号公報
【００１２】
【非特許文献１】
Ｐｏｉｒｉｅｒ Ｙ．，Ｎａｗｒａｔｈ Ｃ．，Ｓｏｍｅｒｖｉｌｌｅ Ｃ，ＢＩＯ／ＴＥＣＨＮＯＬＯＧＹ，１３，１４２−１５０，１９９５年
【００１３】
【発明が解決しようとする課題】
上述したように、ＰＨＢＸの結晶固化速度の向上に結晶核剤としてＰＨＢは効果があるが、両者の混合法に難点があり、ＰＨＢの効果が十分発揮されているとは言い難い。従って、本研究の目的は、従来技術における上記の課題を解決し、従来の方法では得られなかった微粒子でのＰＨＢＸと結晶核剤の混合体の製造法を提供することにある。さらに、本発明は、ＰＨＢＨ樹脂の結晶固化速度を増大させることで、フィルム成形、射出成形、押出成形、紡糸などの成形方法利用の際に、融着性防止、離形性向上、成形サイクル時間の短縮、連続的な樹脂ペレット化、など成形性が改善された生分解性ポリエステル系樹脂組成物の製造方法を提供することを課題とする。
【００１４】
【課題を解決するための手段】
本発明者らは、前期課題の解決のため、鋭意研究を行った結果、３ＨＢと３ＨＢ以外の３ＨＸからなるＰＨＢＸにおいて、ＰＨＢＸ中の３ＨＸモル分率が６％以上で、且つ当該ポリマー中に３ＨＸモル分率が５％以下であるＰＨＢＸあるいはＰＨＢホモポリマーを０．１〜３０％含むＰＨＢＸあるいはＰＨＢＸとＰＨＢの混合物を微粒子状態のまま微生物から得る方法を構築することにより、結晶固化速度の速いＰＨＢＸを得ることに成功し、本発明を完成させるに至った。
【００１５】
すなわち、本発明は、結晶固化速度の向上したポリヒドロキシアルカノエートを微生物により製造する方法であって、当該微生物の培養途中で供給する炭素源の種類を変化させること、あるいは培養途中で炭素源の比基質供給速度を変化させることにより、結晶核剤となるポリヒドロキシアルカノエートを合成させることを特徴とするポリヒドロキシアルカノエートの製造方法に関する。
【００１６】
【発明の実施の形態】
本発明におけるＰＨＢＸとしては、３ＨＢと他の２種以上の３ＨＸとの共重合ポリエステルである。ここで、３ＨＢとの共重合体として３−ヒドロキシプロピオン酸（以下、３ＨＰと略す）、３ＨＸは３−ヒドロキシペンタン酸（以下、３ＨＣと略す）、３−ヒドロキシヘキサン酸（以下、３ＨＨと略す）、３−ヒドロキシオクタン酸（以下、３ＨＯと略す）、３−ヒドロキシノナン酸（以下、３ＨＮと略す）、３−ヒドロキシデカン酸（以下、３ＨＤと略す）、３−ヒドロキシドデカン酸（以下、３ＨＤＤと略す）が挙げられ、この中でも、特に３ＨＸの含有率が８％以上のポリエステルで、非常に幅広くポリエステルの特性が変化するという点で、３ＨＨを含む共重合体が好ましく、従ってＰＨＢＨがより好ましい。また、３ユニットを含む共重合体としては３ＨＢ−３ＨＶ−３ＨＨが好ましい。
【００１７】
これらの共重合体ＰＨＢＸは、結晶核剤として低３ＨＸモル分率の３ＨＢ共重合体（以下、低３ＨＸ−ＰＨＢＸと略す）を一定量含むか、あるいは３ＨＢホモポリマー（ＰＨＢ）を一定量含むことにより、３ＨＢの高い結晶固化速度を利用し、全体としてのＰＨＢＸの結晶固化速度が速くなったポリマーである。
【００１８】
結晶固化速度の速いＰＨＢＸとは、ＰＨＢＸ中の３ＨＸモル分率が、ＰＨＢＸがより柔軟な性質になる点において、好ましくは６％以上のモル分率であり、より好ましくは７％以上、特に好ましくは８％以上である。
【００１９】
当該ＰＨＢＸ中に含まれる低３ＨＸ−ＰＨＢＸ中の３ＨＸ含量は結晶固化速度を速くする点で、好ましくは５％モル分率より低く、より好ましくは４％モル分率以下であり、さらに好ましくは３％以下であり、特に好ましくは０％のホモポリマーＰＨＢである。
【００２０】
これらの低３ＨＸ−ＰＨＢＸあるいはＰＨＢのＰＨＢＸ中の含量は、結晶固化速度を速くする点において、好ましくは０．０１〜３０重量％、より好ましくは０．５〜２０重量％、さらに好ましくは０．５〜５％重量である。
【００２１】
本発明の製造方法において、使用する微生物には特に制限なく、天然から単離された微生物や菌株の寄託機関（例えばＩＦＯ、ＡＴＣＣ等）に寄託されている微生物を使用できる。しかし、本発明の目的から共重合体生産能力と共に、ＰＨＢなどの結晶固化速度の速いホモポリマーを生産する能力を持つ菌株が望ましく、この点において、Ａｌｃａｌｉｇｅｎｅｓ属やＲａｌｓｔｏｎｉａ属、Ａｅｒｏｍｏｎａｓ属、Ｐｓｅｕｄｏｍｏｎａｓ属が好ましいが、より好ましくはＡｌｃａｌｉｇｅｎｅｓ属やＲａｌｓｔｏｎｉａ属である。
【００２２】
また、上記微生物が、野生型の状態では目的とする共重合体を生産できない、もしくはその生産量が低い場合には、上記微生物に、目的とする共重合ポリエステルの重合酵素遺伝子を導入して形質転換し、得られた形質転換微生物を用いることができる。形質転換微生物を作製する場合、ポリエステル重合酵素遺伝子を含む組換えベクターを利用するなどの一般的な方法を用いることができ、該ベクターには、その菌体内で自律的に増殖しうるプラスミドベクターを用いることができる。また、該ポリエステル重合酵素遺伝子を直接宿主の染色体に組み込んでも良い。
【００２３】
本発明の共重合ポリエステルの生産において使用されるポリエステル重合酵素遺伝子としては、特に限定されないが、アエロモナス・キャビエ（Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ）より単離された遺伝子が好ましく、例えば特開平７−２６５０６５号公報に記載されている遺伝子断片を用いることができる。微生物に組換えベクターを導入するには、当業者には周知の方法により行うことができる。本発明に用いられる微生物の一例として、ＰＨＢ非生産株であるＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａに、Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ由来のポリエステル重合酵素遺伝子を導入した、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ ＡＣ３２株として寄託，寄託番号ＦＥＲＭ ＢＰ−６０３８）（Ｔ． Ｆｕｋｕｉ．， Ｙ． Ｄｏｉ．， Ａｐｐｌ Ｍｉｃｒｏｂｉｏｌ Ｂｉｏｔｅｃｈｎｏｌ．， ４９， ３３３−３３６，１９９８）を好ましく用いることができる。 該菌体の場合、増殖速度が速く、油脂を炭素源にＰＨＢＨを効率よく生産する点において好ましい。また、フラクトースを炭素源に用いた場合ＰＨＢが生産できることが知られており、本発明において好適に使用できる。また、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａにはグルコース資化性の菌株も広く使用されており（ＡＴＣＣ Ｎｏ１７６９９、Ｊ ＧｅｎｅｒａｌｌＭｉｃｒｏｂｉｏｌｏｇｙ １１５、１８５−１９２、１９７９）これらを使用することもできる。
【００２４】
本発明の結晶固化速度の速いＰＨＢＸは、上記の菌体を用い、培養の途中で炭素源を連続的あるいは段階的に変化させる、あるいは炭素源を別のものに変更することにより得ることができる。本発明において炭素源として使用する油脂は、大豆油、コーン油、綿実油、パーム油、パーム核油、ヤシ油、落花生油などの比較的安定的に供給される油脂およびこれらの油脂を分別して得られる各画分、例えばパームＷオレイン油（パーム油を２回無溶媒分別した低融点画分）、パーム核油オレイン（パーム核油を１回無溶媒分別した低融点画分）などの分別油脂、またはこれら油脂やその画分を化学的あるいは生物化学的に処理した合成油、さらにはこれらを混合した混合油が使用できる。このなかでも、コストの点からは天然油脂を使用するのが好ましい。さらに具体的にＰＨＢＨの場合を説明すると、油脂の構成脂肪酸としてラウリン酸を含有する油脂、俗にラウリン油脂と称される油脂を使用することが好ましい。例えば、ラウリン油脂を用いた場合、２０％モル分率までの比較的高い３ＨＨ含有率を有するＰＨＢＨを得ることができる。構成脂肪酸としてラウリン油脂としては、パーム核油やヤシ油などの天然油脂、パーム核油オレインなどの分別油脂やそれらラウリン油脂を含む混合油が挙げられる。これらのうち特に３ＨＨモル分率８％以上を達成する点において、パーム核油とパーム核油オレインの使用が好ましい。
【００２５】
一方、大豆油、コーン油、綿実油、パーム油、落花生油あるいはこれらの分別油脂などを用いた場合は１〜１０％モル分率の比較的低い３ＨＨ含有率のＰＨＢＨが得られる。これらを結晶核剤用の低３ＨＸ−ＰＨＢＸを合成するために使用する場合、５％モル分率以下の３ＨＸ−ＰＨＢＸを合成する点において、大豆油、コーン油、綿実油の使用が好ましい。
【００２６】
また、結晶核剤として最も効果の高いホモポリマーＰＨＢを合成する場合には、一般的に糖を用いればよい。糖としては、グルコース、フラクトース、蔗糖、糖蜜が好適に使用できる。中でも特にグルコースは、低コスト及び微生物が効果的に繁殖できる点で好ましい基質である。
【００２７】
本発明の好ましい実施態様では、ラウリン油脂を最初の炭素源に用い、１０％モル分率以上の３ＨＸ組成を持つＰＨＢＸを製造する場合、結晶固化速度を速くするためには、培養途中で炭素源を大豆油、コーン油、綿実油、パーム油、落花生油あるいはこれらの分別油脂などに変更することにより、５％モル分率より低い３ＨＸを含有したＰＨＢＸを生産することができる。あるいは炭素源を油脂や脂肪酸以外の炭水化物、例えば糖やデンプンに変えることによりＰＨＢを産生させることができる。
【００２８】
最初の炭素源に大豆油、コーン油、綿実油、パーム油、落花生油あるいはこれらの分別油脂などを用いた場合には、１０％モル分率以下のＰＨＢＸが生産されるため、培養途中で炭素源を油脂や脂肪酸以外の炭水化物、例えば糖やデンプンに変えることによりＰＨＢを生産させることが望ましい。
【００２９】
本発明者らが炭素源である油脂の最適な添加方法を検討した結果、意外にも油脂の比基質供給速度を調整することによっても、生産されるＰＨＢＸの３ＨＸモル分率を制御できることが明らかとなった。ここで比基質供給速度とは、単位時間に正味の菌体重量あたり供給される油脂の量として定義される培養変数であり、正味の菌体重量とは全菌体重量から含有するポリエステル重量を差し引いた菌体重量（乾燥菌体重量）である。
【００３０】
【００３１】
本発明において、比基質供給速度が小さいほどＰＨＢＸの３ＨＸ含有率が上昇することが見いだされた。従って、３ＨＸ含有率の高いＰＨＢＸを得たい場合には、比基質供給速度を低く設定して培養すればよい。例えば、３ＨＸモル分率が１０％以上のＰＨＢＸを生産する場合に、比基質供給速度は好ましくは０．１０以下、より好ましくは０．０９以下、さらに好ましくは０．０６以下である。また、３ＨＸモル分率が５％以下のＰＨＢＸを得たい場合には、比基質供給速度は好ましくは０．１０より大きく、より好ましくは０．１２以上である。
【００３２】
培養のフェーズによって比基質供給速度を変化させる場合には、培養後半のポリマー蓄積期の適当な時期に変化させるのが望ましい。比基質供給速度は高い値から低い値にする方法と逆の方法があるが、そのどちらも利用できる。
【００３３】
本発明において、油脂の比基質供給速度を制御する場合、培養全期間を菌体増殖期とポリエステル蓄積期の２つのフェーズに分け、それぞれのフェーズにおいて異なる比基質供給速度の値を設定し、それぞれのフェーズ期間内では設定された一定の比基質供給速度となるように制御する、あるいは培養全期間中、比基質供給速度を連続的に変化させる、あるいは、上記フェーズに関わらず、培養途中で比基質供給速度を変化させる方法があり、どの方法を用いても構わない。油脂の比基質供給速度を、培養の特定の期間内で一定値に制御するためには、設定された比基質供給速度の値を満たすように油脂の添加量を連続的にあるいは間欠的に変化させる必要がある。油脂の添加量を連続的に変化させるには、例えばコンピューターを利用して流加量を制御することができる。また間欠的（すなわち段階的）に変化させる場合は、自動で制御することもできるが、手動でも流加量の操作が可能である点で、簡便である。間欠的に変化させる場合、厳密には予め設定された比基質供給速度の値を常に完全に満たすわけではないが、その平均値として上記設定された比基質供給速度の値を満たしていればよい。このように、油脂の比基質供給速度が培養の全期間、或いは培養の特定のフェーズ期間内では、設定されたある一定値となるように制御することで、培養初期に所定量の油脂を全量添加したり、培養期間中一定の油脂添加量で流加培養したり、比基質供給速度に基づかず経験的に油脂の添加量を変化させるといった従来の培養方法では不十分であった共重合ポリエステルの生産性の向上と、モノマーユニットの組成比の制御が初めて達成される。本明細書において培養全期間というのは、本培養における培養開始から培養終了時までの全期間である。
【００３４】
本発明の菌体培養に用いられる窒素源としては、例えばアンモニア、塩化アンモニウム、硫酸アンモニウム、リン酸アンモニウム等のアンモニウム塩である無機窒素源、ペプトン、肉エキス、酵母エキスなどの有機窒素源が挙げられる。無機塩類としては、例えばリン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウムなどが使用される。そのほかの有機栄養源としては、グリシン、アラニン、セリン、スレオニン、プロリンなどアミノ酸、ビタミンＢ１、ビタミンＢ１２、ビタミンＣ等のビタミン類が使用される。しかしながら、生産コスト抑制の観点からは有機栄養源であるペプトン、肉エキス、酵母エキスおよびグリシン、アラニン、セリン、スレオニン、プロリンなどのアミノ酸、ビタミンＢ１、ビタミンＢ１２、ビタミンＣ等のビタミンの使用は最少量とする方が好ましい。
【００３５】
本発明において、ＰＨＢＸを微生物菌体から回収する方法は特に限定されず、公知の溶媒抽出法、物理的破砕法、化学的処理などが採用できる。しかし、微生物内ではＰＨＢＸは通常粒径１ミクロン以下の微粒子として合成されるため、ポリマーを微粒子状態で取り出すことが、ＰＨＢＸと結晶核剤とが均一に微分散した状態で得るために必要である。その結果、得られるポリマーの結晶固化速度が著しく向上する。また、フィルムなどへの加工時に結晶核剤の不均質さから生じる斑や強度の不均一さを無くすことができる。本発明におけるポリマー回収の好ましい実施態様では、（ａ）ＰＨＢＸ含有微生物細胞の水性懸濁液に、攪拌と物理的破砕処理を行いながらアルカリを添加し、該細胞を破砕すると共に、該細胞中のＰＨＢＸ以外の細胞物質を可溶化あるいは乳化させ、次いでＰＨＢＸを懸濁液から分離する工程及び（ｂ）分離されたＰＨＢＸを、酵素及び／又は界面活性化剤で処理し、ＰＨＢＸに付着した不純物を可溶化又は分解後可溶化し、続いて親水性溶媒でＰＨＢＸを洗浄する工程からなる。（ａ）ではＰＨＢＸ含有微生物細胞の水性懸濁液の撹拌と物理的破砕処理を行いながら、該水性懸濁液にアルカリを添加することが重要である。物理的破砕を行わず菌体懸濁液にアルカリを添加すると、微生物細胞からＰＨＢＸと一緒に核酸や菌体細胞壁、細胞膜、不溶性蛋白質などが流出する。本発明者らは、この時、特に遊離した核酸によって懸濁液の粘度が激しく上昇し、条件によっては、懸濁液の攪拌さえできなくなり、ＰＨＢＸの回収が不可能となることを経験した。そこで本発明では、物理的破砕を行いながら徐々にアルカリを添加し、不溶物質の可溶化あるいは乳化を進めることによって、ＰＨＢＸが分解されることなく微粒子状態で不溶物質から容易に分離・回収できるようになる。本発明において、物理的破砕処理を行う装置としては、特に限定されないが、高圧ホモジナイザー、超音波破砕機、乳化分散機、ビーズミル等が挙げられる。特に高圧ホモジナイザーの使用が好ましく、例えば、伊国ニロソアビ社製高圧ホモジナイザーが好ましく用いられる。また、ブランリューベ連続式細胞破砕機（独国Ｂｒａｎ＋Ｌｕｅｂｂｅ社製）、マイクロフルイダイザー（米国Ｍｉｃｒｏｆｌｕｉｄｉｃｓ社製）等も用いることができる。このような装置では必要に応じて、一般の低温恒温循環槽により微生物細胞含有懸濁液を冷却し、温度の上昇を防ぎ、２０〜４０℃での破砕処理を行うのが好ましい。このような比較的低温下で処理を行った場合には、ＰＨＢＸの分子量はほとんど低下しないことが本発明者等により確認されている。
【００３６】
本発明のポリマー回収においては、アルカリ添加時にｐＨをコントロールすることが好ましい。ポリマー以外の菌体由来の不溶物を効果的に可溶化でき、かつＰＨＢＸ自体には悪影響をあまり与えない好ましいｐＨの範囲はｐＨ９〜１３．５，さらに好ましくはｐＨ１０〜１３の範囲である。常にある程度以上のアルカリ条件を維持することで懸濁液を高温にする必要がなくなり、結果、ＰＨＢＸの分子量低下を防ぐことが可能となる。
【００３７】
菌体破砕に使用するアルカリは、ＰＨＢＸ含有微生物の細胞壁を破壊して該細胞中のＰＨＢＸを細胞外に流出できるものであれば特に限定されるものではなく、工業生産に適し、また価格の点で、水酸化ナトリウム、炭酸ナトリウム、水酸化カリウム、水酸化リチウムなどが好ましい。
【００３８】
アルカリ条件下での菌体の物理的破砕後に回収したポリマーを酵素及び界面活性化剤のいずれかあるいはこれらを併用して処理することにより純度の高いポリマーを得ることができる。従来もこれらの処理方法は単独で或いは他のＰＨＢＸ回収方法と併用して用いられていたが、あまり良好な結果は得られていない。ＰＨＢＸ粒子には、普通、蛋白質類、菌体細胞壁成分であるペプチドグリカン、脂質類、多糖類、核酸類、その他の炭水化物類が吸着していると考えられる。
【００３９】
工程（ｂ）において酵素による処理を行う場合、使用される酵素の例としては、蛋白質分解酵素、脂肪酸類分解酵素、細胞壁分解酵素、ＤＮＡ分解酵素などが挙げられる。酵素として、ＰＨＢＸに付着した不溶性蛋白質や不溶性のペプチドグリカンを分解除去する目的においては、蛋白質分解酵素と細胞壁分解酵素から選ばれる１種以上が好ましく、蛋白質分解酵素がより好ましい。蛋白質分解酵素としては、特に限定はしないが、プロテアーゼＡ、プロテアーゼＰ、プロテアーゼＮ（以上、天野エンザイム社製）、アルカラーゼ、ザビナーゼ、エバラーゼ（以上、ノボザイム社製）等が工業的に使用可能であり、分解活性の点からも好適に使用できる。また、細胞壁分解酵素ではリゾチームの使用が好ましいが、これに限られるものではない。酵素処理温度は５０℃以下で行なうのが好ましく、特に２０℃〜５０℃が好ましい。酵素処理時間は所要の処理度を達成するまで維持することによって行なうのが好ましく、この時間は通常０．５〜２時間である。酵素の必要量は、酵素の種類及び活性に依存する。特に制限はされないが、ポリマー重量１００重量部に対して、０．００１〜１０重量部が好ましく、さらにはコストの点から５重量部以下がより好ましい。本発明では、ＰＨＢＸ粒子に付着した不純物を除去するために可溶化剤として界面活性化剤を使用することも可能である。本発明で使用する界面活性化剤としては、陰イオン界面活性化剤、陽イオン界面活性化剤、両性界面活性化剤、非イオン界面活性化剤が挙げられる。洗浄性の点で好ましくは陰イオン界面活性化剤及び又は非イオン界面活性化剤である。蛋白質などを洗浄・除去する目的においては、陰イオン界面活性化剤を用いることが好ましく、また、脂肪酸や油脂を洗浄・除去する目的、あるいは、酵素を併用する場合には、非イオン界面活性化剤を用いることが好ましい。また陰イオン界面活性化剤及び非イオン界面活性化剤の両方を含有してもかまわない。好ましい界面活性化剤としては、ドデシル硫酸ナトリウム、ドデシルベンゼンスルホン酸ナトリウム、コール酸ナトリウム、デオキシコール酸ナトリウム、オレイン酸ナトリウム、ポリエチレングリコール、炭素数１０〜１４のポリオキシエチレンアルキルエーテルなどが価格、使用量や添加効果の点で好ましく、またこれらを２種以上併用して用いることも好ましい。界面活性化剤を酵素処理と併用して使用しても良く、市販の酵素配合洗濯用洗剤は界面活性剤と酵素の混合物であることから、これをそのまま使用することもできる。処理温度と時間は、２０〜５０℃で１分〜２時間程度攪拌することが好ましい。酵素／界面活性化剤処理を行った後、水により洗浄を行い、続いて、脱脂・脱臭・脱色のために、親水性溶媒による洗浄を行うのが好ましい。用いられる親水性溶媒としては特に限定されないが、具体的にはメタノール、エタノール、アセトン、アセトニトリル、テトラヒドロフラン、水などが挙げられる。これらの中で、経済的に安価で洗浄効果のあるメタノールとエタノールが特に好ましく、これらを水と混合して使用しても良い。これらの溶媒で洗浄することにより、より純度の向上したＰＨＢＸを単離することができる。このようにして得られたポリマーは適当な分散剤を用いて懸濁させると平均粒径０．７ミクロン程度の微粒子であることがわかる。このように微分散した状態にあるため、ＰＨＢＸとＰＨＢが均質に混合した状態で回収されることになる。得られたポリマーは、この後凝集やペレット化を行っても均質化は保たれており結晶固化速度の低下はみられない。
【００４０】
本発明の樹脂組成物は、各種繊維、糸、ロープ、織物、編物、不織布、紙、フィルム、シート、チューブ、板、棒、容器、袋、部品、発泡体などの形状に成形できる。また、２軸延伸フィルムにも加工できる。成形品は、農業、漁業、林業、園芸、医学、衛生品、衣料、非衣料、包装、その他の分野に好適に用いることができる。
【００４１】
得られたポリマーのモノマーユニットの分析は、例えば、ガスクロマトグラフ法や核磁気共鳴法などにより行う。
【００４２】
以下、実施例により本発明をさらに具体的に説明する。
【００４３】
以下の実施例においては、いずれも共重合ポリエステルとして、ＰＨＢＨを生産した。もちろん本発明はこれら実施例にその技術範囲を限定するものではなく、ＰＨＢＨに限られるものではない。
【００４４】
【実施例】
実施例で行った培養法、精製法、３ＨＨモル分率解析法、粒度測定法、結晶固化速度測定法は以下の通りである。結果はまとめて表１に示した。
【００４５】
（培養法）
Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ−４／ｐＪＲＤＥＥ３２ｄ１３株（Ｔ． Ｆｕｋｕｉ．， Ｙ． Ｄｏｉ．， Ａｐｐｌ Ｍｉｃｒｏｂｉｏｌ Ｂｉｏｔｅｃｈｎｏｌ．， ４９， ３３３−３３６，（１９９８），受託番号ＦＥＲＭ ＢＰ−６０３８）（以下Ｒｅｄ１３株と略す。）を次のように培養した。
【００４６】
種培地の組成は１ｗ／ｖ％ Ｍｅａｔ−ｅｘｔｒａｃｔ、１ｗ／ｖ％ Ｂａｃｔｏ−Ｔｒｙｐｔｏｎ、０．２ｗ／ｖ％Ｙｅａｓｔ−ｅｘｔｒａｃｔ、０．９ｗ／ｖ％Ｎａ２ＰＯ４・１２Ｈ２Ｏ 、０．１５ｗ／ｖ％ＫＨ２ＰＯ４、（ｐＨ６．８）とした。
【００４７】
前培養培地の組成は１．１ｗ／ｖ％ Ｎａ２ＰＯ４・１２Ｈ２Ｏ、０．１９ｗ／ｖ％ ＫＨ２ＰＯ４、１．２９ ｗ／ｖ％（ＮＨ４）２ＳＯ４ 、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、０．５ｖ／ｖ％ 微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの。）、５×１０−６ｗ／ｖ％ カナマイシンとした。炭素源には、ポリエステル生産用に用いる炭素源と同じものを２．５ｗ／ｖ％の濃度で用いた。
【００４８】
ポリエステル生産培地の組成は０．３８５ｗ／ｖ％ Ｎａ２ＰＯ４・１２Ｈ２Ｏ、０．０６７ｗ／ｖ％ ＫＨ２ＰＯ４、０・２９１ｗ／ｖ％（ＮＨ４）２ＳＯ４ 、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、０．５ｖ／ｖ％ 微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの。）、０．０５ｗ／ｖ％ＢＩＯＳＰＵＲＥＸ２００Ｋ（消泡剤：コグニスジャパン製）、５×１０−６ｗ／ｖ％ カナマイシンとした。
【００４９】
Ｒｅｄ１３株のグリセロールストック（５０μｌ）を種培地（１０ｍｌ）に接種して２４時間培養した。３Ｌの前培養培地を入れた５Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＬ−５００型）に０．２ｖ／ｖ％接種した。運転条件は、培養温度３０℃、攪拌速度５００ｒｐｍ、通気量１．８Ｌ／ｍｉｎとし、ｐＨは６．７〜６．８の間でコントロールしながら２８時間培養した。ｐＨコントロールには７％水酸化アンモニウム水溶液を使用した。
【００５０】
ポリエステル生産培養は６Ｌの生産培地を入れた１０Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＬ−１０００型）にそれぞれ前培養種母を１．０ｖ／ｖ％接種した。運転条件は、培養温度２８℃、攪拌速度４００ｒｐｍ、通気量３．６Ｌ／ｍｉｎとし、ｐＨは６．７から６．８の間でコントロールした。ｐＨコントロールには７％水酸化アンモニウム水溶液を使用した。培養槽は培養時間を６０時間で終了するものと、その後炭素源あるいは比基質供給速度の変更を行いさらに２４時間培養する群に分け行った。
【００５１】
（精製法）
本実施例で行った水系法による培養菌体からのＰＨＢＸの回収方法を以下に示した。
【００５２】
培養後のＲ．ｅｕｔｒｏｐｈａを乾燥菌体重量で２０％の水溶性懸濁液とし、４００ｍｌを１Ｌの攪拌槽に入れた。この懸濁液を約１ｋｗ／ｍ３のスピードで攪拌した。続いて、この反応槽を伊国ニロソアビ社製高圧ホモジナイザーモデルＰＡ２Ｋ型と連結し、懸濁液が攪拌槽と破砕機を循環する形にした。懸濁液を６００〜７００ｋｇｆ／ｃｍ２の圧力でホモジナイズし、４００ｍｌ処理後、菌体懸濁液を最終ｐＨ約１２．５になるまで３Ｎの水酸化ナトリウムを徐々に攪拌槽中の懸濁液に添加した。アルカリ添加槽の温度は２５℃〜３５℃に維持した。破砕循環を懸濁液容量で２０回循環させた後、懸濁液を遠心分離（１２０００ｇ、４０ｍｉｎ）してポリマー画分を回収した。これを水４００ｍｌで２回洗浄し、最後に遠心でポリマー画分を回収した。得られたポリマーを水懸濁液４００ｍｌとし、これにＳＤＳをポリマー乾燥重量当たり５重量％、プロテアーゼＮを０．０８重量％、リゾチーム０．０８重量％を添加した。ｐＨ７．０、４０℃、１時間攪拌し、不純物を分解除去した。ポリマーを遠心により回収し、水４００ｍｌで２回洗浄後、エタノール４００ｍｌで２回洗浄し、最後に遠心でポリマー画分を回収した。これを５０℃で真空乾燥した。
【００５３】
（３ＨＨモル分率解析）
得られた乾燥菌体約８ｇに２５０ｍｌのクロロホルムを加え、６０℃で１時間還流し菌体内のＰＨＢＨを抽出した。菌体残渣をろ別後、エバポレーターで総容量が約１００ｍｌになるまで濃縮した。この溶液をゆっくり攪拌しながらヘプタンを最初の沈殿が現れるまで徐々に加えた。最初に析出するポリマーがＰＨＢあるいは最も低い３ＨＨモル分率を持つＰＨＢＨであるため、最初の沈殿が現れた段階でヘプタンの添加を止め、５時間軽く攪拌しながら放置した。析出した最初の沈殿を２００００ｇ、１０分の遠心で回収した。回収した沈殿は５０℃で真空乾燥した。得られたポリマーについて重量を測定し、含量を算出した。また、３ＨＨモル分率は特開平２００１−３４００７８の実施例１に記載の方法で行った。すなわち、得られた乾燥ポリマー約２０ｍｇに２ｍｌの硫酸−メタノール混液（１５：８５）と２ｍｌのクロロホルムを添加して密栓し、１００℃で１４０分間加熱することでポリエステル分解物のメチルエステルを得た。冷却後、これに１．５ｇの炭酸水素ナトリウムを少しずつを加えて中和し、炭酸ガスの発生がとまるまで放置した。４ｍｌのジイソプロピルエーテルを添加してよく混合した後、遠心して、上清中の３−ヒドロキシブタン酸メチルエステルと３−ヒドロキシヘキサン酸メチルエステルの組成をキャピラリーガスクロマトグラフィーにより分析し、ポリマー中の両モノマーユニットの組成比を算出した。ガスクロマトグラフは島津製作所ＧＣ−１７Ａ、キャピラリーカラムはＧＬサイエンス社製ＮＥＵＴＲＡ ＢＯＮＤ−１（カラム長２５ｍ、カラム内径０．２５ｍｍ、液膜厚０．４μｍ）を用いた。温度条件は、初発温度１００〜２００℃まで８℃／分の速度で昇温、さらに２００〜２９０℃まで３０℃／分の速度で昇温した。
【００５４】
（粒度の測定）
ＰＨＡ粒子の平均粒径は、マイクロトラック粒度計（日機装製、ＦＲＡ）を用いた。ＰＨＢＨを花王社製分散剤デモールに懸濁後、所定濃度に調整し、全粒子の５０％蓄積量に対応する粒径を平均粒径とした。
【００５５】
（結晶固化性の検討）
キャピログラフ（東洋精機製作所製）を用い、１ｍｍφ×１０ｍｍのダイスを使用して、押出温度を１４０及び１５０℃にて実施した。剪断速度１２２ｓｅｃ−１、降下速度１０ｍｍ／ｍｉｎにて樹脂組成物を溶融押出し、押出ストランドが押出吐出口から結晶固化するまでの距離及び時間で、結晶固化性の評価をした。
【００５６】
◎：押出ストランドが、押出吐出口から出てきた時点で、結晶固化する
○：押出吐出口から結晶固化するまでの距離が、０〜約２０ｃｍである
△：押出吐出口から結晶固化するまでの距離が、約２０〜５０ｃｍである
×：押出吐出口から結晶固化するまでの距離が、約５０ｃｍ以上で、かつ、固化時間が、１０分間以上である。
【００５７】
（実施例１）
２基の培養槽を用いて菌体を培養した。パーム核オレインを比基質供給速度０．０６（ｇ−油脂）×（ｇ−正味乾燥菌体重量）−１×（ｈ）−１となるように流加した。６０時間経過後１基は培養を停止し、２基目にはさらに４０％ｗ／ｖフラクトースを２４時間で９７０ｍｌ（４０．６ｍｌ／時間）添加することにより培養を継続した。得られたＰＨＢＸの平均粒径はどちらも０．７ミクロンと微粒子のまま回収されていることが確認できた。３ＨＨモル分率特性、結晶固化性評価を表１に示す。パーム核オレインのみで培養して得られたＰＨＢＸは１４０及び１５０℃における、押出後のストランドはひどく粘着性を帯び、かつ、結晶固化速度は著しく遅く、押出直後から１０分以上の結晶固化時間を要した。フラクトースで継続培養して得られたＰＨＢＸにはＰＨＢが含まれていることが明らかになった。このＰＨＢＨの結晶固化速度は著しく速くなった。
【００５８】
【表１】
パーム核オレインの代わりにヤシ油、フラクトースの代わりに大豆油とコーン油を用いた以外は、実施例１と同様に培養・精製操作を行いＰＨＢＸを得た。得られたＰＨＢＨの３ＨＨモル分率特性、結晶固化性評価を表１に示す。ヤシ油のみで培養して得られたＰＨＢＨは１４０及び１５０℃における、押出後のストランドはひどく粘着性を帯び、かつ、結晶固化速度は著しく遅く、押出直後から１０分以上の結晶固化時間を要した。大豆油とコーン油で継続培養して得られたＰＨＢＨには低３ＨＨ−ＰＨＢＨが含まれていることが明らかになった。１４０及び１５０℃における、結晶固化速度は著しく速くなった。
【００５９】
（実施例３）
パーム核油オレインの代わりに大豆油を用い比基質供給速度０．０６で培養した以外は実施例１と同様に培養・精製操作を行いＰＨＢＨを得た。大豆油のみで培養して得られたＰＨＢＨは、１４０及び１５０℃における押出後のストランドが粘着性を帯び、かつ、結晶固化速度は遅く、押出直後から５分以上の結晶固化時間を要した。フラクトースで継続培養して得られたＰＨＢＨにはＰＨＢが含まれていることが明らかになり、このポリマーの１４０及び１５０℃における、結晶固化速度は著しく速くなった。
【００６０】
（実施例４）
フラクトースの代わりに、パーム核オレイン油を炭素源に用い、最初比基質供給速度を０．０６で培養し、培養５０時間後から０．１２に変更して培養を継続した以外は、実施例１と同様に培養・精製操作を行いＰＨＢＸを得た。比基質供給速度０．０６で培養した時は高３ＨＨモル分率のＰＨＢＸであったが、０．１２に変更したＰＨＢＸには低３ＨＨ−ＰＨＢＨが含まれていることが明らかになった。１４０及び１５０℃における、このポリマーの結晶固化速度は著しく速くなった。
【００６１】
（実施例５）
大豆油の比基質供給速度を５０時間後以降、０．０６から、０．１２へ上げて培養した以外は、実施例１と同様に培養・精製操作を行いＰＨＢＸを得た。比基質供給速度が０．０６のまま培養後得られたＰＨＢＸは中程度の３ＨＨモル分率をもつＰＨＢＨが得られ、１４０及び１５０℃における、押出後のストランドは粘着性を帯び、かつ、結晶固化速度は遅く、押出直後から５分以上の結晶固化時間を要した。比基質供給速度が０．１２に変更することで得られたＰＨＢＨにはより低３ＨＨモル分率のＰＨＢＨが合成されており、１４０及び１５０℃における結晶固化速度は著しく速くなった。
【００６２】
【発明の効果】
本発明の方法により、結晶化速度の著しく向上したポリヒドロキシアルカノエートを、工業的に安価に生産、提供できるようになる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a polyhydroxyalkanoate having a high solidification rate.
[0002]
[Prior art]
Polyhydroxyalkanoate (hereinafter abbreviated as PHBX) is a thermoplastic polyester produced and accumulated as an energy storage substance in cells of many microbial species. PHBX produced by microorganisms using natural organic acids and oils and fats as a carbon source is completely biodegraded by microorganisms in soil and water, and thus is taken into the natural carbon cycle process. Therefore, it can be said that PHBX is an environmentally friendly plastic material that has almost no adverse effect on ecosystems. In recent years, synthetic plastics have become a serious social problem from the viewpoints of environmental pollution, waste treatment and petroleum resources, and PHBX has been attracting attention as an environmentally friendly green plastic, and its practical application has been eagerly desired.
PHBX first discovered in microorganisms is poly-3-hydroxybutyrate (hereinafter abbreviated as PHB), which is a homopolymer of 3-hydroxybutyrate. PHB is hard and brittle due to its high crystallinity and high crystallinity, and has the disadvantages of physical properties such as low melt processability because it rapidly decomposes at a temperature near the melting point (180 ° C.). It is a material that is difficult to put into practical use.
[0003]
Therefore, in order to reduce the crystallinity of PHB and improve brittleness, an attempt has been made to introduce another 3-hydroxyalkanoic acid (hereinafter abbreviated as 3HX) into the PHB skeleton. Copolymer P (3HB-co-3HV) (hereinafter abbreviated as PHBV) obtained by using 3-hydroxyvaleric acid (hereinafter abbreviated as 3HV) as such 3HX (Patent Documents 1 to 3) Has a higher melting point than PHB while maintaining a high solidification rate over a wide 3HV composition range, and is therefore superior in melt processability to PHB having high melting point and easy thermal decomposition. This copolymer has attracted attention as a highly practical polymer material, and was temporarily sold under the name Biopol by Imperial Chemical Industries. However, since the crystallinity of PHBV did not decrease to 50% or less, brittleness could not be improved.
[0004]
Therefore, in order to further improve the brittleness of PHBV, a study was conducted on PHBX in which a hydroxy acid having a side chain larger than 3 HV was introduced into the PHB skeleton. For example, 3-hydroxypropionic acid (hereinafter abbreviated as 3HP), 3-hydroxypentanoic acid (hereinafter abbreviated as 3HC), 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH), 3-hydroxyoctanoic acid (hereinafter abbreviated as 3HO) ), 3-hydroxynonanoic acid (hereinafter abbreviated as 3HN), 3-hydroxydecanoic acid (hereinafter abbreviated as 3HD), 3-hydroxydodecanoic acid (hereinafter abbreviated as 3HDD) and the like as 3HX Has been energetically studied (Non-Patent Document 1). Since these have pentyl or alkyl chains longer than 3 HV, PHBX having these introduced into the skeleton becomes substantially amorphous. It was expected that the properties of these polymers would be very supple and soft, so that the brittleness would be improved, and that they had a low melting point (80 ° C. to 150 ° C.), and that the melt processability would also be improved. However, these polymers have a very low solidification rate, and after heating and melting, they are soft and viscous for a long time even at room temperature after cooling, and because they are sticky, they cannot be taken out of the mold immediately during molding. A new problem arises that actual production cannot be performed continuously. In addition, it became clear that processing equipment used for processing existing general-purpose resins having a high crystallization solidification rate cannot be used for processing biodegradable plastics. In the processing of films, sheets, fibers, foams, molded products, and nonwoven fabrics, the rapid solidification rate of the polymer melt-processed during the production process after cooling makes the production process continuous, thus improving productivity and reducing productivity. Very important because it leads to cost.
[0005]
Therefore, attempts have been made to increase the crystal solidification rate of PHBX. As a general method therefor, a method of adding a crystal nucleating agent has been attempted. For example, in Patent Literature 4, boron nitride is used as a crystal nucleating agent in PHBH to obtain a crystallization promoting effect. However, this is an expensive material and has no biodegradability. For this reason, a less expensive and biodegradable crystal nucleating agent has been studied. Patent Literatures 5 and 6 disclose a technique in which PHB, which has a higher melting temperature than PHBV or PHBH and is biodegradable, is added as a crystal nucleating agent to increase the crystal solidification rate. In these prior arts, as a method of mixing PHBX and PHB, for example, a method of dissolving and mixing PHBX and PHB in hot chloroform and then evaporating chloroform to precipitate a polymer or crystallizing with methanol to recover a mixed polymer, or Attempts have been made to pulverize and mix the two types of polymers while cooling them with dry ice, to mix only PHBX without melting PHB, or to mix by mixing dry polymer powder. However, the method of dissolving and mixing in a solvent requires a very large amount of solvent for dissolving and crystallizing PHBX, which increases the cost. In addition, there is a possibility that crystallization is not performed in a uniformly dispersed state due to a difference in solubility between the polymer and the crystal nucleating agent during crystallization, which is not practical. It is difficult to uniformly mix the polymer by a method of mixing the polymer after pulverization or mixing of the dry powder, and the effect of the crystal nucleating agent is reduced. In particular, the smaller the particle system of PHBX and the crystal nucleating agent, the better the mixing and the higher the number of nucleation sites, so that a high effect can be expected. However, the mixing method using fine particles cannot be expected with the above mixing method. To date, there has been no technique for mixing such fine particles and adjusting the crystallization solidification rate.
[0006]
[Patent Document 1]
JP-A-57-150393
[0007]
[Patent Document 2]
JP-A-59-220192
[0008]
[Patent Document 3]
Japanese Patent Publication No. 11-500008
[0009]
[Patent Document 4]
JP-A-6-157778
[0010]
[Patent Document 5]
Japanese Patent Publication No. Hei 8-510498
[0011]
[Patent Document 6]
WO 02/50156
[0012]
[Non-patent document 1]
Poirier Y. , Nawrath C .; , Somerville C, BIO / TECHNOLOGY, 13, 142-150, 1995
[0013]
[Problems to be solved by the invention]
As described above, PHB is effective as a crystal nucleating agent for improving the crystal solidification rate of PHBX, but it is difficult to say that the effect of PHB is sufficiently exerted due to a difficulty in a method of mixing the two. Therefore, an object of the present study is to solve the above-mentioned problems in the prior art, and to provide a method for producing a mixture of PHBX and a crystal nucleating agent in fine particles which could not be obtained by the conventional method. Furthermore, the present invention increases the crystal solidification rate of the PHBH resin to prevent fusion, improve releasability, and improve molding cycle time when using molding methods such as film molding, injection molding, extrusion molding, and spinning. It is an object of the present invention to provide a method for producing a biodegradable polyester-based resin composition having improved moldability, such as shortening of resin composition and continuous resin pelletization.
[0014]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above-mentioned problem. As a result, in a PHBX composed of 3HB and 3HX other than 3HB, the 3HX molar fraction in PHBX is 6% or more, and 3HX is contained in the polymer. By constructing a method of obtaining PHBX or a mixture of PHBX and PHB containing 0.1 to 30% of PHBX or PHB homopolymer having a mole fraction of 5% or less from a microorganism in a fine particle state, PHBX having a high crystallization solidification rate is obtained. And succeeded in completing the present invention.
[0015]
That is, the present invention is a method for producing a polyhydroxyalkanoate having an improved crystallization solidification rate by a microorganism, wherein the type of carbon source supplied during the culture of the microorganism is changed, or the carbon source is supplied during the culture. The present invention relates to a method for producing a polyhydroxyalkanoate, which comprises synthesizing a polyhydroxyalkanoate serving as a crystal nucleating agent by changing a specific substrate supply rate.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
PHBX in the present invention is a copolymerized polyester of 3HB and two or more other 3HXs. Here, as a copolymer with 3HB, 3-hydroxypropionic acid (hereinafter abbreviated as 3HP) and 3HX are 3-hydroxypentanoic acid (hereinafter abbreviated as 3HC) and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH). 3-hydroxyoctanoic acid (hereinafter abbreviated as 3HO), 3-hydroxynonanoic acid (hereinafter abbreviated as 3HN), 3-hydroxydecanoic acid (hereinafter abbreviated as 3HD), 3-hydroxydodecanoic acid (hereinafter abbreviated as 3HDD) Among them, a copolymer containing 3HH is preferable, and PHBH is more preferable, in particular, a polyester having a content of 3HX of 8% or more, in which the properties of the polyester vary widely. As a copolymer containing three units, 3HB-3HV-3HH is preferable.
[0017]
These copolymers PHBX contain a certain amount of a low 3HX molar fraction 3HB copolymer (hereinafter abbreviated as low 3HX-PHBX) or a certain amount of a 3HB homopolymer (PHB) as a nucleating agent. Thus, it is a polymer in which the crystal solidification rate of PHBX as a whole is increased using the high solidification rate of 3HB.
[0018]
PHBX having a high crystallization solidification rate means that the 3HX mole fraction in PHBX is preferably 6% or more, more preferably 7% or more, and particularly preferably, in that PHBX becomes more flexible. Is 8% or more.
[0019]
The 3HX content in the low 3HX-PHBX contained in the PHBX is preferably lower than 5% by mole, more preferably 4% by mole or less, and still more preferably 3% by mole, from the viewpoint of increasing the crystal solidification rate. % Or less, particularly preferably 0% homopolymer PHB.
[0020]
The content of these low 3HX-PHBX or PHB in PHBX is preferably 0.01 to 30% by weight, more preferably 0.5 to 20% by weight, and still more preferably 0.1 to 30% by weight in terms of increasing the crystal solidification rate. 5-5% by weight.
[0021]
In the production method of the present invention, the microorganism to be used is not particularly limited, and a microorganism isolated from nature or a microorganism deposited at a depository of a strain (eg, IFO, ATCC, etc.) can be used. However, for the purpose of the present invention, a strain having the ability to produce a homopolymer having a high crystallization rate such as PHB together with the ability to produce a copolymer is desirable, and in this regard, Alcaligenes, Ralstonia, Aeromonas, and Pseudomonas are preferred. Preferred, but more preferred are the genus Alcaligenes and the genus Ralstonia.
[0022]
In addition, when the microorganism cannot produce the target copolymer in a wild-type state, or when the production amount is low, the microorganism is introduced with a gene encoding a copolyester of the target copolymerized polyester to form a trait. The transformed microorganism obtained after the transformation can be used. When preparing a transformed microorganism, a general method such as utilizing a recombinant vector containing a polyester synthase gene can be used, and the vector may be a plasmid vector that can grow autonomously in the cells. Can be used. Further, the polyester synthase gene may be directly integrated into the chromosome of the host.
[0023]
The polyester synthase gene used in the production of the copolymerized polyester of the present invention is not particularly limited, but is preferably a gene isolated from Aeromonas caviae, for example, described in JP-A-7-265065. Gene fragments that have been used can be used. In order to introduce a recombinant vector into a microorganism, a method known to those skilled in the art can be used. As an example of the microorganism used in the present invention, Ralstonia eutropha (Alcaligenes eutrophus AC32 strain, deposited as a strain of Alcaligenes eutrophus AC32 (Deposit number: PFM-B38-FERM), which is obtained by introducing a polyester synthase gene derived from Aeromonas caviae into Ralstonia eutropha, a non-PHB-producing strain. T. Fukui., Y. Doi., Appl Microbiol Biotechnol., 49, 333-336, 1998) can be preferably used. Such cells are preferable in that the growth rate is high and PHBH is efficiently produced using oils and fats as a carbon source. It is also known that PHB can be produced when fructose is used as a carbon source, and can be suitably used in the present invention. In addition, glucose-assimilating strains are widely used for Ralstonia eutropha (ATCC No. 17699, J General Microbiology 115, 185-192, 1979), and these can also be used.
[0024]
The PHBX having a high crystallization solidification rate of the present invention can be obtained by using the above-mentioned cells and changing the carbon source continuously or stepwise during the culture, or by changing the carbon source to another one. . Fats and oils used as a carbon source in the present invention can be obtained by fractionating relatively stably supplied fats and oils such as soybean oil, corn oil, cottonseed oil, palm oil, palm kernel oil, coconut oil, peanut oil and the like. Fractions such as palm W olein oil (low melting point fraction obtained by separating palm oil twice without solvent) and palm kernel oil olein (low melting point fraction obtained by once separating palm kernel oil without solvent) Alternatively, synthetic oils obtained by chemically or biochemically treating these fats and oils and their fractions, and mixed oils obtained by mixing these can be used. Among them, it is preferable to use natural fats and oils from the viewpoint of cost. To explain the case of PHBH more specifically, it is preferable to use an oil or fat containing lauric acid as a constituent fatty acid of the oil or fat, or an oil or fat commonly called laurin oil or fat. For example, when lauric fat is used, PHBH having a relatively high 3HH content up to 20% molar fraction can be obtained. Examples of lauric fats and oils as constituent fatty acids include natural fats and oils such as palm kernel oil and coconut oil, fractionated fats and oils such as palm kernel oil olein, and mixed oils containing these lauric fats. Of these, palm kernel oil and palm kernel oil olein are particularly preferred in terms of achieving a 3HH molar fraction of 8% or more.
[0025]
On the other hand, when soybean oil, corn oil, cottonseed oil, palm oil, peanut oil, or a fractionated oil thereof is used, PHBH having a relatively low 3HH content of 1 to 10% by mole can be obtained. When these are used for synthesizing low 3HX-PHBX for a crystal nucleating agent, use of soybean oil, corn oil, and cottonseed oil is preferable in synthesizing 3HX-PHBX having a mole fraction of 5% or less.
[0026]
In addition, when synthesizing a homopolymer PHB having the highest effect as a crystal nucleating agent, sugar may be generally used. As the sugar, glucose, fructose, sucrose and molasses can be suitably used. Above all, glucose is a preferable substrate in terms of low cost and effective propagation of microorganisms.
[0027]
In a preferred embodiment of the present invention, when PHBX having a 3HX composition of 10% mol fraction or more is produced using lauric fat as an initial carbon source, in order to increase the crystallization solidification rate, a carbon source must be added during the culturing. By changing soybean oil, corn oil, cottonseed oil, palm oil, peanut oil, or fractionated oils and fats thereof, PHBX containing 3HX lower than 5% by mole can be produced. Alternatively, PHB can be produced by changing the carbon source to a carbohydrate other than fats and oils and fatty acids, for example, sugar and starch.
[0028]
When soybean oil, corn oil, cottonseed oil, palm oil, peanut oil, or their fractionated fats and oils are used as the first carbon source, PHBX having a molar fraction of 10% or less is produced. It is desirable to produce PHB by converting glycerol into a carbohydrate other than fats and oils and fatty acids, such as sugar and starch.
[0029]
The present inventors have studied the optimal method for adding fats and oils as a carbon source, and as a result, it is clear that the 3HX mole fraction of PHBX produced can be controlled by adjusting the specific substrate supply rate of the fats and fats. It became. Here, the specific substrate supply rate is a culture variable defined as the amount of fats and oils supplied per unit cell weight of the net cell weight per unit time, and the net cell weight is the weight of the polyester contained from the total cell weight. This is the cell weight after subtraction (dry cell weight).
[0030]
[0031]
In the present invention, it has been found that the lower the specific substrate supply rate, the higher the 3HX content of PHBX. Therefore, when it is desired to obtain PHBX having a high 3HX content, the culture may be performed with the specific substrate supply rate set low. For example, when producing PHBX having a 3HX molar fraction of 10% or more, the specific substrate supply rate is preferably 0.10 or less, more preferably 0.09 or less, and further more preferably 0.06 or less. When it is desired to obtain PHBX having a 3HX molar fraction of 5% or less, the specific substrate supply rate is preferably larger than 0.10, and more preferably 0.12 or more.
[0032]
When the specific substrate supply rate is changed depending on the culture phase, it is desirable to change the specific substrate supply rate at an appropriate time during the polymer accumulation phase in the latter half of the culture. The specific substrate supply rate can be changed from a high value to a low value in a reverse manner, but both methods can be used.
[0033]
In the present invention, when controlling the specific substrate supply rate of fats and oils, the entire culture period is divided into two phases of a cell growth phase and a polyester accumulation phase, and a different specific substrate supply rate value is set in each phase. During the whole period of the culture, the specific substrate supply rate is controlled to be constant, or the specific substrate supply rate is continuously changed during the entire culture period. There is a method of changing the substrate supply rate, and any method may be used. In order to control the specific substrate supply rate of fats and oils to a constant value within a specific period of culture, the amount of fats and oils added is continuously or intermittently changed so as to satisfy the set specific substrate supply rate. Need to be done. In order to continuously change the added amount of fats and oils, for example, a fed amount can be controlled by using a computer. In the case of intermittent (that is, stepwise) change, automatic control can be performed, but it is simple in that the feed rate operation can be performed manually. In the case of intermittent change, strictly, it does not always completely satisfy the value of the specific substrate supply speed set in advance, but it is sufficient if the value of the specific substrate supply speed set as the average value is satisfied. . As described above, by controlling the specific substrate supply rate of the fat or oil to be a set constant value during the whole period of the culture or within a specific phase of the culture, a predetermined amount of the fat or oil can be completely removed at the beginning of the culture. Copolyesters that were insufficient with conventional cultivation methods such as adding, feeding and culturing with a fixed amount of fat or oil during the culturing period, or changing the amount of fat or oil empirically irrespective of the specific substrate supply rate For the first time, and control of the monomer unit composition ratio is achieved for the first time. In the present specification, the entire culture period is the entire period from the start of culture to the end of culture in main culture.
[0034]
Examples of the nitrogen source used in the cell culture of the present invention include inorganic nitrogen sources which are ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, and organic nitrogen sources such as peptone, meat extract and yeast extract. . As the inorganic salts, for example, potassium monophosphate, potassium diphosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like are used. As other organic nutrients, amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C are used. However, the use of organic nutrients such as peptone, meat extract, yeast extract, and amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C are the most desirable from the viewpoint of controlling production costs. It is preferable to use a small amount.
[0035]
In the present invention, the method of recovering PHBX from microbial cells is not particularly limited, and a known solvent extraction method, physical crushing method, chemical treatment, or the like can be employed. However, since PHBX is usually synthesized as microparticles having a particle size of 1 micron or less in microorganisms, it is necessary to take out the polymer in a fine particle state in order to obtain PHBX and a crystal nucleating agent in a uniformly finely dispersed state. . As a result, the crystallization speed of the obtained polymer is significantly improved. Further, unevenness and unevenness in strength caused by inhomogeneity of the crystal nucleating agent during processing into a film or the like can be eliminated. In a preferred embodiment of the polymer recovery according to the present invention, (a) an alkali is added to the aqueous suspension of PHBX-containing microbial cells while performing stirring and physical crushing treatment to crush the cells, A step of solubilizing or emulsifying cellular substances other than PHBX, and then separating PHBX from the suspension; and (b) treating the separated PHBX with an enzyme and / or a surfactant to remove impurities attached to the PHBX. Solubilization or decomposition followed by solubilization, followed by washing the PHBX with a hydrophilic solvent. In (a), it is important to add an alkali to the aqueous suspension of the PHBX-containing microbial cells while stirring and physically disrupting the aqueous suspension. When an alkali is added to the cell suspension without physical disruption, nucleic acids, cell walls, cell membranes, insoluble proteins, etc., flow out of the microbial cells together with PHBX. At this time, the present inventors have experienced that the viscosity of the suspension was drastically increased, particularly due to the released nucleic acid, and under some conditions, the suspension could not even be stirred, and the PHBX could not be recovered. Therefore, in the present invention, by gradually adding alkali while performing physical crushing and promoting solubilization or emulsification of the insoluble substance, PHBX can be easily separated and recovered from the insoluble substance in a fine particle state without being decomposed. become. In the present invention, the apparatus for performing the physical crushing treatment is not particularly limited, and examples thereof include a high-pressure homogenizer, an ultrasonic crusher, an emulsifying and dispersing machine, and a bead mill. In particular, the use of a high-pressure homogenizer is preferable, and for example, a high-pressure homogenizer manufactured by Nichiso Nishiso Aviation Co., Ltd. is preferably used. In addition, a Bran-Lube continuous cell disrupter (manufactured by Bran + Luebbe, Germany), a microfluidizer (manufactured by Microfluidics, USA) and the like can also be used. In such an apparatus, if necessary, it is preferable to cool the suspension containing microbial cells by a general low-temperature constant-temperature circulating tank to prevent the temperature from rising, and to perform a crushing treatment at 20 to 40 ° C. It has been confirmed by the present inventors that the molecular weight of PHBX hardly decreases when the treatment is performed at such a relatively low temperature.
[0036]
In the polymer recovery of the present invention, it is preferable to control the pH at the time of adding the alkali. A preferred pH range that can effectively solubilize insolubles derived from bacterial cells other than the polymer and does not significantly affect PHBX itself is pH 9 to 13.5, and more preferably pH 10 to 13. It is not necessary to keep the suspension at a high temperature by always maintaining the alkaline conditions to a certain degree or more. As a result, it is possible to prevent a decrease in the molecular weight of PHBX.
[0037]
The alkali used for crushing the cells is not particularly limited as long as it can break the cell wall of the PHBX-containing microorganism and allow PHBX in the cells to flow out of the cell, and is suitable for industrial production and in terms of price. Preferred are sodium hydroxide, sodium carbonate, potassium hydroxide, lithium hydroxide and the like.
[0038]
A polymer having high purity can be obtained by treating the polymer recovered after physical disruption of the cells under alkaline conditions with either an enzyme or a surfactant or a combination thereof. Conventionally, these treatment methods have been used alone or in combination with other PHBX recovery methods, but have not obtained very good results. It is generally considered that proteins, peptidoglycans, lipids, polysaccharides, nucleic acids, and other carbohydrates, which are cell wall components, are adsorbed on PHBX particles.
[0039]
When the treatment with an enzyme is performed in step (b), examples of the enzyme used include a protease, a fatty acid-decomposing enzyme, a cell wall-degrading enzyme, and a DNA-degrading enzyme. For the purpose of decomposing and removing insoluble proteins and insoluble peptidoglycan attached to PHBX, one or more enzymes selected from proteolytic enzymes and cell wall degrading enzymes are preferable, and proteolytic enzymes are more preferable. Proteolytic enzymes are not particularly limited, but protease A, protease P, protease N (manufactured by Amano Enzyme Co., Ltd.), alcalase, savinase, evalase (manufactured by Novozyme) and the like can be used industrially. It can be used preferably also from the viewpoint of decomposition activity. In addition, although lysozyme is preferably used as a cell wall degrading enzyme, it is not limited thereto. The enzyme treatment is preferably performed at a temperature of 50 ° C. or lower, particularly preferably 20 ° C. to 50 ° C. The enzyme treatment is preferably performed by maintaining the treatment time until a required treatment degree is achieved, and this time is usually 0.5 to 2 hours. The required amount of the enzyme depends on the type and activity of the enzyme. Although not particularly limited, it is preferably 0.001 to 10 parts by weight, more preferably 5 parts by weight or less from the viewpoint of cost, based on 100 parts by weight of the polymer. In the present invention, it is also possible to use a surfactant as a solubilizing agent to remove impurities attached to PHBX particles. The surfactant used in the present invention includes an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant. Anionic surfactants and / or nonionic surfactants are preferred from the viewpoint of detergency. For the purpose of washing and removing proteins and the like, it is preferable to use an anionic surfactant, and for the purpose of washing and removing fatty acids and fats or when using an enzyme in combination, it is preferable to use a nonionic surfactant. It is preferable to use an agent. Further, both an anionic surfactant and a nonionic surfactant may be contained. Preferred surfactants include sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium cholate, sodium deoxycholate, sodium oleate, polyethylene glycol, and polyoxyethylene alkyl ethers having 10 to 14 carbon atoms. It is preferable in terms of the amount and the effect of addition, and it is also preferable to use two or more of these in combination. A surfactant may be used in combination with the enzyme treatment, and since a commercially available enzyme-containing laundry detergent is a mixture of a surfactant and an enzyme, it can be used as it is. The treatment temperature and time are preferably stirred at 20 to 50 ° C. for about 1 minute to 2 hours. After performing the enzyme / surfactant treatment, it is preferable to wash with water, and then to wash with a hydrophilic solvent for degreasing, deodorizing, and decoloring. The hydrophilic solvent used is not particularly limited, but specific examples include methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, water and the like. Of these, methanol and ethanol, which are economically inexpensive and have a cleaning effect, are particularly preferable, and they may be used by mixing with water. By washing with these solvents, PHBX with higher purity can be isolated. It can be seen that the polymer thus obtained is fine particles having an average particle size of about 0.7 μm when suspended using a suitable dispersant. Since it is in a finely dispersed state, PHBX and PHB are collected in a homogeneously mixed state. The homogeneity of the obtained polymer is maintained even after subsequent aggregation or pelletization, and no decrease in the crystal solidification rate is observed.
[0040]
The resin composition of the present invention can be formed into various fibers, yarns, ropes, woven fabrics, knitted fabrics, nonwoven fabrics, papers, films, sheets, tubes, plates, bars, containers, bags, parts, foams, and the like. It can also be processed into a biaxially stretched film. The molded article can be suitably used in agriculture, fishing, forestry, horticulture, medicine, sanitary goods, clothing, non-clothing, packaging, and other fields.
[0041]
The analysis of the monomer unit of the obtained polymer is performed by, for example, a gas chromatography method or a nuclear magnetic resonance method.
[0042]
Hereinafter, the present invention will be described more specifically with reference to examples.
[0043]
In the following examples, PHBH was produced as a copolymerized polyester. Of course, the present invention does not limit the technical scope to these embodiments, and is not limited to PHBH.
[0044]
【Example】
The culture method, purification method, 3HH mole fraction analysis method, particle size measurement method, and crystal solidification rate measurement method performed in the examples are as follows. The results are summarized in Table 1.
[0045]
(Culture method)
Ralstonia eutropha PHB-4 / pJRDEE32d13 strain (T. Fukui., Y. Doi., Appl Microbiol Biotechnol., 49, 333-336, (1998); Culture was performed as follows.
[0046]
The composition of the seed medium is 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / v% Na 2 PO 4 ・ 12H 2 O 2, 0.15 w / v% KH 2 PO 4 , (PH 6.8).
[0047]
The composition of the preculture medium is 1.1 w / v% Na 2 PO 4 ・ 12H 2 O, 0.19 w / v% KH 2 PO 4 , 1.29 w / v% (NH 4 ) 2 SO 4 , 0.1w / v% MgSO 4 -7H2O, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid) 3 ・ 6H 2 O, 1w / v% CaCl 2 ・ 2H 2 O, 0.02 w / v% CoCl 2 ・ 6H 2 O, 0.016 w / v% CuSO 4 ・ 5H 2 O, 0.012 w / v% NiCl 2 ・ 6H 2 What melted O. ) 5 × 10 −6 w / v% kanamycin. As the carbon source, the same carbon source used for the production of polyester was used at a concentration of 2.5 w / v%.
[0048]
The composition of the polyester production medium is 0.385 w / v% Na 2 PO 4 ・ 12H 2 O, 0.067 w / v% KH 2 PO 4 , 0.129 w / v% (NH 4 ) 2 SO 4 , 0.1w / v% MgSO 4 ・ 7H 2 O, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid) 3 ・ 6H 2 O, 1w / v% CaCl 2 ・ 2H 2 O, 0.02 w / v% CoCl 2 ・ 6H 2 O, 0.016 w / v% CuSO 4 ・ 5H 2 O, 0.012 w / v% NiCl 2 ・ 6H 2 What melted O. ), 0.05 w / v% BIOSPUREX200K (antifoaming agent: manufactured by Cognis Japan), 5 × 10 −6 w / v% kanamycin.
[0049]
A glycerol stock of Red 13 strain (50 μl) was inoculated into a seed medium (10 ml) and cultured for 24 hours. 0.2 v / v% was inoculated into a 5 L jar fermenter (MDL-500, manufactured by Marubishi Bioengine) containing 3 L of the preculture medium. The operating conditions were a culture temperature of 30 ° C., a stirring speed of 500 rpm, an aeration rate of 1.8 L / min, and culture for 28 hours while controlling the pH between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control.
[0050]
For polyester production culture, a 10 L jar fermenter (MDL-1000, manufactured by Marubishi Bioengine) containing 6 L of a production medium was inoculated with 1.0 v / v% of the precultured seed. The operating conditions were a culture temperature of 28 ° C., a stirring speed of 400 rpm, an aeration rate of 3.6 L / min, and the pH was controlled between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control. The culturing tanks were divided into groups in which the culturing time was completed in 60 hours, and those in which the carbon source or the specific substrate supply rate was changed after that, and the cells were further cultured for 24 hours.
[0051]
(Purification method)
The method for recovering PHBX from cultured cells by the aqueous method performed in this example is described below.
[0052]
R. after culture Eutropha was made into a 20% aqueous suspension by weight of the dry cells, and 400 ml of the suspension was put into a 1 L stirring tank. About 1 kw / m 3 At a speed of. Subsequently, this reaction tank was connected to a high-pressure homogenizer model PA2K model manufactured by Niroso Aviation Incorporated, and the suspension was circulated through a stirring tank and a crusher. 600-700 kgf / cm of suspension 2 After treatment with 400 ml, the bacterial cell suspension was gradually added with 3N sodium hydroxide to the suspension in the stirring tank until the final pH was about 12.5. The temperature of the alkali addition tank was maintained at 25 ° C to 35 ° C. After circulating the crushing circulation 20 times in the suspension volume, the suspension was centrifuged (12000 g, 40 min) to collect a polymer fraction. This was washed twice with 400 ml of water, and finally the polymer fraction was collected by centrifugation. The obtained polymer was made into 400 ml of a water suspension, and 5% by weight of SDS, 0.08% by weight of protease N, and 0.08% by weight of lysozyme were added to the dry weight of the polymer. The mixture was stirred at pH 7.0 and 40 ° C. for 1 hour to decompose and remove impurities. The polymer was collected by centrifugation, washed twice with 400 ml of water, twice with 400 ml of ethanol, and finally, a polymer fraction was collected by centrifugation. This was vacuum dried at 50 ° C.
[0053]
(3HH mole fraction analysis)
250 ml of chloroform was added to about 8 g of the obtained dried cells, and the mixture was refluxed at 60 ° C. for 1 hour to extract PHBH in the cells. After the cell residue was filtered off, it was concentrated by an evaporator until the total volume became about 100 ml. While slowly stirring the solution, heptane was added slowly until the first precipitate appeared. Since the polymer precipitated first is PHB or PHBH having the lowest 3HH mole fraction, the addition of heptane was stopped when the first precipitate appeared, and the mixture was left under light stirring for 5 hours. The first precipitate that precipitated was collected by centrifugation at 20,000 g for 10 minutes. The collected precipitate was vacuum-dried at 50 ° C. The weight of the obtained polymer was measured, and the content was calculated. The 3HH molar fraction was determined by the method described in Example 1 of JP-A-2001-340078. That is, 2 ml of a mixture of sulfuric acid and methanol (15:85) and 2 ml of chloroform were added to about 20 mg of the obtained dry polymer, and the mixture was sealed and heated at 100 ° C. for 140 minutes to obtain a methyl ester of a polyester decomposition product. . After cooling, the mixture was neutralized by adding 1.5 g of sodium bicarbonate little by little, and allowed to stand until the generation of carbon dioxide gas ceased. After 4 ml of diisopropyl ether was added and mixed well, the mixture was centrifuged, and the composition of methyl 3-hydroxybutanoate and methyl 3-hydroxyhexanoate in the supernatant was analyzed by capillary gas chromatography. The composition ratio of the monomer unit was calculated. The gas chromatograph used was Shimadzu GC-17A, and the capillary column used was NU Science's NEUTRA BOND-1 (column length: 25 m, column inner diameter: 0.25 mm, liquid film thickness: 0.4 μm). The temperature was raised from the initial temperature of 100 to 200 ° C. at a rate of 8 ° C./min, and further from 200 to 290 ° C. at a rate of 30 ° C./min.
[0054]
(Measurement of particle size)
The average particle size of the PHA particles was measured using a Microtrac particle sizer (Nikkiso FRA). After PHBH was suspended in Kao's dispersant Demol, the concentration was adjusted to a predetermined concentration, and the particle size corresponding to the 50% accumulation of all particles was defined as the average particle size.
[0055]
(Examination of crystal solidification)
The extrusion was carried out at a temperature of 140 and 150 ° C. using a 1 mmφ × 10 mm die using a Capillograph (manufactured by Toyo Seiki Seisakusho). Shear speed 122 sec -1 The resin composition was melt-extruded at a descent speed of 10 mm / min, and the crystal solidification was evaluated based on the distance and time until the extruded strand crystallized from the extrusion discharge port.
[0056]
◎: Crystallized when the extruded strand comes out of the extrusion discharge port
：: The distance from the extrusion outlet to solidification of the crystal is 0 to about 20 cm.
Δ: The distance from the extrusion outlet to solidification of the crystal is about 20 to 50 cm.
X: The distance from the extrusion discharge port to solidification of the crystal is about 50 cm or more, and the solidification time is 10 minutes or more.
[0057]
(Example 1)
Cells were cultured using two culture tanks. Palm kernel olein is supplied at a specific substrate supply rate of 0.06 (g-oil) x (g-net dry cell weight). -1 × (h) -1 Was fed. After a lapse of 60 hours, the culture of one group was stopped, and the culture of the second group was continued by further adding 970 ml (40.6 ml / hour) of 40% w / v fructose over 24 hours. The average particle size of the obtained PHBX was 0.7 μm in both cases, and it was confirmed that the particles were collected as fine particles. Table 1 shows the 3HH mole fraction characteristics and crystal solidification evaluation. PHBX obtained by culturing only with palm nucleus olein has a very sticky post-extrusion strand at 140 and 150 ° C., and has a remarkably low crystallization solidification speed. Cost me. It became clear that PHBX obtained by continuous culturing with fructose contained PHB. The crystal solidification rate of this PHBH was significantly increased.
[0058]
[Table 1]
PHBX was obtained by culturing and purifying in the same manner as in Example 1, except that palm oil olein was used instead of coconut oil, and fructose was used instead of soybean oil and corn oil. Table 1 shows the 3HH mole fraction characteristics and the crystal solidification evaluation of the obtained PHBH. PHBH obtained by culturing only with coconut oil has a very sticky post-extrusion strand at 140 and 150 ° C., has a remarkably low crystallization solidification speed, and requires a crystallization solidification time of at least 10 minutes immediately after extrusion. did. It was revealed that PHBH obtained by continuous culturing with soybean oil and corn oil contained low 3HH-PHBH. The crystal solidification rate at 140 and 150 ° C. was significantly increased.
[0059]
(Example 3)
PHBH was obtained by culturing and purifying in the same manner as in Example 1 except that soybean oil was used instead of palm kernel oil olein, and culture was performed at a specific substrate supply rate of 0.06. The PHBH obtained by culturing only with soybean oil had sticky strands after extrusion at 140 and 150 ° C., had a low crystallization solidification rate, and required a crystallization solidification time of 5 minutes or more immediately after extrusion. It was revealed that PHBH obtained by continuous culture with fructose contained PHB, and the solidification rate of the polymer at 140 and 150 ° C. was remarkably increased.
[0060]
(Example 4)
Example 1 Example 1 was repeated except that palm kernel olein oil was used as a carbon source instead of fructose, and the specific substrate supply rate was initially cultivated at 0.06, and cultivation was changed to 0.12 after 50 hours of cultivation. Culture and purification operations were performed in the same manner as in the above to obtain PHBX. When the culture was carried out at a specific substrate supply rate of 0.06, PHBX had a high 3HH molar fraction, but it was revealed that PHBX changed to 0.12 contained low 3HH-PHBXH. The crystal solidification rate of the polymer at 140 and 150 ° C. was significantly faster.
[0061]
(Example 5)
PHBX was obtained by culturing and purifying in the same manner as in Example 1, except that the specific substrate supply rate of soybean oil was raised from 0.06 to 0.12 after 50 hours, and then cultured. The PHBX obtained after culturing at a specific substrate supply rate of 0.06 yields PHBXH with a moderate 3HH mole fraction, and the extruded strands at 140 and 150 ° C. are sticky and crystallized. The solidification rate was slow, and a crystallization solidification time of 5 minutes or more was required immediately after extrusion. PHBH having a lower 3HH mole fraction was synthesized in PHBH obtained by changing the specific substrate supply rate to 0.12, and the crystal solidification rate at 140 and 150 ° C. was significantly increased.
[0062]
【The invention's effect】
According to the method of the present invention, a polyhydroxyalkanoate having a significantly improved crystallization rate can be industrially produced and provided at low cost.
Claims (17)
（ａ）ポリヒドロキシアルカノエート含有微生物細胞の水性懸濁液に、攪拌と物理的破砕処理を行いながらアルカリを添加し、該細胞を破砕すると共に、該細胞中のポリヒドロキシアルカノエート以外の細胞物質を可溶化あるいは乳化させ、次いでポリヒドロキシアルカノエートを懸濁液から分離する工程；
（ｂ）分離されたポリヒドロキシアルカノエートを、酵素及び／又は界面活性化剤で処理し、ポリヒドロキシアルカノエートに含まれる不純物を可溶化又は分解後可溶化し、続いて親水性溶媒でポリヒドロキシアルカノエートを洗浄する工程。17. A method for recovering polyhydroxyalkanoate from a polyhydroxyalkanoate-containing microbial cell, comprising the following steps (a) and (b): polyhydroxyalkanoate according to any one of claims 1 to 16. Production method.
(A) An alkali is added to an aqueous suspension of polyhydroxyalkanoate-containing microbial cells while stirring and physically crushing the cells to crush the cells, and cell substances other than polyhydroxyalkanoate in the cells. Solubilizing or emulsifying and then separating the polyhydroxyalkanoate from the suspension;
(B) treating the separated polyhydroxyalkanoate with an enzyme and / or a surfactant to solubilize or decompose impurities contained in the polyhydroxyalkanoate, and then solubilizing the polyhydroxyalkanoate with a hydrophilic solvent; Washing the alkanoate.
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