JP4007580B2 - Method and apparatus for producing polyhydroxyalkanoate - Google Patents

Description

(２)生理学的性質
カタラーゼ :陽性
オキシダーゼ :陽性
Ｏ/Ｆ試験 :酸化型(非発酵性)
硝酸塩の還元 :陰性
インドールの生成 :陽性
ブドウ糖酸性化 :陰性
アルギニンジヒドロラーゼ :陰性
ウレアーゼ :陰性
エスクリン加水分解 :陰性
ゼラチン加水分解 :陰性
β-ガラクトシダーゼ :陰性
Ｋing's Ｂ寒天での蛍光色素産生 :陽性
４％ＮaＣlでの生育 :陽性(弱い生育)
ポリ-β-ヒドロキシ酪酸の蓄積 :陰性(＊)
Ｔｗeen 80 の加水分解 :陽性
＊ nutrient agar培養コロニーをズダンブラックで染色することで判定。
(３)基質資化能
ブドウ糖 :陽性
Ｌ-アラビノース :陽性
Ｄ-マンノース :陰性
Ｄ-マンニトール :陰性
Ｎ-アセチル-Ｄ-グルコサミン :陰性
マルトース :陰性
グルコン酸カリウム :陽性
n-カプリン酸 :陽性
アジピン酸 :陰性
dl-リンゴ酸 :陽性
クエン酸ナトリウム :陽性
酢酸フェニル :陽性
ＴＬ２株は、特許手続上の微生物の寄託の国際的承認に関するブタペスト条約に基づき、寄託番号「ＦＥＲＭ ＢＰ-6913」として、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(旧経済産業省産業技術総合研究所生命工学工業技術研究所特許微生物寄託センター)に国際寄託されている。
【００３４】
本発明の方法では、これらの細胞中に生産された不水溶性のＰＨＡは、粒子状の形状で蓄積されるが、このＰＨＡ粒子を固-液分離により回収する際、その他の細胞構成成分からなる挟雑不純物を次亜塩素酸塩による処理で可溶化させることで、不水溶性のＰＨＡ粒子のみを固相成分として分離している。
【００３５】
即ち、本発明の方法においては、培養後、ＰＨＡを蓄積した細胞を培養液から回収し、この細胞を次亜塩素酸塩を含む処理液中に懸濁し、この処理液中で一定時間、一定条件下で反応させることによって、ＰＨＡ以外の細胞構成成分を可溶化させて除去し、該細胞からＰＨＡ粒子を分離する手段を用いる。
【００３６】
本発明の方法で用いる次亜塩素酸塩としては、次亜塩素酸カリウム(ＫＣlＯ)、次亜塩素酸カルシウム(Ｃa(ＯＣl)₂)、次亜塩素酸ナトリウム(ＮaＣlＯ)等の中から、適宜選択して用いることができる。
また、処理液中に溶解させる次亜塩素酸塩の濃度は、処理液中の有効塩素濃度として、0.5％から 12.0％、望ましくは 1.5％から 5.0％の範囲内に選択するのが好ましい。例えば、次亜塩素酸塩として市販の次亜塩素酸ナトリウム水溶液を用いる場合は、次亜塩素酸ナトリウムの濃度は４％(有効塩素濃度として約1.7％)から６％(有効塩素濃度として約2.5％)程度が望ましい。また、細胞の乾燥重量１g当たり、処理液の液量を 50ｍlから 300ｍlの範囲内に選択して、処理を行なうことが好ましい。処理温度は、室温(20℃程度)以上になると、ＰＨＡ自体にも作用して部分的に加水分解等を起こし、ＰＨＡの分子量低下を招く恐れが有る為、処理温度を０℃から 10℃の範囲内に選択して処理を行なうことが望ましい。なお、細胞に次亜塩素酸ナトリウムが接触した際に発熱することが有るので、その際は特に注意が必要である。前記の次亜塩素酸塩の濃度、温度条件を選択する際、処理時間は、１時間から５時間の範囲内、通常、２時間程度に選択すると十分に可溶化処理が達成できる。１時間未満であるとＰＨＡ成分以外の細胞成分の可溶化が不十分となり、５時間以上処理を行なうとＰＨＡの分子量低下の原因となるので注意が必要である。
【００３７】
前記の可溶化処理の反応を終了した後、処理液中のＰＨＡ粒子を回収する手段としては、ＰＨＡ粒子を、共存する可溶化された細胞成分と効果的に分離できる限り、いかなる固-液分離手段をも利用することができるが、例えば、遠心分離法を利用して、ＰＨＡ粒子を可溶性画分と分離することができる。この時点でのＰＨＡの様態は水溶液に懸濁された微粒子状になっているが、その後の脱塩素処理の効率が高くなるようにＰＨＡ微粒子の様態を保ったまま遠心分離操作を行なう事が必要である。つまり、遠心分離の際の温度は０℃から 10℃程度、好ましくは０℃から５℃で行なうことが望ましい。
更に、遠心分離法で回収したＰＨＡ粒子を脱イオン水、蒸留水、純水等の精製水で更に洗浄し、僅かに混入する可能性を有する挟雑成分の十分な除去を図る工程を加えることがより望ましい。前記の洗浄する工程は２回から３回程度行なうことが望ましい。
【００３８】
特に高純度のＰＨＡを得る必要が有る際には、ＰＨＡの分離及び洗浄操作の後に、例えば、酵素、酸化剤、界面活性剤またはこれらを組み合わせた化学的処理等を行なうこともできる。
【００３９】
前記の次亜塩素酸塩による処理の工程の前に、細胞を破砕する工程を更に加えることで、ＰＨＡを更に効果的に分離することができる。この細胞を破砕する工程では、超音波破砕法、ホモジナイザー法、圧力破砕法、ビーズ衝撃法、摩砕法、擂潰法(ガラス粉末やアルミナ粉末等の助剤を加えて乳鉢中で磨り潰す方法)、凍結融解法等の薬剤を使用しない方法を用いることが望ましい。更に、この再懸濁液について、遠心分離等の方法により固体成分と可溶成分とを分離し、ＰＨＡ成分が含まれている固体成分を次亜塩素酸塩で処理する。
【００４０】
ところで、前記した通り、本発明においては、ＰＨＡ以外の細胞構成成分を可溶化させる酸化剤として次亜塩素酸塩を好適に用いることができる。この次亜塩素酸塩による処理では、消費される次亜塩素酸イオンに起因する塩素が副生され、その一部が不水溶性のＰＨＡ粒子表面に付着して分離される。弱く付着する部分は、次亜塩素酸塩による処理を行ない,固-液分離した後、水洗浄をおこなうことで溶出除去されるものの、強固に付着している一部の塩素は残留塩素となり、加熱を伴う加工を行なう際に初めて離脱するものとなる。この様な残留塩素は、得られたＰＨＡの利用を考える上で問題となり得る。
【００４１】
この様な残留塩素を除去するために、本発明においては、前記の次亜塩素酸塩による処理とその後の固-液分離工程の後に、以下の様な、塩素が可溶な液中で洗浄処理を更に施す工程を付け加えた。液中での洗浄処理は、懸濁、攪拌など工程として簡便で、かつ洗浄後の回収が容易であり、回収率や純度を低下させないので、微小な粒子の処理方法として適格である。洗浄液としては、塩素が可溶であるほか、ＰＨＡを溶解しないことが要求されることは言うまでもない。残留塩素を除去する洗浄処理として、得られたＰＨＡを更に精製水に懸濁し、30℃から 60℃に昇温し攪拌しながら、回収したＰＨＡに残存する塩素を脱離させる手段を用いることができる。この場合の温水の量は脱離した塩素が十分溶解し得る量、すなわちＰＨＡ１g(乾燥重量)当たり 50ｍl以上であることが望ましく、処理時間は６時間以上(処理温度を低くするにつれて処理時間を長くすることが望ましい)が望ましく、更に本処理は温水を新たなものに換えて２回以上行なう事が望ましい。
【００４２】
上記洗浄の終了後、洗浄液中のＰＨＡ粒子を回収する方法としては、ＰＨＡ粒子を、共存成分から効果的に分離精製し得る方法であれば、いかなる方法をも用いることができるが、例えば、遠心分離法を用いることができる。
【００４３】
残留塩素を除去する別の洗浄処理として、下記の如き残留塩素をチオ硫酸塩水溶液で洗浄して低減させる手段を用いることができる。
【００４４】
チオ硫酸塩としては、チオ硫酸カリウム(Ｋ₂Ｓ₂Ｏ₃)、チオ硫酸カリウム・３水和物(Ｋ₂Ｓ₂Ｏ₃・３Ｈ₂Ｏ)、チオ硫酸カルシウム・６水和物(ＣaＳ₂Ｏ₃・６Ｈ₂Ｏ)、チオ硫酸第一鉄(ＦeＳ₂Ｏ₃)、チオ硫酸ナトリウム(Ｎa₂Ｓ₂Ｏ₃)、チオ硫酸ナトリウム・５水和物(Ｎa₂Ｓ₂Ｏ₃・５Ｈ₂Ｏ)、チオ硫酸アンモニウム((ＮＨ₄)₂Ｓ₂Ｏ₃)等からなる群より適宜選択して用いることができる。また、上記のチオ硫酸塩を含む溶液や混合物を用いることもできる。
【００４５】
チオ硫酸塩のうち、チオ硫酸ナトリウム・５水和物(Ｎa₂Ｓ₂Ｏ₃・５Ｈ₂Ｏ、通称「ハイポ」)は、従来、主に水道水の「カルキ抜き剤」として使用されてきた。チオ硫酸ナトリウム・５水和物は水溶液中で塩素を吸収する作用を有しており、そのメカニズムは、下記式[１]に示す通りである。
【００４６】
Ｎa₂Ｓ₂Ｏ₃＋４Ｃl₂＋５Ｈ₂Ｏ→２ＮaＣl＋２Ｈ₂ＳＯ₄＋６ＨＣl[１]
チオ硫酸塩水溶液でＰＨＡを洗浄処理することによって、当該ＰＨＡに保持された残留塩素は,上記[１]の反応によって除去される。
【００４７】
チオ硫酸塩による洗浄処理における温度は、当該ＰＨＡの物性等に応じて適宜設定するものではあるが、通常、０℃から 100℃、望ましくは 20℃から 70℃に設定するのが良い。また、本洗浄処理における洗浄時間は、５分間から 24時間、望ましくは、１時間から３時間とするのが良い。
【００４８】
終了後の、洗浄液中のＰＨＡ粒子を回収する方法としては、ＰＨＡ粒子を、共存成分から効果的に分離精製し得る方法であれば、いかなる方法をも用いることができるが、例えば、遠心分離法を用いることができる。また、本洗浄処理で洗浄したＰＨＡ粒子を、精製水等で更に洗浄する処理を加えれば、なお望ましい。
【００４９】
また、残留塩素を除去するさらに別の洗浄処理として、下記の如き残留塩素を有機極性溶媒で洗浄して低減させる手段を用いることができる。
【００５０】
この極性溶媒溶液による洗浄処理は、ＰＨＡ粒子表面に付着している残留塩素を、目的のＰＨＡ自体に対する溶解性を有しない有機極性溶媒を全体積の 50％以上の濃度で含む極性溶媒溶液で洗浄して、この極性溶媒溶液中に溶出させ低減させるものである。
【００５１】
不水溶性のＰＨＡ粒子表面に強固に付着している一部の塩素に対して、前記の如き有機極性溶媒を利用する洗浄を施すことで、その大部分を除去することができる。なお、本方法で得られるＰＨＡ粒子においては、残留塩素の絶対量が大幅に低減されている為、かかるＰＨＡ粒子を原材料として加工品を作製する際、熱を加えても、放出される塩素量は、作業環境汚染等の要因とはならない程度に抑制できる。
【００５２】
この極性溶媒溶液による洗浄処理に用いる有機極性溶媒としては、アルコール、ケトン等を用いることができる。より具体的には、例えば、メタノール、エタノール、イソプロパノール、イソブタノール、アセトン等の中から適宜選択して用いることができる。なお、これら有機極性溶媒は、単独で利用することが一般的であるが、複数種を含む混合溶媒を用いることもできる。更に、前記アルコール、ケトン等の有機極性溶媒は水との高い親和性を有するので、水と均一に混和させた混合溶媒として用いることもできる。水と均一に混和させた混合溶媒として用いる際には、有機極性溶媒の含有率(濃度)を高くすることが望ましく、例えば、含有率(濃度)を 99％以上とすると、単一溶媒を用いる際と遜色の無い洗浄効果が達成できる。
【００５３】
この極性溶媒溶液による洗浄処理における温度は、目的とするＰＨＡの物性、例えば、有機極性溶媒に対する溶解度、軟化点、融点等に応じて適宜選択するものでもあるが、通常、利用する有機極性溶媒が液体状態であり、かつ、その蒸散が少ない温度領域、例えば、０℃から 50℃の範囲に選択することが望ましい。また、この洗浄処理における洗浄時間は、用いる有機極性溶媒の種類や温度に依存するものの、５分間から 24時間の範囲内、好ましくは、１時間から３時間の範囲内に選択することで、十分な洗浄・除去が達成できる。
【００５４】
この極性溶媒溶液を用いる洗浄処理を終了した後、有機極性溶媒を含む液中から洗浄処理済みのＰＨＡ粒子を回収する手段としては、ＰＨＡ粒子と、有機極性溶媒を含む極性溶媒溶液中に溶解している共存成分とを効果的に分離できる限り、いかなる固-液分離方法をも用いることができる。例えば、遠心分離法を用いて、有機極性溶媒を含む液に溶解する成分と、ＰＨＡ粒子との分離を図ることができる。また、有機極性溶媒を用いる洗浄処理後、回収したＰＨＡ粒子を、再度精製水等で洗浄し、有機極性溶媒の残留分を除去する処理を加えれば、なお望ましい。
【００５５】
なお、前記の極性溶媒溶液による洗浄処理における洗浄効果は、ＰＨＡ粒子の種類に依存せず、汎用性を有し、また、再現性も高いことが見出されている。また、ＰＨＡ粒子の量は、元の細胞自体の嵩に比較すると格段に少ないので、前記の有機極性溶媒を利用する洗浄に用いる有機極性溶媒は、有機溶媒抽出法で使用される有機溶媒の量とは比較にならない程僅かな量である。しかも、ＰＨＡ自体は溶解させず、その表面に付着する塩素のみを溶出させ、固-液分離法を利用して、洗浄されたＰＨＡ粒子を再回収する為、かかる工程において、揮発・蒸散する有機溶媒量を極めて少なくすることも可能となる。加えて、有機溶媒抽出のように、水層中にも若干量の有機溶媒が残留しその回収処理が必要となることや、有機相からＰＨＡを回収する際に溶媒留去等で排除する多量の有機溶媒の回収処理を行なう必要が有ること等、規模が増すにつれてますます困難となる有機溶媒の回収作業も、本発明の方法ではより簡単に実施できる。
【００５６】
以上説明したように,本発明の洗浄とは,通常用いられると同様の意味であって,液中にＰＨＡ粒子を懸濁させ,攪拌し,洗浄液とＰＨＡ粒子を分離する一連の工程である。しかし,同様の洗浄効果が得られるものであれば,攪拌を省略したり,分離がなく,洗浄液を連続的に排出・追加するものであったり,必要に応じて,この工程を変化させてもよい。
【００５７】
上記の残留塩素を除去する工程で得られたＰＨＡ粒子を乾燥する工程においては、自然乾燥、真空乾燥法、凍結乾燥法等、当該ＰＨＡの物性や取得しようとする形態等に応じて適宜選択するものとする。
【００５８】
前記の方法でＰＨＡを製造する為の装置としては、通常の微生物生産や化学合成等に用いる装置を単独または複数組み合わせて用いることができるが、更に、下記の様な構成を備えたＰＨＡの製造装置を、本発明のＰＨＡの製造方法に最適な製造装置の一形態として例示することができる。
【００５９】
ＰＨＡを含有する細胞を、前記の次亜塩素酸塩で処理して、ＰＨＡ以外の細胞構成成分を除去する手段と、該処理された細胞を水溶性画分と水不溶性画分(ＰＨＡ)とに分離する手段と、該水不溶性画分から残留塩素を除去する洗浄手段とが備わっている。残留塩素を除去する手段としては、該水不溶性画分を温水で洗浄処理する手段、該水不溶性画分をチオ硫酸塩水溶液で洗浄処理する手段、該水不溶性画分を、前記ＰＨＡ自体に対する溶解性を有しない有機極性溶媒を含有してなる極性溶媒溶液で洗浄処理する手段からなる群より選択される、少なくとも一つ以上の手段よりなる。更に必要に応じて、前記の次亜塩素酸塩で処理する手段の前に、ＰＨＡを含有する細胞を破砕して破砕物を得る手段と、該破砕物を水溶性画分と水不溶性画分とに分離する手段とを備えた構成の製造装置とすることもできる。
【００６０】
【実施例】
以下に、実施例を示して、本発明を更に具体的に説明する。これら実施例は本発明の最良の実施形態の一例ではあるものの、本発明はこれらの実施例に限定されるものではない。なお、以下の実施例における「％」は特に標記した以外は重量基準である。また、各実施例で用いたＭ９培地は下記の組成を有するものである。
【００６１】
[Ｍ９培地]
Ｎa₂ＨＰＯ₄ 6.2g
ＫＨ₂ＰＯ₄ 3.0g
ＮaＣl 0.5g
ＮＨ₄Ｃl 1.0g
(培地１リットル中、pＨ7.0)
(実施例１)
リンゴ酸ナトリウム 0.1％を含有するＭ９寒天培地上のＴＢ64株のコロニーを、500ｍl容振盪フラスコ中の、リンゴ酸ナトリウム 0.5％を含有するＭ９培地 50ｍlに植菌し、30℃で振盪培養した。24時間後、培養液５ｍlを、窒素源であるＮＨ₄Ｃlのみ１/10 濃度に調製したＭ９培地にリンゴ酸ナトリウム 0.5％を含有した培地１リットルに加え、同様に振盪して菌体にＰＨＢを蓄積させた。52時間後、ＰＨＢ蓄積菌体を遠心分離によって収穫し、蒸留水 30ｍlに再懸濁して３等分した(各 10ｍl)。これらに１から３まで番号を付け、以下の処理を行なった。
【００６２】
１:対照:更に遠心分離後メタノールで洗浄し、凍結乾燥して秤量した後、クロロホルムで 60℃、24時間抽出操作を行ない、抽出液を濾過、濃縮後、メタノールで再沈殿させ、減圧乾燥して対照ポリマー(試料１とする)を得た。
【００６３】
２:４℃に冷却した脱イオン水を 30ｍl加え、４℃に冷却した次亜塩素酸ナトリウム溶液(キシダ化学社製；次亜塩素酸ナトリウム 12％(有効塩素濃度５％以上)含有)を 20ｍl加えて、４℃で２時間反応させた。反応終了後、反応液に４℃に冷却した脱イオン水 140ｍlを加え、４℃、29400ｍ/s²(3000×g)で 30分間遠心分離を行なった。得られた沈殿物に４℃に冷却した脱イオン水 80ｍlを加え、超音波処理にて粒子を十分分散させた後、遠心分離を行なった(４℃、29400ｍ/s²(3000×g)で 30分間)。更にこの操作を２回繰り返し、得られた沈殿物を凍結乾燥した。これを試料２とする。
【００６４】
３:２の操作の脱イオン水による洗浄を行なった試料を、50ｍlの脱イオン水に懸濁し、50℃で６時間攪拌したのち、上澄を遠心分離(４℃、29400ｍ/s²(3000×g)で 30分間)によって除去した。この温水洗浄処理をもう１度繰り返した。得られた沈殿物に４℃に冷却した脱イオン水 40ｍlを加え、超音波処理にて粒子を十分分散させた後、遠心分離を行なった(４℃、29400ｍ/s²(3000×g)で 30分間)。更にこの操作を１回繰り返し、得られた沈殿物を凍結乾燥した。これを試料３とする。
【００６５】
＜残留塩素の測定＞
試料２及び試料３については以下の様にして残留塩素を測定した。
【００６６】
図１に示す１:三角フラスコ(全容量 637ｍl)と、２a:中心部に穴をあけた外蓋(スクリューキャップ)、２b:中心部に穴をあけたテフロン製内蓋、及び２c:内蓋と同じ大きさのアルミ箔で構成された蓋をオーブン中で予め 150℃に加熱しておき、３:アルミ箔上に載せた４:試料 0.3gを入れて蓋を密閉し、同じく 150℃で加熱した。３分後、蓋の穴から６:検知管(ガステック社製；塩素ガス検知管・品番８Ｌa；検出範囲濃度:0.5ppｍから８ppｍ或いは、品番８Ｈ；検出範囲濃度:0.5ppｍから８ppｍ)を差込み、100ｍlを吸引して塩素濃度を測定した。
【００６７】
その結果、温水処理を行なっていない試料２の塩素濃度が約50ppｍであったのに対し、温水処理を行なった試料３では約４ppｍであった。試料３は試料２に比べて塩素低減の効果が見られる。
【００６８】
次に、以下に示す「回収率」及び「純度」を評価する為、次の操作を行なった。
【００６９】
乾燥した１から３までの試料にクロロホルム 30ｍlを加え、60℃で 24時間攪拌抽出操作を行なった。ＰＨＢが抽出されたクロロホルム溶液を 0.45μｍのＰＴＦＥフィルターで濾過し、ロータリーエバポレーターで濃縮して 10倍量のメタノールに注加しＰＨＢを沈殿、回収した。これらを減圧乾燥して秤量した。
【００７０】
対照である１に対する、２及び３の試料のクロロホルム抽出によって得られたＰＨＢの質量比を「回収率」とし、各試料の、クロロホルム抽出前の試料に対するクロロホルム抽出によって得られたＰＨＢの質量比を「純度」として、表１に示す。
【００７１】
【表１】

【００７２】
試料３は、温水処理をしているにもかかわらず、回収率及び純度は、温水処理を行なっていない試料２とほぼ同等であった。したがって、温水処理は、回収率と純度を高く保ったまま、残留塩素を減少させる効果がある。
【００７３】
(実施例２)
n-ノナン酸 0.1％を含有するＭ９寒天培地上のシュードモナス・オレオボランスのコロニーを、n-ノナン酸 0.3％を含有するＭ９培地 50ｍlに植菌し、30℃で振盪培養した。40時間後、培養液５ｍlをn-ノナン酸 0.2％及び５-フェニル吉草酸 0.05％を含有するＭ９培地１リットルに加え、振盪培養した。更に 36時間後、菌体を遠心分離によって収穫し、窒素源であるＮＨ₄Ｃlを含まず、n-ノナン酸 0.05％及び５-フェニル吉草酸 0.2％を含有するＭ９培地１リットルに再懸濁し、同様に振盪して菌体に３-ヒドロキシノナン酸、３-ヒドロキシヘプタン酸、３-ヒドロキシ吉草酸、及び３-ヒドロキシ-５-フェニル吉草酸をユニットとして含むＰＨＡを蓄積させた。48時間後、ＰＨＡ蓄積菌体を遠心分離によって収穫し、以下の処理を行なった。
【００７４】
４:対照:更に遠心分離後メタノールで洗浄し、凍結乾燥して秤量した後クロロホルムで 60℃、24時間抽出操作を行ない、抽出液を濾過、濃縮後、メタノールで再沈殿させ、減圧乾燥して対照ポリマー(試料４とする)を得た。
【００７５】
５:４℃に冷却した脱イオン水を 30ｍl加え、４℃に冷却した次亜塩素酸ナトリウム溶液(キシダ化学社製；次亜塩素酸ナトリウム 12％(有効塩素濃度５％以上)含有)を 20ｍl加えて、４℃で２時間反応させた。反応終了後反応液に４℃に冷却した脱イオン水 140ｍlを加え、４℃、29400ｍ/s²(3000×g)で 30分間遠心分離を行なった。得られた沈殿物に４℃に冷却した脱イオン水 80ｍlを加え、超音波処理にて粒子を十分分散させた後、遠心分離を行なった(４℃、29400ｍ/s²(3000×g)で 30分間)。更にこの操作を２回繰り返し、得られた沈殿物を凍結乾燥し,試料５を得た。
【００７６】
６:５の操作の脱イオン水による洗浄を行なった試料を、50ｍlの脱イオン水に懸濁し、30℃で 12時間攪拌した。更に上澄を遠心分離(４℃、29400ｍ/s²(3000×g)で 30分間)によって除去し、50ｍlの脱イオン水に再懸濁し、30℃で 12時間攪拌した。処理終了後上澄を遠心分離(４℃、29400ｍ/s²(3000×g)で 30分間)によって除去し、得られた沈殿物に４℃に冷却した脱イオン水 40ｍlを加え、超音波処理にて粒子を十分分散させた後、遠心分離を行なった(４℃、29400ｍ/s²(3000×g)で 30分間)。更にこの操作を１回繰り返し、得られた沈殿物を凍結乾燥し、試料６を得た。
【００７７】
試料５及び試料６は実施例１と同様に方法で残留塩素を測定した。その結果、温水処理を行なっていない試料５の塩素濃度が約20ppｍ(検知管８Ｌaの 30ｍl引きから換算)であったのに対し、温水処理を行なった試料６では約２ppｍであった。試料６は試料５に比べて塩素低減の効果が見られる。
【００７８】
更にこれらの試料を実施例１と同様にクロロホルム抽出し、回収率及び純度を求めた。結果を表２に示す。
【００７９】
【表２】

【００８０】
温水処理試料６の回収率及び純度は、温水処理を行なっていない試料５とほぼ同等であった。実施例１のＰＨＢに対すると同様,本実施例のような芳香環を有するＰＨＡに対しても、温水処理は、回収率と純度を高く保ったまま、残留塩素を減じさせる効果がある。
【００８１】
(実施例３)チオ硫酸ナトリウム水溶液による洗浄(１)
酵母エキス(オリエンタル酵母工業(株))0.5％を含むＭ９培地 200ｍlに、ＹＮ２株を植菌し、500ｍl容振盪フラスコ中で 30℃、125 ストローク/分の条件下で振盪培養した。８時間後、前記培養液を、ポリペプトン(日本製薬(株))0.5％と５-チオフェノキシ吉草酸 0.1％とを含むＭ９培地 25リットルに添加し、50リットル容ジャーファーメンター中で、30℃で 48時間通気攪拌培養した。
【００８２】
上記培養液のうち 200ｍlを遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)して微生物細胞を回収し、冷メタノールで一度洗浄して真空乾燥した。この真空乾燥ペレットを 20ｍlのクロロホルムに懸濁し、60℃で 20時間攪拌してＰＨＡを抽出した。抽出液を孔径 0.45μｍのメンブレンフィルターで濾過した後、ロータリーエバポレーターで濃縮し、濃縮液を冷メタノール中で再沈殿させ、更に沈殿のみを回収して真空乾燥してＰＨＡを得た。このＰＨＡについて、常法に従ってメタノリシスを行ない、ガスクロマトグラフィー-質量分析装置(ＧＣ-ＭＳ,島津ＱＰ-5050、ＥI法)で分析してＰＨＡモノマーユニットのメチルエステル化物の同定を行なった。その結果、当該培養液中の微生物細胞には表３に示すモノマーユニット組成を有するＰＨＡが含まれていることがわかった。
【００８３】
【表３】

【００８４】
上記の分析操作に用いた以外の培養液より、遠心分離によって微生物細胞を回収した。得られた細胞は精製水 1.6リットルに懸濁し、次亜塩素酸ナトリウム溶液(有効塩素濃度５％以上)0.8リットルを添加した。これを４℃で２時間振盪してＰＨＡ以外の細胞構成成分を可溶化させた後、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収した。得られたＰＨＡを精製水 200ｍlに懸濁し、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収する操作を３回繰り返して水洗浄を行ない、ＰＨＡ粒子(試料７とする)を得た。
【００８５】
当該ＰＨＡ粒子に保持された残留塩素は、以下の方法で評価した。
【００８６】
(１)官能評価
室温放置されたＰＨＡ粒子の塩素臭を官能的に判定し、塩素臭の強いものから順に、
＋＋＋(強),＋＋(中),＋(低),±(微弱),−(なし)
の５段階で評価した。
【００８７】
(２)濃度測定
500ｍl容蓋付きフラスコ中に上記のＰＨＡ粒子を入れて密封し、150℃雰囲気中で１時間加熱して、残留塩素を放出させた後、ガス検知管を用いてフラスコ内の塩素濃度を測定した。
【００８８】
残留塩素の評価の結果を表４に示した。表４より、当該ＰＨＡ粒子における塩素の残留が認められた。
【００８９】
【表４】

【００９０】
上記の方法で得られる、乾燥重量で 1.0gに相当するＰＨＡ粒子を、精製水または 10％チオ硫酸ナトリウム・５水和物水溶液各 50ｍlに懸濁した。これらの懸濁液を 50℃で６時間攪拌した後、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収した。得られたＰＨＡを精製水 40ｍlに懸濁し、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収する操作を３回繰り返した。得られたＰＨＡを精製水 10ｍlに懸濁して凍結乾燥し、ＰＨＡの洗浄粒子を得た。精製水を用いた洗浄ＰＨＡ粒子を試料８、10％チオ硫酸ナトリウム・５水和物水溶液を用いた洗浄ＰＨＡ粒子を試料９とする。
【００９１】
当該洗浄粒子に保持された残留塩素は、以下の方法で評価した。
【００９２】
(１)官能評価
室温放置されたＰＨＡ粒子の塩素臭を官能的に判定し、塩素臭の強いものから順に、
＋＋＋(強),＋＋(中),＋(低),±(微弱),−(なし)
の５段階で評価した。
【００９３】
(２)濃度測定
500ｍl容蓋付きフラスコ中に上記の洗浄粒子を入れて密封し、150℃雰囲気中で１時間加熱して、残留塩素を放出させた後、ガス検知管を用いてフラスコ内の塩素濃度を測定した。
【００９４】
残留塩素の評価の結果を表５に示した。表４と表５の比較により、精製水による温水洗浄でも塩素濃度減少効果はあるが、チオ硫酸ナトリウム水溶液による洗浄によって、ＰＨＡの洗浄粒子の残留塩素がさらに低減されることが明らかになった。
【００９５】
【表５】

【００９６】
(実施例４)チオ硫酸ナトリウム水溶液による洗浄(２)
実施例３の方法で得られた試料７の、乾燥重量で 1.0gに相当するＰＨＡ粒子を、０％、0.1％、1.0％、または、10％のチオ硫酸ナトリウム・５水和物水溶液各 50ｍlに懸濁した。これら４種類の懸濁液を 50℃で６時間攪拌した後、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収した。得られたＰＨＡを精製水 50ｍlに懸濁し、更に 50℃で６時間攪拌した後、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収した。得られたＰＨＡを精製水 40ｍlに懸濁し、遠心分離(29400ｍ/s²(3000×g)、４℃、30分間)によりＰＨＡを回収する操作を３回繰り返した。得られたＰＨＡを精製水 10ｍlに懸濁して凍結乾燥し、ＰＨＡの洗浄粒子を得た。チオ硫酸ナトリウム水溶液の濃度が０％、0.1％、1.0％、10％であったものをそれぞれ、試料10，11，12，13 とする。
【００９７】
当該洗浄粒子に保持された残留塩素は、以下の方法で評価した。
【００９８】
(１)官能評価
室温放置された洗浄粒子の塩素臭を官能的に判定し、塩素臭の強いものから順に、
＋＋＋(強),＋＋(中),＋(低),±(微弱),−(なし)
の５段階で評価した。
【００９９】
(２)濃度測定
500ｍl容蓋付きフラスコ中に上記の洗浄粒子を入れて密封し、150℃雰囲気中で１時間加熱して、残留塩素を放出させた後、ガス検知管を用いてフラスコ内の塩素濃度を測定した。
【０１００】
残留塩素の評価の結果を表６に示した。表４と表６により、精製水(０％チオ硫酸ナトリウム水溶液)の温水洗浄でも,塩素濃度を減少させる効果はあるが,チオ硫酸ナトリウム水溶液による洗浄によって、官能評価による微妙な差異であるが,ＰＨＡの洗浄粒子の残留塩素が低減されること,その効果も,チオ硫酸ナトリウムの濃度が高いほど大きいことが明らかになった。
【０１０１】
【表６】

【０１０２】
(実施例５) アルコールによる洗浄処理
乳酸ナトリウム 0.5％を添加したＭ９培地 1.6リットルに、アルカリゲネス属(Ａlcaligenes sp.)・ＴＬ２株を植菌し、30℃、125 ストローク/分で振盪培養した。48時間後、培地から細胞を遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)によって回収した。
【０１０３】
上記の培養操作により得られた細胞を精製水 80ｍlに懸濁し、次亜塩素酸ナトリウム溶液(有効塩素濃度５％以上)40ｍlを添加した。この懸濁液を４℃で２時間振盪して、ＰＨＡ以外の細胞構成成分を可溶化した後、遠心分離(59000ｍ/s²(6000×g)、４℃、20分間)によりＰＨＡを回収した。回収したＰＨＡを再び精製水 40ｍlに懸濁し、遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)によりＰＨＡ粒子を回収する操作を、計２回繰り返して、ＰＨＡの水洗浄処理を行なって、ＰＨＡ粒子を得た。
【０１０４】
得られたＰＨＡの一部は、凍結乾燥した後、常法に従ってメタノリシスを行ない、ガスクロマトグラフィー-質量分析装置(ＧＣ-ＭＳ,島津ＱＰ-5050、ＥI法)で分析して、ＰＨＡを構成するモノマーユニットのメチルエステル化物の同定を行なった。その同定の結果、得られたＰＨＡは、３-ヒドロキシ酪酸をモノマーユニットとするＰＨＢであった。
【０１０５】
上記工程で得られた、乾燥重量で 0.3gに相当するＰＨＡ粒子を、精製水、メタノール、エタノール、並びに、イソブタノール 各 40ｍlにそれぞれ懸濁した。この懸濁液を４℃で１時間振盪した後、遠心分離(29400ｍ/s²(3000×g)、４℃、20分間)によりＰＨＡ粒子を回収した。回収したＰＨＡ粒子を再び精製水 40ｍlに懸濁し、遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)によりＰＨＡ粒子を回収した。この洗浄処理を終えたＰＨＡ粒子を精製水 10ｍlに懸濁して凍結乾燥し、ＰＨＡの洗浄処理済み粒子を得た。上記洗浄液が、精製水、メタノール、エタノール、イソブタノールであったＰＨＡ粒子を、それぞれ、試料14，15，16，17 とする。
【０１０６】
室温放置時と加熱時について、前記洗浄処理済みＰＨＡ粒子(試料14-17)から放出される塩素量を、それぞれ以下の方法で測定した。50ｍlバイアル瓶に前記の洗浄処理済み粒子を入れて密封し、室温で１晩放置した後、ガス検知管を用いてバイアル瓶内の気相中に含まれる塩素量を測定した。次に、当該バイアル瓶をホットプレート上で 150℃、２分間加熱した後、ガス検知管を用いてバイアル瓶内の気相中に含まれる塩素量を測定した。
【０１０７】
表７に、その測定結果を示す。表７に示す結果より、アルコールによる洗浄を施した際(試料15-17)には、精製水による洗浄を施した場合(試料14)と比較して、洗浄処理済みのＰＨＡ粒子から放出される塩素量が、室温放置下、加熱下、いずれにおいても約半分またはそれ以上に減少していることが明らかになった。
【０１０８】
【表７】

【０１０９】
(実施例６) ケトンによる洗浄処理
乳酸ナトリウム 0.5％を添加したＭ９培地 1.6リットルに、アルカリゲネス属(Ａlcaligenes sp.)・ＴＬ２株を植菌し、30℃、125ストローク/分で振盪培養した。48時間後、培地から細胞を遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)によって回収した。
【０１１０】
上記の培養操作により得られた細胞を精製水 80ｍlに懸濁し、次亜塩素酸ナトリウム溶液(有効塩素濃度５％以上)40ｍlを添加した。この懸濁液を４℃で２時間振盪して、ＰＨＡ以外の細胞構成成分を可溶化した後、遠心分離(59000ｍ/s²(6000×g)、４℃、20分間)によりＰＨＡを回収した。回収したＰＨＡを再び精製水 40ｍlに懸濁し、遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)によりＰＨＡ粒子を回収する操作を、計２回繰り返して、ＰＨＡの水洗浄処理を行なって、ＰＨＡ粒子を得た。
【０１１１】
得られたＰＨＡの一部は、凍結乾燥した後、常法に従ってメタノリシスを行ない、ガスクロマトグラフィー-質量分析装置(ＧＣ-ＭＳ,島津ＱＰ-5050、ＥI法)で分析して、ＰＨＡを構成するモノマーユニットのメチルエステル化物の同定を行なった。その同定の結果、得られたＰＨＡは、３-ヒドロキシ酪酸をモノマーユニットとするＰＨＢであった。
【０１１２】
上記工程で得られた、乾燥重量で 0.3gに相当するＰＨＡ粒子を、精製水またはアセトン 40ｍlに懸濁した。この懸濁液を４℃で１時間振盪した後、遠心分離(29400ｍ/s²(3000×g)、４℃、20分間)によりＰＨＡ粒子を回収した。回収されたＰＨＡ粒子を再び精製水 40ｍlに懸濁し、遠心分離(78000ｍ/s²(8000×g)、４℃、10分間)により、ＰＨＡ粒子を回収した。この洗浄処理を終えたＰＨＡ粒子は精製水 10ｍlに懸濁して凍結乾燥し、ＰＨＡの洗浄処理済み粒子を得た。上記２回の洗浄が、ともに精製水洗浄であったものを試料18、１回目がアセトン洗浄であったものを試料19 とする。
【０１１３】
室温放置時と加熱時について、前記洗浄処理済みＰＨＡ粒子から放出される塩素量を、それぞれ以下の方法で測定した。50ｍlバイアル瓶に前記の洗浄処理済み粒子を入れて密封し、室温で１晩放置した後、ガス検知管を用いてバイアル瓶内の気相中に含まれる塩素量を測定した。次に、当該バイアル瓶をホットプレート上で 150℃、２分間加熱した後、ガス検知管を用いてバイアル瓶内の気相中に含まれる塩素量を測定した。
【０１１４】
表８に、その測定結果を示す。表８に示す結果より、アセトンによる洗浄を施した際(試料19)には、精製水による洗浄を施した場合(試料18)と比較して、洗浄処理済みのＰＨＡ粒子から放出される塩素量が格段に少なくなっており、即ち、洗浄処理済みＰＨＡ粒子になお残留している塩素量が大幅に低減されていることが明らかになった。
【０１１５】
【表８】

【０１１６】
(実施例７)破砕処理
リンゴ酸ナトリウム 0.1％を含有するＭ９寒天培地上のＴＢ64株のコロニーを、500ｍl容振盪フラスコ中の、リンゴ酸ナトリウム 0.5％を含有するＭ９培地 50ｍlに植菌し、30℃で振盪培養した。24時間後、培養液５ｍlを、窒素源であるＮＨ₄Ｃlのみ１/10 濃度に調製したＭ９培地にリンゴ酸ナトリウム 0.5％を含有した培地１リットルに加え、同様に振盪して菌体にＰＨＢを蓄積させた。48時間後、ＰＨＢ蓄積菌体を遠心分離によって収穫し、蒸留水 50ｍlに再懸濁して５等分した(各10ｍl)。これらに以下の 20-24 の５種類の処理を行なった。
【０１１７】
20:対照:上記５等分した懸濁液の１つを遠心分離後メタノールで洗浄し、凍結乾燥して秤量した後クロロホルムで 60℃、24時間抽出操作を行ない、抽出液を濾過、濃縮後、メタノールで再沈殿させ、減圧乾燥して対照ポリマー(試料 20 とする)を得た。
【０１１８】
21:上記５等分した懸濁液の他の１つに 31％過酸化水素水(三菱瓦斯化学社製；ＪIＳ Ｋ-8230)を 40ｍl添加し、80℃で１時間処理を行なった。
【０１１９】
22:上記５等分した中のまた別の懸濁液を蒸留水で 50ｍlにメスアップし、フレンチプレス(大岳製作所社製:フレンチプレス 5501)を行なった後、４℃、29400ｍ/s²(3000×g)で30分間遠心分離を行なった。その後更に蒸留水 40ｍlを加え、４℃、29400ｍ/s²(3000×g)で 30分間遠心分離を行なって洗浄した。
【０１２０】
23:上記５等分した中のさらに別の懸濁液に 22 と同様の操作を行なった後、沈殿部分を 10ｍlの蒸留水に懸濁し、31％過酸化水素水を 40ｍl添加し、80℃で１時間処理を行なった。
【０１２１】
24:上記５等分した懸濁液の最後の１つに 22 と同様の操作を行なった後、沈殿部分を 10ｍlの蒸留水に懸濁し、５ｍlの次亜塩素酸ナトリウム溶液(キシダ化学社製；次亜塩素酸ナトリウム 12％(有効塩素濃度５％以上)含有)を加えて、４℃で２時間処理を行なった。
【０１２２】
番号 21 から 24 の処理をおこなったそれぞれを、遠心分離(４℃、29400ｍ/s²(3000×g)で 30分間)し、更に再度４℃に冷却した後蒸留水 40ｍlを加え良く攪拌した後、同条件で２回遠心分離し、沈殿物を凍結乾燥して秤量した。これをそれぞれ試料 21 から 24 とする。
【０１２３】
以下に示す「回収率」及び「純度」を評価する為、次の操作を行なった。
【０１２４】
凍結乾燥した 20 から 24 までの試料にクロロホルム 30ｍlを加え、60℃で 24時間攪拌抽出操作を行なった。ＰＨＢが抽出されたクロロホルム溶液を 0.45μｍのＰＴＦＥフィルターで濾過し、ロータリーエバポレーターで濃縮して 10倍量のメタノールに注加しＰＨＢを沈殿、回収した。これらを減圧乾燥して秤量した。
【０１２５】
対照である 20 に対する、21 から 24 の試料のクロロホルム抽出によって得られたＰＨＢの質量比を「回収率」とし、各試料の、クロロホルム抽出前の試料に対するクロロホルム抽出によって得られたＰＨＢの質量比を「純度」として、表９に示す。
【０１２６】
【表９】

【０１２７】
回収率はそれ程変わらないが、純度は,フレンチプレスを経ないで過酸化水素水処理した試料 21 と,フレンチプレスのみ行ない過酸化水素水処理をしなかった試料 22 に比べて,フレンチプレス処理とその後の過酸化水素処理を行なった試料 23,及びフレンチプレス処理とその後の次亜塩素酸処理を行なった試料 24 が良好であった。破砕処理と酸化剤処理を組み合わせることで純度が改善されることがわかる。
【０１２８】
得られたＰＨＢは、ゲルパーミエーションクロマトグラフイー(ＧＰＣ；東ソーＨＬＣ-8020、カラム:ポリマーラボラトリーＰＬgel ＭＩＸＥＤ-Ｃ(５μｍ)、溶媒:クロロホルム、ポリスチレン換算)により分子量を測定した。結果を表 10 に示す。
【０１２９】
【表１０】

【０１３０】
各試料の分子量の差は殆ど見受けられなかった。従来法のクロロホルム抽出に比べて,破砕処理や酸化剤処理による分子量変化はない事がわかる。
【０１３１】
試料24 は、さらに、残留塩素を低減する工程を経ることにより、塩素濃度を減少させ、最終的なＰＨＡ粒子を得る。塩素低減工程としては、実施例１と同様に、温水洗浄を用いることができるが、その他の実施例２ないし６のいずれかの塩素低減工程を用いてもよい。
【０１３２】
本発明の方法により、微生物等の細胞内に蓄積されたポリヒドロキシアルカノエートを、簡便な方法で、かつ本来の分子量をほぼ保ったままで、高い回収率で回収することが可能となった。また、本発明のＰＨＡの製造方法を用いることをにより、有機溶媒抽出の手段を利用しなくとも、残留塩素を低減させたＰＨＡ粒子を工業的規模で効率的に製造できる。
【０１３３】
本発明の方法により得られるポリヒドロキシアルカノエートの粒子は、生分解性素材、生体適合性素材、各種機能性材料等として有用な原材料となり、デバイス材料や医用材料等の各分野への応用が期待できる。
【図面の簡単な説明】
【図１】実施例１及び実施例２における塩素濃度測定方法を表す。
【符号の説明】
１:三角フラスコ
２:蓋
２a:外蓋
２b:内蓋
２c:アルミ箔
３:アルミ箔
４:試料
５:検知管吸引部
６:検知管[0001]
BACKGROUND OF THE INVENTION
The present invention relates to microorganisms capable of producing polyhydroxyalkanoate (hereinafter abbreviated as PHA) and accumulating it in cells, plant cells capable of producing PHA by incorporating a PHA synthesis gene, etc. The present invention relates to a method for producing PHA using higher organisms. More specifically, the PHA-containing cells are treated with hypochlorite to remove cell constituents other than PHA, and then the obtained PHA is washed with an aqueous thiosulfate solution. The present invention relates to a method for producing PHA, characterized in that residual chlorine retained in PHA is reduced.
[0002]
The present invention also relates to an apparatus capable of implementing the above-described method for producing PHA.
[0003]
[Background]
Until now, it has been reported that many microorganisms produce poly-3-hydroxybutyric acid (hereinafter sometimes abbreviated as PHB) or other PHA and accumulate it in cells ("Biodegradable Plastic Handbook"). , Edited by Biodegradable Plastics Research Group, NTS, P178-197). These polymers can be used for production of various products by melt processing or the like, as in the case of conventional plastics. Furthermore, because it is biodegradable, it has the advantage of being completely degraded by microorganisms in nature. Therefore, unlike many synthetic polymer compounds used in the past, they do not remain in the natural environment and cause pollution, and it is not necessary to incinerate them. From the viewpoint of preventing air pollution and global warming Can also be useful materials. Furthermore, it is excellent in biocompatibility and is expected to be applied as a soft medical member.
[0004]
It is known that a microorganism-producing PHA can have various compositions and structures depending on the type of microorganisms used in the production, medium composition, culture conditions, and so on, mainly from the viewpoint of improving the physical properties of PHA. Studies on the control of such composition and structure have been made.
[0005]
For example, Alkaligenes eutrophus H16 (ALCaligenes eutrophus H16, ATCC No. 17699) and its mutant strains are produced by changing the carbon source at the time of culturing, so that the co-weight of 3-hydroxybutyric acid and 3-hydroxyvaleric acid can be reduced. It has been reported that coalescence is produced in various composition ratios (Japanese Patent Publication No. 6-15604, Japanese Patent Publication No. 7-14352, Japanese Patent Publication No. 8-19227, etc.). In JP-A-5-74492, microorganisms of the genus Methylobacterium sp., Paracoccus sp., Alcaligenes sp., Pseudomonas sp. To 7 to produce a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid. In JP-A-9-191893, Comamonas acidovorans IFO 13852 (Comamonas acidovorans IFO 13852) was obtained by culturing with gluconic acid and 1,4-butanediol as a carbon source. The production of polyesters having hydroxybutyric acid units is disclosed.
[0006]
On the other hand, in Japanese Patent Publication No. 2642937, Pseudomonas oleovorans ATCC 29347 (Pseudomonas oleovorans ATCC 29347) is provided with a non-cyclic aliphatic hydrocarbon as a carbon source, thereby producing a 3-hydroxyalkane having 6 to 12 carbon atoms. It is disclosed to produce PHA having acid units. In JP-A-5-93049 and JP-A-7-265065, 3-hydroxybutyric acid and 3-hydroxyhexane are obtained by culturing Aeromonas caviae using oleic acid or olive oil as a carbon source. The production of binary copolymers with acids is disclosed.
[0007]
These are all PHA composed of a monomer unit having an alkyl group in the side chain, that is, “usual PHA”, which is synthesized by β-oxidation of hydrocarbons or the like by microorganisms or fatty acid synthesis from sugar.
[0008]
On the other hand, it has been reported that certain microorganisms produce PHA in which various substituents other than alkyl groups are introduced into the side chain, that is, “unusual PHA”. Attempts have also been made to improve physical properties. Furthermore, “unusual PHA” is extremely useful when considering a wider range of applications of microbially produced PHA, for example, application as a functional polymer. Examples of the substituent include those containing an aromatic ring (phenyl group, phenoxy group, benzoyl group, etc.), unsaturated hydrocarbon, ester group, allyl group, cyano group, halogenated hydrocarbon, epoxide and the like. Among these, particularly, PHA having an aromatic ring has been actively studied.
[0009]
(a) including a phenyl group or a partially substituted product thereof
In Makromol. Chem., 191, 1957-1965 (1990) and Macromoles, 24, 5256-5260 (1991), Pseudomonas oleovorans uses 3-phenyl-5-valeric acid as a substrate and 3-hydroxy-5 -It has been reported to produce PHA containing phenylvaleric acid as a unit. In Macromolecules, 29, 1762-1766 (1996), Pseudomonas oleovorans contains 5- (4'-tolyl) valeric acid as a substrate and 3-hydroxy-5- (4'-tolyl) valeric acid as a unit. It has been reported to produce PHA. In Macromolecules, 32, 2889-2895 (1999), Pseudomonas oleovorans is 3-hydroxy-5- (2 ', 4'-dinitrophenyl) using 5- (2', 4'-dinitrophenyl) valeric acid as a substrate. It has been reported to produce PHA containing valeric acid and 3-hydroxy-5- (4′-nitrophenyl) valeric acid as units.
[0010]
(b) Those containing a phenoxy group or a partially substituted product thereof
In Macromol. Chem. Phys., 195, 1665-1672 (1994), Pseudomonas oleovorans uses 3-hydroxy-5-phenoxyvaleric acid unit and 3-hydroxy-9-phenoxy with 11-phenoxyundecanoic acid as a substrate. It has been reported to produce PHA copolymers with nonanoic acid units. Japanese Patent Publication No. 2989175 comprises 3-hydroxy-5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) units or 3-hydroxy-5- (difluorophenoxy) pentanoate (3H5 (DFP) P) units. Inventions relating to homopolymers, copolymers containing at least 3H5 (MFP) P units or 3H5 (DFP) P units, Pseudomonas putidas for synthesizing these polymers, and methods for producing the aforementioned polymers using Pseudomonas are disclosed. As its effect, it is said that stereoregularity and water repellency can be provided while maintaining good processability with a high melting point. Furthermore, JP 2000-72865 A reports that Pseudomonas putida 27N01 (Pseudomonas putida 27N01) produces PHA containing various 3-hydroxyfluorophenoxyvaleric acid units.
[0011]
In addition to such fluorine group-substituted products, studies on substituted products of cyano groups and nitro groups have also been made. Can. J. Microbiol., 41, 32-43 (1995) and Polymer International, 39, 205-213 (1996) use Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442. Production of PHA containing 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit using acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as a substrate Has been.
[0012]
These reports are useful for obtaining a polymer having a physical property derived from an aromatic ring in the side chain of PHA, unlike general PHA in which the side chain is an alkyl group.
[0013]
In addition, as a new category, research is being conducted not only to change physical properties but also to produce PHA having an appropriate functional group in the side chain and to create a new function using the functional group.
[0014]
For example, Macromolecules, 31, 1480-1486 (1996) and Journal of Polymer Science: Part A: Polymer Chemistry, 36, 2381-2387 (1998), etc., synthesize PHA containing a unit having a vinyl group at the end of the side chain. After that, it was reported that a PHA containing a highly reactive epoxy group at the end of the side chain was synthesized by epoxidation with an oxidizing agent. In addition to the vinyl group, as a synthesis example of PHA containing a unit having a thioether that is expected to have high reactivity, Macromolcules, 32, 8315-8318 (1999), Pseudomonas putida 27N01 is 11-thiophenoxy. It has been reported that PHA copolymer composed of 3-hydroxy-5-thiophenoxyvaleric acid unit and 3-hydroxy-7-thiophenoxyheptanoic acid unit is produced using valeric acid as a substrate.
[0015]
By the way, it is known that microorganisms accumulate the produced PHA in the cells in the form of particles. As a method for separating and purifying PHA accumulated in the cells from microbial cells, PHA is extracted with a chlorinated organic solvent such as chloroform or dichloromethane, and cell components other than PHA are solubilized and removed. There is a method for obtaining PHA particles. The former solvent extraction method is an excellent method capable of extracting and separating PHA simply and with high purity, but at the industrial production level, as a result of the increased amount of processing, it is necessary to use a large amount of organic solvent. (Chlorinated organic solvents such as chloroform used have been pointed out to cause environmental and health problems when volatilized or evaporated). Moreover, when it is desired to obtain PHA in the form of particles, a method using this solvent extraction cannot be employed.
[0016]
On the other hand, as a method of solubilizing and removing cell constituents other than the latter PHA, cell components other than PHA are solubilized by treatment with chemicals such as hypochlorite and hydrogen peroxide, and solid-liquid A method for recovering insoluble PHA particles by separation is known. For example, J. Gen. Microbiology, 19, 198-209 (1958) reports a method for separating and purifying a polymer by treating microbial cells with an alkaline solution of sodium hypochlorite. Japanese Patent Publication No. 8-508881 discloses a method for separating PHA from bacterial cells by treating PHA accumulating bacterial cells with proteolytic enzyme, then treating with an appropriate chelating agent, and further treating with hydrogen peroxide. Has been. As another method, JP-A-57-174094 discloses a method for separating PHA from microbial cells by heating and pressurizing PHA accumulating cells and releasing the pressure to disrupt the cells. It is disclosed. Japanese Patent Application Laid-Open No. 63-226291 describes a method of separating the uppermost PHA formed after transforming bacterial cells into spheroplasts, crushing them by sonic vibration treatment, and centrifuging them. .
[0017]
These methods are also extremely effective when it is desired to obtain PHA in the form of particles.
[0018]
In particular, the treatment method using hypochlorite is milder in treatment conditions and simpler than the method using other treatment agents, and it is less contaminated with cell-derived impurities. It is an excellent method having many advantages such as being able to obtain PHA of purity in the form of particles and being practically low cost.
[0019]
[Problems to be solved by the invention]
However, the method of recovering PHA accumulated in the cells in the form of particles by using cell treatment with hypochlorite such as sodium hypochlorite may reduce the molecular weight depending on the structure of PHA. In addition, it has been considered that it is not suitable for practical use because a non-negligible amount of chlorine remains in the recovered PHA (see, for example, JP-A-7-177894).
[0020]
According to the study by the present inventors, it has been found that this residual chlorine is rapidly released when the PHA is heated. Therefore, when PHA particles obtained by using such hypochlorite treatment are used as a desired processed product, if the amount of chlorine released in the heat treatment step is large, factors such as work environment contamination There are strong concerns about becoming. In addition, as a result of investigations by the present inventors, it has been found that the residual chlorine contained in the PHA particles cannot be sufficiently removed by a normal treatment of washing with water. That is, it was concluded that although the residual chlorine was firmly held in the PHA particles, it was released at a time by performing heat treatment, and as a result, a large amount of chlorine was observed.
[0021]
Therefore, in the PHA manufacturing method that uses treatment with hypochlorite in the manufacturing process, the development of means for reducing residual chlorine firmly held in the PHA particles is the development and use of PHA on a commercial scale. In particular, in order to obtain and use PHA in the form of particles, it is an extremely important issue.
[0022]
Accordingly, an object of the present invention is to produce chlorine that remains in the recovered PHA particles with a simple means with good reproducibility when producing PHA using treatment with an oxidizing agent containing hypochlorite. The object of the present invention is to provide a method of manufacturing a PHA provided with a processing step that can be removed and reduced.
[0023]
[Means for Solving the Problems]
The present inventors diligently investigated a method for producing PHA for obtaining high-purity PHA in a high yield by removing cell components other than PHA from PHA-containing cells with a small number of steps at low cost and efficiency. As a result, the following invention has been achieved.
[0024]
That is, the present invention is a method for producing PHA comprising a step of treating cells containing PHA with an oxidizing agent to remove cell constituents other than PHA, comprising at least hypochlorite. Treating the cells with an oxidizing agent, separating the treated cells into a water-soluble fraction and a water-insoluble fraction, and reducing chlorine remaining in the water-insoluble fraction. It is characterized by. Further preferably, the step of reducing chlorine is a step of washing the water-insoluble fraction in a solution in which chlorine is soluble.
[0025]
Specifically, one embodiment of the present invention is a method for producing PHA including a step of treating cells containing PHA with an oxidizing agent to remove cell components other than PHA, and comprises hypochlorite. A step of treating the cells with an oxidant containing at least, a step of separating the treated cells into a water-soluble fraction and a water-insoluble fraction, and a step of washing the water-insoluble fraction with warm water It is characterized by including.
[0026]
Another embodiment of the present invention is a method for producing PHA including a step of treating cells containing PHA with an oxidizing agent to remove cell constituents other than PHA, and the method includes at least hypochlorite. A step of treating the cells with an oxidizing agent, and a step of washing the obtained PHA with an aqueous thiosulfate solution.
[0027]
Another embodiment of the present invention is a method for producing PHA including a step of treating cells containing PHA with an oxidizing agent to remove cell constituents other than PHA, and the method includes at least hypochlorite. And the step of treating the cells with an oxidizing agent, and the step of washing the obtained PHA with a polar solvent solution containing at least an organic polar solvent in which the PHA is not soluble. To do.
[0028]
Another embodiment of the present invention is a method for producing PHA including a step of treating a cell containing PHA with an oxidizing agent to remove cell components other than PHA, wherein the cell containing polyhydroxyalkanoate Crushing to obtain a crushed product, and separating the crushed product into a water-soluble fraction and a water-insoluble fraction, and the water-insoluble fraction contains at least the hypochlorite. It is used for the process processed with the oxidizing agent formed.
[0029]
DETAILED DESCRIPTION OF THE INVENTION
In the method for producing PHA of the present invention, the target PHA is not only a microorganism having such PHA producing ability, but also the PHA synthesis gene possessed by the microorganism is introduced using genetic recombination technology. Utilizing higher organisms such as plant cells that have become possible, they can be biosynthesized and accumulated in the cells. Accordingly, the term “cell (also referred to as biological cell or biological cell)” in the present invention refers to not only a microbial cell but also a cell of a higher organism such as a plant cell capable of producing PHA by incorporating a PHA synthetic gene. Also included.
[0030]
As the microorganism having PHA-producing ability as described above, any microorganism can be used as long as it has a desired PHA-producing ability. For example, Ralstonia, which is a PHA-producing bacterium used in the examples of the present invention.・ Utotropa ・ TB64 (Ralstonia eutropha TB64), Pseudomonas oleovorans ・ ATCC 29347 (Pseudomonas oleovorans ATCC 29347, sold from American Type Culture Collection), Pseudomonas chicoryai ・ YN2 sp.TL2) and the like can be used.
[0031]
The TB64 strain is disclosed in Japanese Patent Application Laid-Open No. 2000-166587, and under the deposit number “FERM BP-6933”, the National Institute of Advanced Industrial Science and Technology is based on the Budapest Treaty on the International Approval of Deposit of Microorganisms in Patent Procedures. Has been deposited internationally at the Institute for Patent Biological Deposits (formerly the Ministry of Economy, Trade and Industry's National Institute of Advanced Industrial Science and Technology, Biotechnology Institute of Technology's Patent Microorganism Depositary Center).
[0032]
The YN2 strain has the deposit number “FERM BP-7375” and is based on the Budapest Treaty on the International Approval of Deposits of Microorganisms in Patent Procedures. It has been deposited internationally at the Research Institute for Biotechnology and Industrial Technology (Patent Microbiology Deposit Center). The mycological properties of the YN2 strain are listed as follows.
[0033]
<Mycological properties of YN2 strain>
(1) Morphological properties

(2) Physiological properties
Catalase: positive
Oxidase: Positive
O / F test: Oxidized type (non-fermentable)
Reduction of nitrate: negative
Indole formation: Positive
Glucose acidification: Negative
Arginine dihydrolase: Negative
Urease: Negative
Esculin hydrolysis: Negative
Gelatin hydrolysis: negative
β-galactosidase: negative
Fluorescent dye production on King's B agar: Positive
Growth with 4% NaCl: Positive (weak growth)
Accumulation of poly-β-hydroxybutyric acid: Negative (*)
Tween 80 hydrolysis: positive
* Judged by staining nutrient agar culture colonies with Sudan Black.
(3) Substrate utilization ability
Glucose: Positive
L-arabinose: positive
D-Mannose: Negative
D-mannitol: Negative
N-acetyl-D-glucosamine: negative
Maltose: Negative
Potassium gluconate: positive
n-Capric acid: Positive
Adipic acid: Negative
dl-malic acid: positive
Sodium citrate: positive
Phenyl acetate: positive
Based on the Budapest Treaty on the International Approval of Deposits of Microorganisms in Patent Procedures, the TL2 strain has the deposit number “FERM BP-6913” as an independent administrative agency, National Institute of Advanced Industrial Science and Technology, Deposited internationally at the Research Center for Biotechnology and Industrial Technology, Patent Microbial Deposit Center).
[0034]
In the method of the present invention, the water-insoluble PHA produced in these cells is accumulated in the form of particles. When this PHA particle is recovered by solid-liquid separation, it is removed from other cell components. In order to solubilize the interstitial impurities by treatment with hypochlorite, only water-insoluble PHA particles are separated as solid phase components.
[0035]
That is, in the method of the present invention, after culturing, the cells that have accumulated PHA are recovered from the culture solution, suspended in a treatment solution containing hypochlorite, and in this treatment solution for a certain period of time. By reacting under conditions, cell constituents other than PHA are solubilized and removed, and means for separating PHA particles from the cells is used.
[0036]
Hypochlorite used in the method of the present invention includes potassium hypochlorite (KClO), calcium hypochlorite (Ca (OCl)). ₂ ), Sodium hypochlorite (NaClO), and the like.
The concentration of hypochlorite dissolved in the treatment liquid is preferably selected from the range of 0.5% to 12.0%, preferably 1.5% to 5.0% as the effective chlorine concentration in the treatment liquid. For example, when using a commercially available sodium hypochlorite aqueous solution as hypochlorite, the concentration of sodium hypochlorite ranges from 4% (about 1.7% as effective chlorine concentration) to 6% (about 2.5% as effective chlorine concentration). %) Is desirable. Further, it is preferable to perform the treatment by selecting the amount of the treatment solution within a range of 50 ml to 300 ml per 1 g of dry cell weight. If the processing temperature is higher than room temperature (about 20 ° C), it may also affect the PHA itself, causing partial hydrolysis and the like, leading to a decrease in the molecular weight of the PHA. It is desirable to select and perform processing within the range. In addition, since heat may be generated when sodium hypochlorite comes into contact with cells, special attention is required in that case. When selecting the hypochlorite concentration and temperature conditions, the treatment time can be sufficiently achieved by selecting the treatment time within the range of 1 to 5 hours, usually about 2 hours. If it is less than 1 hour, solubilization of cellular components other than the PHA component is insufficient, and if it is treated for 5 hours or more, it will cause a decrease in the molecular weight of PHA, so care must be taken.
[0037]
After completion of the solubilization reaction, as a means for recovering the PHA particles in the treatment liquid, any solid-liquid separation is possible as long as the PHA particles can be effectively separated from the coexisting solubilized cell components. Means can also be used, but for example, PHA particles can be separated from the soluble fraction using a centrifugation method. The state of PHA at this time is in the form of fine particles suspended in an aqueous solution, but it is necessary to perform a centrifugal separation operation while maintaining the state of the PHA fine particles so that the efficiency of the subsequent dechlorination treatment is increased. It is. That is, it is desirable that the temperature at the time of centrifugation is about 0 ° C. to 10 ° C., preferably 0 ° C. to 5 ° C.
In addition, a step of further washing the PHA particles recovered by the centrifugal separation method with purified water such as deionized water, distilled water, pure water, etc., and sufficiently removing interstitial components that may possibly be mixed in is added. Is more desirable. It is desirable to perform the washing step twice to three times.
[0038]
In particular, when it is necessary to obtain high-purity PHA, for example, an enzyme, an oxidizing agent, a surfactant, or a chemical treatment combining these may be performed after the separation and washing operation of PHA.
[0039]
The PHA can be more effectively separated by further adding a step of disrupting the cells before the step of treatment with hypochlorite. In the process of crushing the cells, ultrasonic crushing method, homogenizer method, pressure crushing method, bead impact method, crushing method, crushing method (a method of crushing in a mortar with the aid of glass powder or alumina powder) It is desirable to use a method that does not use a drug, such as a freeze-thaw method. Further, for this resuspension, the solid component and the soluble component are separated by a method such as centrifugation, and the solid component containing the PHA component is treated with hypochlorite.
[0040]
By the way, as described above, in the present invention, hypochlorite can be suitably used as an oxidizing agent that solubilizes cell components other than PHA. In this treatment with hypochlorite, chlorine resulting from the consumed hypochlorite ions is by-produced, and a part thereof adheres to the surface of the water-insoluble PHA particles and is separated. The weakly adhering part is treated with hypochlorite, separated and separated by solid-liquid separation and then washed with water, but some of the strongly adhering chlorine becomes residual chlorine, It will be detached for the first time when processing with heating. Such residual chlorine can be a problem when considering the use of the obtained PHA.
[0041]
In order to remove such residual chlorine, in the present invention, after the treatment with hypochlorite and the subsequent solid-liquid separation step, the following washing in a chlorine-soluble liquid is performed. A process for further processing was added. The washing process in the liquid is simple as a process such as suspending and stirring, is easy to collect after washing, and does not reduce the collection rate and purity, and therefore is suitable as a method for treating fine particles. It goes without saying that the cleaning liquid is required not only to dissolve chlorine but also not to dissolve PHA. As a cleaning treatment for removing residual chlorine, a means for further suspending the obtained PHA in purified water and desorbing the chlorine remaining in the recovered PHA while heating to 30 ° C. to 60 ° C. and stirring. it can. In this case, the amount of warm water is desirably 50 ml or more per 1 g (dry weight) of PHA, in which the detached chlorine can be sufficiently dissolved, and the treatment time is 6 hours or more (the treatment time increases as the treatment temperature is lowered). In addition, it is desirable to perform this treatment twice or more by replacing the hot water with a new one.
[0042]
Any method can be used as a method for recovering the PHA particles in the cleaning liquid after the completion of the cleaning as long as the PHA particles can be effectively separated and purified from the coexisting components. Separation methods can be used.
[0043]
As another cleaning treatment for removing residual chlorine, the following means for reducing residual chlorine by washing with an aqueous thiosulfate solution can be used.
[0044]
As thiosulfate, potassium thiosulfate (K ₂ S ₂ O _Three ), Potassium thiosulfate trihydrate (K ₂ S ₂ O _Three ・ 3H ₂ O), calcium thiosulfate hexahydrate (CaS) ₂ O _Three ・ 6H ₂ O), ferrous thiosulfate (FeS) ₂ O _Three ), Sodium thiosulfate (Na) ₂ S ₂ O _Three ), Sodium thiosulfate pentahydrate (Na ₂ S ₂ O _Three ・ 5H ₂ O), ammonium thiosulfate ((NH _Four ) ₂ S ₂ O _Three ) And the like. Moreover, the solution and mixture containing said thiosulfate can also be used.
[0045]
Of the thiosulfates, sodium thiosulfate pentahydrate (Na ₂ S ₂ O _Three ・ 5H ₂ O, commonly known as “Hypo”) has been used mainly as a “decolorizer” for tap water. Sodium thiosulfate pentahydrate has an action of absorbing chlorine in an aqueous solution, and the mechanism is as shown in the following formula [1].
[0046]
Na ₂ S ₂ O _Three + 4Cl ₂ + 5H ₂ O → 2NaCl + 2H ₂ SO _Four + 6HCl [1]
By washing the PHA with a thiosulfate aqueous solution, the residual chlorine retained in the PHA is removed by the reaction [1] above.
[0047]
The temperature in the washing treatment with thiosulfate is appropriately set according to the physical properties of the PHA, but is usually set to 0 ° C to 100 ° C, preferably 20 ° C to 70 ° C. The cleaning time in the main cleaning process is 5 minutes to 24 hours, preferably 1 hour to 3 hours.
[0048]
As a method for recovering the PHA particles in the cleaning liquid after completion, any method can be used as long as it can effectively separate and purify the PHA particles from the coexisting components. Can be used. Further, it is more desirable to add a treatment for further washing the PHA particles washed by this washing treatment with purified water.
[0049]
Further, as another cleaning treatment for removing residual chlorine, the following means for reducing residual chlorine by washing with an organic polar solvent can be used.
[0050]
In this cleaning treatment with a polar solvent solution, residual chlorine adhering to the surface of the PHA particles is washed with a polar solvent solution containing an organic polar solvent having no solubility in the target PHA itself at a concentration of 50% or more of the total volume. Then, it is eluted and reduced in this polar solvent solution.
[0051]
Most of the chlorine adhering firmly to the surface of the water-insoluble PHA particles can be removed by washing using an organic polar solvent as described above. In addition, since the absolute amount of residual chlorine is greatly reduced in the PHA particles obtained by this method, the amount of chlorine released even when heat is applied when manufacturing a processed product using such PHA particles as a raw material. Can be suppressed to the extent that it does not cause factors such as contamination of the working environment.
[0052]
As the organic polar solvent used for the washing treatment with the polar solvent solution, alcohol, ketone or the like can be used. More specifically, for example, it can be appropriately selected from methanol, ethanol, isopropanol, isobutanol, acetone and the like. These organic polar solvents are generally used alone, but a mixed solvent containing a plurality of types can also be used. Furthermore, since the organic polar solvents such as alcohol and ketone have a high affinity with water, they can also be used as a mixed solvent mixed uniformly with water. When used as a mixed solvent uniformly mixed with water, it is desirable to increase the content (concentration) of the organic polar solvent. For example, when the content (concentration) is 99% or more, a single solvent is used. A cleaning effect comparable to that of the other can be achieved.
[0053]
The temperature in the washing treatment with the polar solvent solution is appropriately selected according to the physical properties of the target PHA, for example, the solubility in the organic polar solvent, the softening point, the melting point, etc. It is desirable to select a temperature range that is in a liquid state and less transpiration, for example, a range of 0 ° C to 50 ° C. The cleaning time in this cleaning process depends on the type and temperature of the organic polar solvent used, but it is sufficient to select it within the range of 5 minutes to 24 hours, preferably within the range of 1 hour to 3 hours. Cleaning and removal can be achieved.
[0054]
After the cleaning treatment using the polar solvent solution is completed, as a means for recovering the washed PHA particles from the liquid containing the organic polar solvent, the PHA particles are dissolved in the polar solvent solution containing the organic polar solvent. Any solid-liquid separation method can be used as long as the coexisting components can be effectively separated. For example, it is possible to separate a component dissolved in a liquid containing an organic polar solvent from PHA particles by using a centrifugal separation method. Further, it is more desirable that after the washing treatment using the organic polar solvent, the recovered PHA particles are washed again with purified water or the like to remove the residual organic polar solvent.
[0055]
It has been found that the cleaning effect in the cleaning treatment with the polar solvent solution is not dependent on the type of PHA particles, has versatility, and has high reproducibility. In addition, since the amount of PHA particles is much smaller than the original cell volume, the organic polar solvent used for washing using the organic polar solvent is the amount of organic solvent used in the organic solvent extraction method. Is so small that it cannot be compared. Moreover, the PHA itself is not dissolved, only the chlorine adhering to its surface is eluted, and the washed PHA particles are recovered again by using a solid-liquid separation method. It is also possible to extremely reduce the amount of solvent. In addition, a small amount of organic solvent remains in the aqueous layer as in organic solvent extraction, and it is necessary to recover it, or a large amount that is eliminated by removing the solvent when recovering PHA from the organic phase. Organic solvent recovery operations that become increasingly difficult as the scale increases, such as the necessity of performing a recovery process for the organic solvent, can be more easily performed with the method of the present invention.
[0056]
As described above, the cleaning according to the present invention has the same meaning as that usually used, and is a series of steps in which PHA particles are suspended in the liquid and stirred to separate the cleaning liquid and the PHA particles. However, if the same cleaning effect can be obtained, stirring can be omitted, the separation can be performed continuously, and the cleaning liquid can be discharged and added continuously, or this process can be changed as necessary. Good.
[0057]
In the step of drying the PHA particles obtained in the above-mentioned step of removing residual chlorine, natural drying, vacuum drying, freeze drying, etc. are appropriately selected according to the physical properties of the PHA, the form to be obtained, etc. Shall.
[0058]
As an apparatus for producing PHA by the above-described method, apparatuses used for normal microorganism production, chemical synthesis, and the like can be used alone or in combination. Further, production of PHA having the following configuration is used. An apparatus can be illustrated as one form of the manufacturing apparatus optimal for the manufacturing method of PHA of this invention.
[0059]
Means for treating cells containing PHA with the above hypochlorite to remove cell components other than PHA, and treating the treated cells with a water-soluble fraction and a water-insoluble fraction (PHA). And a cleaning means for removing residual chlorine from the water-insoluble fraction. Means for removing residual chlorine include means for washing the water-insoluble fraction with warm water, means for washing the water-insoluble fraction with an aqueous thiosulfate solution, and dissolving the water-insoluble fraction in the PHA itself. It comprises at least one means selected from the group consisting of means for washing with a polar solvent solution containing an organic polar solvent having no properties. Furthermore, if necessary, before the means for treating with hypochlorite, means for crushing PHA-containing cells to obtain a crushed product, the crushed product with a water-soluble fraction and a water-insoluble fraction. It is also possible to provide a manufacturing apparatus having a configuration including a separating means.
[0060]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. These examples are examples of the best mode of the present invention, but the present invention is not limited to these examples. In the following examples, “%” is based on weight unless otherwise specified. The M9 medium used in each example has the following composition.
[0061]
[M9 medium]
Na ₂ HPO _Four 6.2g
KH ₂ PO _Four 3.0g
NaCl 0.5g
NH _Four Cl 1.0g
(PH 7.0 in 1 liter of medium)
(Example 1)
A colony of the TB64 strain on M9 agar medium containing 0.1% sodium malate was inoculated into 50 ml of M9 medium containing 0.5% sodium malate in a 500 ml shake flask and cultured at 30 ° C. with shaking. After 24 hours, 5 ml of the culture solution was added to NH as a nitrogen source. _Four Only Cl was added to 1 liter of a medium containing 0.5% sodium malate in M9 medium prepared to a concentration of 1/10, and similarly shaken to accumulate PHB in the cells. After 52 hours, the PHB-accumulating cells were harvested by centrifugation, resuspended in 30 ml of distilled water and divided into three equal parts (10 ml each). These were numbered from 1 to 3, and the following processing was performed.
[0062]
1: Control: After centrifugation, washed with methanol, freeze-dried, weighed, extracted with chloroform at 60 ° C for 24 hours, filtered, concentrated, reprecipitated with methanol, and dried under reduced pressure. A control polymer (referred to as sample 1) was obtained.
[0063]
2: 30 ml of deionized water cooled to 4 ° C was added, and 20 ml of sodium hypochlorite solution (made by Kishida Chemical Co .; containing 12% sodium hypochlorite (effective chlorine concentration of 5% or more)) cooled to 4 ° C In addition, the mixture was reacted at 4 ° C. for 2 hours. After completion of the reaction, 140 ml of deionized water cooled to 4 ° C was added to the reaction solution, and 4 ° C, 29400 m / s. ² Centrifugation was performed at (3000 × g) for 30 minutes. 80 ml of deionized water cooled to 4 ° C. was added to the resulting precipitate, and the particles were sufficiently dispersed by ultrasonic treatment, followed by centrifugation (4 ° C., 29400 m / s). ² (3000 × g) for 30 minutes). This operation was further repeated twice, and the resulting precipitate was lyophilized. This is designated as Sample 2.
[0064]
The sample washed with deionized water of 3: 2 was suspended in 50 ml of deionized water, stirred at 50 ° C for 6 hours, and then the supernatant was centrifuged (4 ° C, 29400 m / s). ² (3000 × g for 30 minutes). This warm water washing treatment was repeated once more. 40 ml of deionized water cooled to 4 ° C. was added to the resulting precipitate, and the particles were sufficiently dispersed by sonication, followed by centrifugation (4 ° C., 29400 m / s). ² (3000 × g) for 30 minutes). This operation was further repeated once, and the resulting precipitate was lyophilized. This is designated as Sample 3.
[0065]
<Measurement of residual chlorine>
For sample 2 and sample 3, residual chlorine was measured as follows.
[0066]
1: Erlenmeyer flask (total volume 637 ml), 2a: outer lid with a hole in the center (screw cap), 2b: Teflon inner lid with a hole in the center, and 2c: inner lid A lid made of aluminum foil of the same size as in the above was heated to 150 ° C in an oven in advance, 3: placed on the aluminum foil, 4: 0.3g of sample was put in, and the lid was sealed. Heated. Three minutes later, 6: detector tube (made by Gastec; chlorine gas detector tube, product number 8La; detection range concentration: 0.5ppm to 8ppm or product number 8H; detection range concentration: 0.5ppm to 8ppm) is inserted through the hole in the lid. The chlorine concentration was measured by aspirating 100 ml.
[0067]
As a result, the chlorine concentration of sample 2 that was not subjected to the hot water treatment was about 50 ppm, whereas that of sample 3 that was subjected to the hot water treatment was about 4 ppm. Sample 3 is more effective in reducing chlorine than sample 2.
[0068]
Next, in order to evaluate the “recovery rate” and “purity” shown below, the following operation was performed.
[0069]
Chloroform 30 ml was added to the dried samples 1 to 3, followed by stirring and extraction at 60 ° C. for 24 hours. The chloroform solution from which PHB was extracted was filtered through a 0.45 μm PTFE filter, concentrated by a rotary evaporator, poured into 10 times the amount of methanol, and PHB was precipitated and collected. These were dried under reduced pressure and weighed.
[0070]
The mass ratio of PHB obtained by chloroform extraction of samples 2 and 3 with respect to 1 as the control is defined as “recovery rate”, and the mass ratio of PHB obtained by chloroform extraction of each sample to the sample before chloroform extraction is The “purity” is shown in Table 1.
[0071]
[Table 1]

[0072]
Although Sample 3 was subjected to hot water treatment, the recovery rate and purity were almost the same as Sample 2 that was not subjected to hot water treatment. Therefore, the hot water treatment has an effect of reducing residual chlorine while maintaining a high recovery rate and purity.
[0073]
(Example 2)
Pseudomonas oleovorans colonies on M9 agar medium containing 0.1% n-nonanoic acid were inoculated into 50 ml of M9 medium containing 0.3% n-nonanoic acid and cultured at 30 ° C. with shaking. After 40 hours, 5 ml of the culture solution was added to 1 liter of M9 medium containing 0.2% n-nonanoic acid and 0.05% 5-phenylvaleric acid, and cultured with shaking. After another 36 hours, the cells were harvested by centrifugation, and NH, the nitrogen source, was collected. _Four Cl-free, resuspended in 1 liter of M9 medium containing 0.05% n-nonanoic acid and 0.2% 5-phenylvaleric acid, and shaken in the same manner to give 3-hydroxynonanoic acid, 3-hydroxyheptanoic acid to the cells. PHA containing 3-hydroxyvaleric acid and 3-hydroxy-5-phenylvaleric acid as a unit was accumulated. After 48 hours, the PHA-accumulating cells were harvested by centrifugation and subjected to the following treatment.
[0074]
4: Control: Centrifugation, washing with methanol, lyophilization, weighing, chloroform extraction at 60 ° C for 24 hours, filtration, concentration, reprecipitation with methanol, drying under reduced pressure A control polymer (referred to as sample 4) was obtained.
[0075]
5: Add 30 ml of deionized water cooled to 4 ° C and add 20 ml of sodium hypochlorite solution (made by Kishida Chemical Co .; containing 12% sodium hypochlorite (effective chlorine concentration of 5% or more)) cooled to 4 ° C. In addition, the mixture was reacted at 4 ° C. for 2 hours. After completion of the reaction, 140 ml of deionized water cooled to 4 ° C is added to the reaction solution, and 4 ° C, 29400 m / s. ² Centrifugation was performed at (3000 × g) for 30 minutes. 80 ml of deionized water cooled to 4 ° C. was added to the resulting precipitate, and the particles were sufficiently dispersed by ultrasonic treatment, followed by centrifugation (4 ° C., 29400 m / s). ² (3000 × g) for 30 minutes). Further, this operation was repeated twice, and the resulting precipitate was lyophilized to obtain Sample 5.
[0076]
The sample washed with deionized water in the 6: 5 operation was suspended in 50 ml of deionized water and stirred at 30 ° C. for 12 hours. Centrifuge the supernatant (4 ° C, 29400m / s ² (3000 × g for 30 minutes), resuspended in 50 ml deionized water and stirred at 30 ° C. for 12 hours. After completion of the treatment, the supernatant is centrifuged (4 ° C, 29400 m / s ² (3000 × g for 30 minutes) and 40 ml of deionized water cooled to 4 ° C. was added to the resulting precipitate, and the particles were sufficiently dispersed by sonication, followed by centrifugation ( 4 ℃, 29400m / s ² (3000 × g) for 30 minutes). This operation was further repeated once, and the resulting precipitate was freeze-dried to obtain Sample 6.
[0077]
For samples 5 and 6, residual chlorine was measured by the same method as in Example 1. As a result, the chlorine concentration of the sample 5 not subjected to the hot water treatment was about 20 ppm (converted from the 30 ml subtraction of the detector tube 8La), whereas the sample 6 subjected to the hot water treatment was about 2 ppm. Sample 6 is more effective in reducing chlorine than sample 5.
[0078]
Further, these samples were extracted with chloroform in the same manner as in Example 1 to obtain the recovery rate and purity. The results are shown in Table 2.
[0079]
[Table 2]

[0080]
The recovery rate and purity of the hot water treated sample 6 were almost the same as those of the sample 5 not subjected to the hot water treatment. Similar to the PHB of Example 1, for the PHA having an aromatic ring as in this example, the hot water treatment has the effect of reducing the residual chlorine while keeping the recovery rate and purity high.
[0081]
(Example 3) Washing with aqueous sodium thiosulfate solution (1)
YN2 strain was inoculated into 200 ml of M9 medium containing 0.5% yeast extract (Oriental Yeast Industry Co., Ltd.) and cultured under shaking in a 500 ml shake flask at 30 ° C. and 125 strokes / minute. After 8 hours, the culture solution was added to 25 liters of M9 medium containing 0.5% of polypeptone (Nippon Pharmaceutical Co., Ltd.) and 0.1% of 5-thiophenoxyvaleric acid, and 30 ° C in a 50 liter jar fermenter. And aerated and agitated for 48 hours.
[0082]
Centrifuge 200ml of the above culture broth (78000m / s ² (8000 × g), 4 ° C., 10 minutes), the microbial cells were collected, washed once with cold methanol, and vacuum-dried. This vacuum-dried pellet was suspended in 20 ml of chloroform and stirred at 60 ° C. for 20 hours to extract PHA. The extract was filtered through a membrane filter having a pore size of 0.45 μm and then concentrated with a rotary evaporator. The concentrate was reprecipitated in cold methanol, and only the precipitate was collected and dried in vacuo to obtain PHA. This PHA was subjected to methanolysis according to a conventional method, and analyzed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) to identify the methyl esterified product of the PHA monomer unit. As a result, it was found that the microbial cells in the culture broth contained PHA having the monomer unit composition shown in Table 3.
[0083]
[Table 3]

[0084]
Microbial cells were collected from the culture solution other than that used in the above analytical operation by centrifugation. The obtained cells were suspended in 1.6 liters of purified water, and 0.8 liter of sodium hypochlorite solution (effective chlorine concentration of 5% or more) was added. This was shaken at 4 ° C. for 2 hours to solubilize cell components other than PHA, and then centrifuged (29400 m / s ² (3000 × g), 4 ° C., 30 minutes) to recover PHA. The obtained PHA is suspended in 200 ml of purified water and centrifuged (29400 m / s ² The operation of recovering PHA by (3000 × g), 4 ° C., 30 minutes) was repeated 3 times to perform water washing to obtain PHA particles (referred to as sample 7).
[0085]
Residual chlorine retained in the PHA particles was evaluated by the following method.
[0086]
(1) Sensory evaluation
Sensitively determine the chlorine odor of PHA particles left at room temperature, in order from the one with the strong chlorine odor,
++++ (strong), ++ (medium), + (low), ± (weak),-(none)
It was evaluated in five stages.
[0087]
(2) Concentration measurement
The above PHA particles were put in a 500 ml lid flask and sealed, heated in an atmosphere of 150 ° C. for 1 hour to release residual chlorine, and then the chlorine concentration in the flask was measured using a gas detector tube. .
[0088]
The results of evaluation of residual chlorine are shown in Table 4. From Table 4, the residual chlorine in the PHA particles was observed.
[0089]
[Table 4]

[0090]
PHA particles corresponding to 1.0 g in dry weight obtained by the above method were suspended in 50 ml of purified water or 10% sodium thiosulfate pentahydrate aqueous solution. These suspensions were stirred at 50 ° C. for 6 hours and then centrifuged (29400 m / s ² (3000 × g), 4 ° C., 30 minutes) to recover PHA. The obtained PHA is suspended in 40 ml of purified water and centrifuged (29400 m / s ² The operation of recovering PHA by (3000 × g), 4 ° C., 30 minutes) was repeated three times. The obtained PHA was suspended in 10 ml of purified water and lyophilized to obtain PHA washed particles. Washed PHA particles using purified water are referred to as Sample 8, and washed PHA particles using 10% sodium thiosulfate · pentahydrate aqueous solution are referred to as Sample 9.
[0091]
The residual chlorine retained in the cleaning particles was evaluated by the following method.
[0092]
(1) Sensory evaluation
Sensitively determine the chlorine odor of PHA particles left at room temperature, in order from the one with the strong chlorine odor,
++++ (strong), ++ (medium), + (low), ± (weak),-(none)
It was evaluated in five stages.
[0093]
(2) Concentration measurement
The above cleaning particles were placed in a 500 ml lid flask and sealed, heated in an atmosphere at 150 ° C. for 1 hour to release residual chlorine, and then the chlorine concentration in the flask was measured using a gas detector tube. .
[0094]
The results of evaluation of residual chlorine are shown in Table 5. Comparison of Tables 4 and 5 revealed that warm water washing with purified water has an effect of reducing chlorine concentration, but washing with aqueous sodium thiosulfate solution further reduces residual chlorine in the PHA washing particles.
[0095]
[Table 5]

[0096]
(Example 4) Washing with aqueous sodium thiosulfate solution (2)
The PHA particles corresponding to 1.0 g in dry weight of Sample 7 obtained by the method of Example 3 were mixed with 50 ml each of 0%, 0.1%, 1.0%, or 10% sodium thiosulfate pentahydrate aqueous solution. It was suspended in. These four types of suspensions were stirred for 6 hours at 50 ° C and then centrifuged (29400 m / s ² (3000 × g), 4 ° C., 30 minutes) to recover PHA. The obtained PHA was suspended in 50 ml of purified water and further stirred at 50 ° C. for 6 hours, followed by centrifugation (29400 m / s ² (3000 × g), 4 ° C., 30 minutes) to recover PHA. The obtained PHA is suspended in 40 ml of purified water and centrifuged (29400 m / s ² The operation of recovering PHA by (3000 × g), 4 ° C., 30 minutes) was repeated three times. The obtained PHA was suspended in 10 ml of purified water and lyophilized to obtain PHA washed particles. Samples 10, 11, 12, and 13 having concentrations of an aqueous sodium thiosulfate solution of 0%, 0.1%, 1.0%, and 10%, respectively.
[0097]
The residual chlorine retained in the cleaning particles was evaluated by the following method.
[0098]
(1) Sensory evaluation
Sensitively determine the chlorine odor of the cleaning particles left at room temperature, in order from the one with the strong chlorine odor,
++++ (strong), ++ (medium), + (low), ± (weak),-(none)
It was evaluated in five stages.
[0099]
(2) Concentration measurement
The above cleaning particles were placed in a 500 ml lid flask and sealed, heated in an atmosphere at 150 ° C. for 1 hour to release residual chlorine, and then the chlorine concentration in the flask was measured using a gas detector tube. .
[0100]
The results of evaluation of residual chlorine are shown in Table 6. According to Table 4 and Table 6, warm water washing of purified water (0% sodium thiosulfate aqueous solution) has the effect of reducing the chlorine concentration, but there is a subtle difference due to sensory evaluation by washing with sodium thiosulfate aqueous solution. It was clarified that the residual chlorine in the cleaning particles of PHA was reduced and the effect was larger as the concentration of sodium thiosulfate was higher.
[0101]
[Table 6]

[0102]
(Example 5) Cleaning with alcohol
Alcaligenes sp. TL2 strain was inoculated into 1.6 liter of M9 medium supplemented with 0.5% sodium lactate, and cultured with shaking at 30 ° C. and 125 strokes / min. After 48 hours, the cells are centrifuged from the medium (78000 m / s ² (8000 × g), 4 ° C., 10 minutes).
[0103]
Cells obtained by the above culturing operation were suspended in 80 ml of purified water, and 40 ml of sodium hypochlorite solution (effective chlorine concentration of 5% or more) was added. This suspension was shaken at 4 ° C. for 2 hours to solubilize cell components other than PHA, and then centrifuged (59000 m / s ² (6000 × g), 4 ° C., 20 minutes) to recover PHA. The recovered PHA is suspended again in 40 ml of purified water and centrifuged (78000 m / s ² The operation of recovering the PHA particles by (8000 × g), 4 ° C., 10 minutes) was repeated twice in total to perform the PHA water washing treatment to obtain PHA particles.
[0104]
A part of the obtained PHA is freeze-dried, then subjected to methanolysis according to a conventional method, and analyzed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) to constitute PHA. The methyl esterified product of the monomer unit was identified. As a result of the identification, the obtained PHA was PHB having 3-hydroxybutyric acid as a monomer unit.
[0105]
The PHA particles corresponding to 0.3 g in dry weight obtained in the above step were suspended in 40 ml each of purified water, methanol, ethanol, and isobutanol. The suspension was shaken at 4 ° C. for 1 hour and then centrifuged (29400 m / s ² The PHA particles were recovered by (3000 × g), 4 ° C., 20 minutes). The recovered PHA particles are suspended again in 40 ml of purified water and centrifuged (78000 m / s ² (8000 × g), 4 ° C., 10 minutes) to recover the PHA particles. The washed PHA particles were suspended in 10 ml of purified water and freeze-dried to obtain washed particles of PHA. The PHA particles in which the washing liquid is purified water, methanol, ethanol, and isobutanol are referred to as Samples 14, 15, 16, and 17, respectively.
[0106]
The amount of chlorine released from the washed PHA particles (Sample 14-17) was measured by the following method when left at room temperature and when heated. The washed particles were placed in a 50 ml vial, sealed, allowed to stand overnight at room temperature, and then the amount of chlorine contained in the gas phase in the vial was measured using a gas detector tube. Next, after the vial was heated on a hot plate at 150 ° C. for 2 minutes, the amount of chlorine contained in the gas phase in the vial was measured using a gas detection tube.
[0107]
Table 7 shows the measurement results. From the results shown in Table 7, when washed with alcohol (sample 15-17), compared with the case of washed with purified water (sample 14), it is released from the washed PHA particles. It was revealed that the amount of chlorine was reduced to about half or more in both standing at room temperature and heating.
[0108]
[Table 7]

[0109]
(Example 6) Cleaning treatment with ketone
Alcaligenes sp. TL2 strain was inoculated into 1.6 liters of M9 medium supplemented with 0.5% sodium lactate and cultured with shaking at 30 ° C. and 125 strokes / min. After 48 hours, the cells are centrifuged from the medium (78000 m / s ² (8000 × g), 4 ° C., 10 minutes).
[0110]
Cells obtained by the above culturing operation were suspended in 80 ml of purified water, and 40 ml of sodium hypochlorite solution (effective chlorine concentration of 5% or more) was added. This suspension was shaken at 4 ° C. for 2 hours to solubilize cell components other than PHA, and then centrifuged (59000 m / s ² (6000 × g), 4 ° C., 20 minutes) to recover PHA. The recovered PHA is suspended again in 40 ml of purified water and centrifuged (78000 m / s ² The operation of recovering the PHA particles by (8000 × g), 4 ° C., 10 minutes) was repeated twice in total to perform the PHA water washing treatment to obtain PHA particles.
[0111]
A part of the obtained PHA is freeze-dried, then subjected to methanolysis according to a conventional method, and analyzed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) to constitute PHA. The methyl esterified product of the monomer unit was identified. As a result of the identification, the obtained PHA was PHB having 3-hydroxybutyric acid as a monomer unit.
[0112]
The PHA particles corresponding to 0.3 g in dry weight obtained in the above step were suspended in 40 ml of purified water or acetone. The suspension was shaken at 4 ° C. for 1 hour and then centrifuged (29400 m / s ² The PHA particles were recovered by (3000 × g), 4 ° C., 20 minutes). The recovered PHA particles are suspended again in 40 ml of purified water and centrifuged (78000 m / s ² (8000 × g), 4 ° C., 10 minutes), PHA particles were recovered. After the washing treatment, the PHA particles were suspended in 10 ml of purified water and lyophilized to obtain PHA washed particles. Sample 18 is the one in which the above two washings were purified water washing, and sample 19 is the first washing in acetone.
[0113]
The amount of chlorine released from the washed PHA particles was measured by the following method when left at room temperature and when heated. The washed particles were placed in a 50 ml vial, sealed, allowed to stand overnight at room temperature, and then the amount of chlorine contained in the gas phase in the vial was measured using a gas detector tube. Next, after the vial was heated on a hot plate at 150 ° C. for 2 minutes, the amount of chlorine contained in the gas phase in the vial was measured using a gas detection tube.
[0114]
Table 8 shows the measurement results. From the results shown in Table 8, the amount of chlorine released from the cleaned PHA particles when washed with acetone (sample 19) compared to when washed with purified water (sample 18) It has become clear that the amount of chlorine remaining in the washed PHA particles is greatly reduced.
[0115]
[Table 8]

[0116]
(Example 7) Crushing treatment
A colony of the TB64 strain on M9 agar medium containing 0.1% sodium malate was inoculated into 50 ml of M9 medium containing 0.5% sodium malate in a 500 ml shake flask and cultured at 30 ° C. with shaking. After 24 hours, 5 ml of the culture solution was added to NH as a nitrogen source. _Four Only Cl was added to 1 liter of a medium containing 0.5% sodium malate in M9 medium prepared to a concentration of 1/10, and similarly shaken to accumulate PHB in the cells. After 48 hours, PHB accumulating cells were harvested by centrifugation, resuspended in 50 ml of distilled water and divided into 5 equal portions (10 ml each). These were subjected to the following 20-24 treatments.
[0117]
20: Control: Centrifugation, washing with methanol after centrifuging one of the above five suspensions, lyophilizing and weighing, followed by extraction with chloroform at 60 ° C for 24 hours, filtering and concentrating the extract Then, it was reprecipitated with methanol and dried under reduced pressure to obtain a control polymer (referred to as Sample 20).
[0118]
21: 40 ml of 31% aqueous hydrogen peroxide (manufactured by Mitsubishi Gas Chemical Co., Inc .; JIS K-8230) was added to the other one of the suspensions divided into 5 equal parts and treated at 80 ° C. for 1 hour.
[0119]
22: Another suspension of the above 5 divided portions was made up to 50 ml with distilled water and subjected to a French press (manufactured by Otake Manufacturing Co., Ltd .: French press 5501), then 4 ° C., 29400 m / s ² Centrifugation was performed at (3000 × g) for 30 minutes. After that, add 40ml of distilled water, 4 ℃, 29400m / s ² Washing was performed by centrifugation at (3000 × g) for 30 minutes.
[0120]
23: After carrying out the same operation as 22 in another suspension obtained by dividing the above into 5 equal parts, the precipitate was suspended in 10 ml of distilled water, 40 ml of 31% hydrogen peroxide was added, and For 1 hour.
[0121]
24: After the same operation as 22 was performed on the last one of the above 5 divided suspensions, the precipitate was suspended in 10 ml of distilled water, and 5 ml of sodium hypochlorite solution (manufactured by Kishida Chemical Co., Ltd.). Sodium 12 chlorite (containing an effective chlorine concentration of 5% or more) was added, and the mixture was treated at 4 ° C. for 2 hours.
[0122]
Each of the treatments Nos. 21 to 24 was centrifuged (4 ° C, 29400 m / s ² (30 minutes at 3000 × g), and after cooling again to 4 ° C., 40 ml of distilled water was added and stirred well, then centrifuged twice under the same conditions, and the precipitate was lyophilized and weighed. These are designated as samples 21 to 24, respectively.
[0123]
In order to evaluate the “recovery rate” and “purity” shown below, the following operations were performed.
[0124]
30 ml of chloroform was added to the lyophilized samples 20 to 24, followed by stirring and extraction at 60 ° C. for 24 hours. The chloroform solution from which PHB was extracted was filtered through a 0.45 μm PTFE filter, concentrated by a rotary evaporator, poured into 10 times the amount of methanol, and PHB was precipitated and collected. These were dried under reduced pressure and weighed.
[0125]
The mass ratio of PHB obtained by chloroform extraction of samples 21 to 24 with respect to 20 as the control is defined as “recovery rate”, and the mass ratio of PHB obtained by chloroform extraction of each sample to the sample before chloroform extraction is defined as “recovery rate”. The “purity” is shown in Table 9.
[0126]
[Table 9]

[0127]
Although the recovery rate does not change that much, the purity is higher than that of Sample 21 treated with hydrogen peroxide without passing through a French press and Sample 22 treated only with French press and not treated with hydrogen peroxide. Sample 23, which was treated with hydrogen peroxide after that, and Sample 24, which was treated with French press and then treated with hypochlorous acid, were good. It turns out that purity is improved by combining a crushing process and an oxidizing agent process.
[0128]
The molecular weight of the obtained PHB was measured by gel permeation chromatography (GPC; Tosoh HLC-8020, column: polymer laboratory PLgel MIXED-C (5 μm), solvent: chloroform, converted to polystyrene). The results are shown in Table 10.
[0129]
[Table 10]

[0130]
There was almost no difference in the molecular weight of each sample. Compared with the conventional chloroform extraction, it can be seen that there is no change in molecular weight due to crushing or oxidizing agent treatment.
[0131]
The sample 24 is further subjected to a process of reducing residual chlorine, thereby reducing the chlorine concentration and obtaining final PHA particles. As the chlorine reduction step, hot water washing can be used as in the first embodiment, but any of the chlorine reduction steps in any of the second to sixth embodiments may be used.
[0132]
According to the method of the present invention, polyhydroxyalkanoate accumulated in cells such as microorganisms can be recovered at a high recovery rate by a simple method and while maintaining the original molecular weight. Further, by using the method for producing PHA of the present invention, it is possible to efficiently produce PHA particles with reduced residual chlorine on an industrial scale without using an organic solvent extraction means.
[0133]
The polyhydroxyalkanoate particles obtained by the method of the present invention are useful raw materials as biodegradable materials, biocompatible materials, various functional materials, etc., and are expected to be applied to various fields such as device materials and medical materials. it can.
[Brief description of the drawings]
1 represents a chlorine concentration measurement method in Example 1 and Example 2. FIG.
[Explanation of symbols]
1: Erlenmeyer flask
2: Lid
2a: outer lid
2b: Inner lid
2c: Aluminum foil
3: Aluminum foil
4: Sample
5: Detector tube suction part
6: Detector tube

Claims (5)

Treating cells containing polyhydroxyalkanoate with an oxidizing agent containing at least hypochlorite;
Separating the treated cells into a water soluble fraction and a water insoluble fraction;
A step of reducing chlorine remaining in the water-insoluble fraction by washing the water-insoluble fraction with an aqueous thiosulfate solution at a washing treatment temperature of 20 ° C. to 70 ° C. Method for producing hydroxyalkanoate.   The method according to claim 1, wherein the concentration of the hypochlorite is in the range of 1.5% to 5.0% as an effective chlorine concentration.   The manufacturing method according to claim 1, wherein the hypochlorite is sodium hypochlorite. The polyhydroxyalkanoate The production method according to any one of claims 1 to 3, characterized in that a poly-3-hydroxybutyrate. Means for treating cells containing polyhydroxyalkanoate with an oxidizing agent containing at least hypochlorite;
Means for separating the treated cells into a water soluble fraction and a water insoluble fraction;
And a means for reducing chlorine remaining in the water-insoluble fraction by washing the water-insoluble fraction with an aqueous thiosulfate solution at a washing treatment temperature of 20 ° C. to 70 ° C. An apparatus for producing polyhydroxyalkanoate.

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