JP2022152571A - シトクロムの製造方法 - Google Patents
シトクロムの製造方法 Download PDFInfo
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Landscapes
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Abstract
Description
シトクロムを製造する方法であって、
ハロモナス属に属する好塩菌を培地中で好気培養して、当該好塩菌にポリ3-ヒドロキシ酪酸を蓄積させるPHB蓄積工程と、
前記培地から前記シトクロムを回収する回収工程と、を含む点にある。
前記PHB蓄積工程において、10時間以上好気培養を行う点にある。
前記PHB蓄積工程後に、前記好塩菌を培地中でpHを調整しながら微好気培養して、当該好塩菌の菌体内に蓄積されたポリ3-ヒドロキシ酪酸を3-ヒドロキシ酪酸として培地中に分泌させる3HB分泌工程を行う点にある。
前記シトクロムがシトクロムcである点にある。
前記好塩菌が、ハロモナス・エスピー(Halomonas sp.)KM-1株である点にある。
本発明のシトクロムの製造方法は、ハロモナス属に属する好塩菌を培養して、培地中にシトクロムを放出させ、培地からシトクロムを回収する方法である。
(1)ハロモナス属に属する好塩菌を培地中で好気培養して、当該好塩菌にポリ3-ヒドロキシ酪酸を蓄積させる工程(PHB蓄積工程)と、
(2)培地からシトクロムを回収する回収工程。
(3)PHB蓄積工程後に、好塩菌を培地中でpHを調整しながら微好気培養して、当該好塩菌の菌体内に蓄積されたポリ3-ヒドロキシ酪酸を3-ヒドロキシ酪酸として培地中に分泌させる工程(3HB分泌工程)。
本発明の製造方法におけるPHB蓄積工程は、ハロモナス属に属する好塩菌(以下、単に好塩菌ともいう)を培地中で好気培養して、この好塩菌にポリ3-ヒドロキシ酪酸を蓄積させる工程である。
本発明の製造方法における3HB分泌工程は、PHB蓄積工程後に、好塩菌を培地中でpHを調整しながら微好気培養して、当該好塩菌の菌体内に蓄積されたポリ3-ヒドロキシ酪酸を3-ヒドロキシ酪酸として培地中に分泌させる工程である。
本発明の製造方法における回収工程は、PHB蓄積工程又は3HB分泌工程後の培地中から、シトクロムを回収する工程である。
容積90Lのジャーファーメンターに上記スクロース水溶液を追加した培地を50L張り込み、ハロモナス・エスピーKM-1株を40時間好気培養した(PHB蓄積工程)。その後、適切な分子量分画の膜分離を組み合わせることで、濃縮液側にヘム鉄を含む赤色成分が分離された(回収工程)。この濃縮液について、Native PAGE(登録商標)によるタンパク質分離を行い、赤色成分が存在するバンドを切り出してアミノ酸配列を決定したところ、シトクロムc5(Accession Number WP_010626206.1)であると同定された。
濃縮液中のタンパク質1g当たりの鉄量をICPで測定したところ、50mgであることが判明し、実施例1では、スクロース1kgからシトクロムcとして1.1g、ヘム鉄として55mgを製造できることを確認した。
容積90Lのジャーファーメンターに上記スクロース水溶液を追加した培地を50L張り込み、ハロモナス・エスピーKM-1株を40時間好気培養した(PHB蓄積工程)。その後、曝気を止めて、pHを調整しながら5時間微好気培養し、菌体内に蓄積されたPHBを3HBとして菌体外に分泌させた(3HB分泌工程)。続いて、実施例1と同様に、膜分離を行い、濃縮液を回収した(回収工程)。濃縮液について、紫外可視吸収スペクトルを測定したところ、ヘム鉄に特有の450nm付近のOD値が実施例1の約8倍程度であった。このことから、3HB分泌工程を行うことで、シトクロムcの放出量が顕著に増大することが確認できた。
また、濃縮液中のタンパク質1g当たりの鉄量をICPで測定したところ、440mgであることが判明し、実施例2では、スクロース1kgからシトクロムcとして10g、ヘム鉄として4400mgを製造できることが確認できた。
容積90Lのジャーファーメンターに上記スクロース水溶液を追加した培地を50L張り込み、ハロモナス・エスピーKM-1株を10時間好気培養した。その後、MF膜を用いた膜分離により菌体を除去した。ついで、MF膜の透過液に対してUF膜による膜分離を行ったところ、濃縮液及び透過液のいずれでも赤色成分が検出されなかった。
このことから、ハロモナス属に属する好塩菌を好気培養しても、PHBの蓄積が起こらなければ培地中にシトクロムが放出されないことが確認できた。
Claims (5)
- シトクロムを製造する方法であって、
ハロモナス属に属する好塩菌を培地中で好気培養して、当該好塩菌にポリ3-ヒドロキシ酪酸を蓄積させるPHB蓄積工程と、
前記培地から前記シトクロムを回収する回収工程と、を含むシトクロムの製造方法。 - 前記PHB蓄積工程において、10時間以上好気培養を行う請求項1に記載のシトクロムの製造方法。
- 前記PHB蓄積工程後に、前記好塩菌を培地中でpHを調整しながら微好気培養して、当該好塩菌の菌体内に蓄積されたポリ3-ヒドロキシ酪酸を3-ヒドロキシ酪酸として培地中に分泌させる3HB分泌工程を行う請求項1又は2に記載のシトクロムの製造方法。
- 前記シトクロムがシトクロムcである請求項1~3のいずれか一項に記載のシトクロムの製造方法。
- 前記好塩菌が、ハロモナス・エスピー(Halomonas sp.)KM-1株である請求項4に記載のシトクロムの製造方法。
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