Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration

Toshiki Furuya, Takaaki Kanno, Hiroaki Yamamoto, Norihiro Kimoto, Akinobu Matsuyama, Kuniki Kino

    Research output: Contribution to journalArticle

    1 Citation (Scopus)

    Abstract

    5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.

    Original languageEnglish
    Pages (from-to)111-118
    Number of pages8
    JournalJournal of Molecular Catalysis B: Enzymatic
    Volume94
    DOIs
    Publication statusPublished - 2013

    Fingerprint

    Camphor 5-Monooxygenase
    NAD
    Glucose
    Regeneration
    Glucose 1-Dehydrogenase
    Cells
    Escherichia coli
    Metabolism
    Washing
    Adamantane
    adamantanone
    Catalysts
    Mixed Function Oxygenases
    Glycerol
    Catalysis
    Buffers
    Oxidoreductases

    Keywords

    • Glucose dehydrogenase
    • Monooxygenase
    • NADH regeneration
    • P450cam
    • Whole-cell catalyst

    ASJC Scopus subject areas

    • Biochemistry
    • Bioengineering
    • Catalysis
    • Process Chemistry and Technology

    Cite this

    Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration. / Furuya, Toshiki; Kanno, Takaaki; Yamamoto, Hiroaki; Kimoto, Norihiro; Matsuyama, Akinobu; Kino, Kuniki.

    In: Journal of Molecular Catalysis B: Enzymatic, Vol. 94, 2013, p. 111-118.

    Research output: Contribution to journalArticle

    Furuya, Toshiki ; Kanno, Takaaki ; Yamamoto, Hiroaki ; Kimoto, Norihiro ; Matsuyama, Akinobu ; Kino, Kuniki. / Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration. In: Journal of Molecular Catalysis B: Enzymatic. 2013 ; Vol. 94. pp. 111-118.
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    AU - Furuya, Toshiki

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    AU - Kimoto, Norihiro

    AU - Matsuyama, Akinobu

    AU - Kino, Kuniki

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    N2 - 5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.

    AB - 5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.

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    KW - Monooxygenase

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    KW - P450cam

    KW - Whole-cell catalyst

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