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 Jan 1

Keywords

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

ASJC Scopus subject areas

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology

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