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Biotechnol J. 2016 Jul;11(7):890-8. doi: 10.1002/biot.201500495. Epub 2016 Mar 10.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

Author information

1
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
2
Chemistry and Biotechnology, Hochschule Darmstadt, Darmstadt, Germany.
3
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany. Kolmar@biochemie-tud.de.

Abstract

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes.

KEYWORDS:

Alcohol dehydrogenase; Cofactor regenerating system; Coiled-coil interaction; Enzyme immobilization; Multienzyme cascade

PMID:
26901842
DOI:
10.1002/biot.201500495
[Indexed for MEDLINE]

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