title: Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.
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.
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Valerie
authors.initials: V
authors.lastname: Spieler
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Valldorf
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Maaß
authors.affiliation: Chemistry and Biotechnology, Hochschule Darmstadt, Darmstadt, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kleinschek
authors.affiliation: Chemistry and Biotechnology, Hochschule Darmstadt, Darmstadt, Germany.
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Hüttenhain
authors.affiliation: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany. Kolmar@biochemie-tud.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001592:Benzyl Alcohols / Q000737:chemistry; D055162:Biocatalysis; D002175:Candida / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D002479:Inclusion Bodies / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology; D002721:omega-Chloroacetophenone / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D001592:Benzyl Alcohols; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011994:Recombinant Proteins; D009243:NAD; D002721:omega-Chloroacetophenone; C017838:4-chloroacetophenone; C002017:methylphenyl carbinol; D000426:Alcohol Dehydrogenase; D005560:Formate Dehydrogenases
keywords: Alcohol dehydrogenase; Cofactor regenerating system; Coiled-coil interaction; Enzyme immobilization; Multienzyme cascade