Design and application of a bi-functional redox biocatalyst through covalent co-immobilization of ene-reductase and glucose dehydrogenase

• Published in: Journal of biotechnology 2020-11, Vol.323, p.246-253
• Main Authors: Nagy, Flóra, Gyujto, Imre, Tasnádi, Gábor, Barna, Bence, Balogh-Weiser, Diána, Faber, Kurt, Poppe, László, Hall, Mélanie
• Format: Article
• Language: English
• Subjects: Asymmetric bioreduction; Covalent immobilization; Ene-reductase; Glucose dehydrogenase; Nicotinamide regeneration; Old yellow enzyme
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2020.08.005

[Display omitted] •A bi-functional redox biocatalyst was obtained by co-immobilization of ene-reductase and glucose dehydrogenase.•The bi-functional biocatalyst showed increased conversion compared to the combined use of individual catalysts.•Excellent reusability of the co-immobilized biocatalyst was shown over 10 cycles.•Preparative-scale synthesis allowed access to important chemical building blocks in high enantiopurity. An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions.