Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation

Viktor Varga; Vladimír Štefuca; Lenka Mihálová; Zdenko Levarski; Eva Struhárňanská; Jaroslav Blaško; Robert Kubinec; Pavel Farkaš; Vladimír Sitkey; Ján Turňa; Michal Rosenberg; Stanislav Stuchlík

doi:10.3389/fmicb.2021.684640

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation

Front Microbiol. 2021 Jun 24:12:684640. doi: 10.3389/fmicb.2021.684640. eCollection 2021.

Authors

Affiliations

¹ Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
² Institute of Biotechnology, Faculty of Food and Chemical Technology, Slovak University of Technology, Bratislava, Slovakia.
³ Science Park of Comenius University, Bratislava, Slovakia.
⁴ Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
⁵ Axxence Slovakia, s.r.o., Bratislava, Slovakia.

Abstract

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn²⁺ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn²⁺ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni²⁺, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

Keywords: acetophenone reduction; alcohol dehydrogenase; biotransformation; formate dehydrogenase; green notes; immobilized enzyme regeneration; recombinant protein production and purification.