Whole‐Cell Teabag Catalysis for the Modularisation of Synthetic Enzyme Cascades in Micro‐Aqueous Systems

Abstract

Combining enzymes to form multi‐step enzyme cascades has great potential to replace existing chemical routes with high atom‐efficient and eco‐efficient synthesis strategies as well as to grant access to new products, especially those with multi‐stereogenic centres. However, easy solutions and tools for setting up appropriate reaction conditions and process modes are hardly available. The utilisation of teabags filled with whole cells has several advantages, such as 1) simplified handling and recovery of catalyst, 2) easy combination of various catalysts from catalyst toolboxes, 3) fast testing of different operating modes during cascadation and 4) simplified downstream processing. One of the main advantages is that lyophilised whole‐cell catalysts can be applied in micro‐aqueous media, allowing high substrate loads (also of poorly water‐soluble substrates) and concomitantly enabling high catalyst stability. This was demonstrated herein for a synthetic two‐step cascade towards chiral 1,2‐diols starting from cheap aldehydes. The carboligation of two aldehydes using Pseudomonas fluorescens benzaldehyde lyase and subsequent oxidoreduction with Ralstonia sp. alcohol dehydrogenase yielded 1‐phenylpropane‐1,2‐diol [(1R,2R)‐PPD] in concentrations of up to 339 mM and excellent enantiomeric and diastereomeric excesses >99 %. Therefore, the combination of whole‐cell catalysis and teabag modularisation allows cheap, easy‐to‐apply and efficient catalyst preparation to test enzyme combinations and optimal reaction conditions up to the preparative scale. By circumventing catalyst purification and immobilisation, and enabling high substrate loadings compared to those in aqueous systems, efficient production of a chiral diol with extraordinarily high product concentrations can be achieved.

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

“…The catalytic 'teabags' were not only shown to properly retain active catalyst, but also showed excellent recyclability even under challenging micro-aqueous conditions and extraordinarily high substrate loads (400 mM) [57].…”

Modularized Biocatalysis: Immobilization of Whole Cells for Preparative Applications in Microaqueous Organic Solvents

“…In combination with microaqueous solvents, high concentrations of poorly water‐soluble compounds can be applied as the reaction medium is mainly composed of a suitable organic solvent. The organic solvents are supplemented with small amounts of buffer, just enough to restore the enzyme activity within the lyophilized cells . Besides the high substrate loads, these media enable a facilitated downstream processing simply by solvent evaporation, which renders extraction steps needless.…”

Biocatalytic Racemization Employing TeSADH: Substrate Scope and Organic Solvent Compatibility for Dynamic Kinetic Resolution

“…To compartmentalize the dehydrogenase from the lipase, we placed the E. coli /TeSADH WIC in a TeaBag made of PVDF membrane. Such TeaBags have been applied in μAq conditions previously . This approach applied here significantly reduced the hydrolysis, although results were still poor owing to the low racemization rate.…”

“…DKR reference products were obtained by Fisher esterification. TeaBags were prepared in accordance with an already described procedure . For more detailed experimental information see the Supporting Information.…”