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

Abstract

The use of whole‐cell biocatalysts enables catalyst application in microaqueous reaction systems, in which the liquid phase consists of high substrate loadings in organic solvents, to enable access to high concentrations of easy‐to‐purify product. One current research focus is the modularization of single reaction steps to (i) enable flexible combinations into multi‐step enzyme reactions, (ii) investigate ideal reaction conditions, and (iii) facilitate catalyst handling and recycling. Therefore, we published the easy‐to‐apply encapsulation of a lyophilized whole‐cell catalyst in a polymeric membrane recently. These catalytic “teabags” were demonstrated to enable flexible catalyst combinations for multi‐step reactions and excellent recyclability during repeated batch experiments. We now describe the applicability of these “teabags” on a larger scale by using the new SpinChem reactor and a classical stirred‐tank reactor model. As an alternative, we investigate the described alginate entrapment approach and compare the results. The carboligation reaction towards (R)‐benzoin, using lyophilized E. coli that enclose Pseudomonas fluorescens benzaldehyde lyase (EC 4.1.2.38), served as a model reaction. It was demonstrated that the catalytic “teabags” are scalable and perform equally on the investigatory 5 mL scale and the preparative 140 mL reactor scale. Tested in a more advanced application, the “teabags” were proven to be useful in a one‐pot two‐step reaction for the gram‐scale production of 1‐phenylpropane‐1,2‐diol by using the SpinChem reactor, which allowed to reach an industrially relevant product concentration (32.9 g L−1) and space–time yield (8.2 g L−1 d−1).

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

“…As an alternative to the entrapment of whole cell catalyst into alginate beads, we recently demonstrated the application of catalytic 'teabags' inside the SpinChem reactor, exhibiting the same reaction rate as in a 140 mL model stirred tank reactor and 5 mL vial scale. Furthermore, the simple catalyst exchange when using the SpinChem reactor makes it particularly appealing for set-up and optimization of biocatalytic multi-step reactions [75].…”

“…The flow streams through four immobilizate-filled compartments and is extruded to the outside. Alternatively to immobilizate particles polymeric membrane bags including lyophilized whole cell catalyst can be used (b, top left)[74,75].…”

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

“…Such TeaBags have been appliedi nmAq conditions previously. [40,41] This approach applied here significantly reduced the hydrolysis, although results were stillp oor owing to the lowr acemization rate. As the mAq approach was not satisfactory,abiphasic migratory DKR system was tested, in which simultaneous racemization and kinetic resolution are separated between the phases, so that racemization takes place in the aqueous phase catalyzed by the ADH and the esterification occurs at the same time in the organic phase catalyzed by the lipase compartmentalized in aT eaBag.…”

“…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.…”

Whole‐cell biocatalysis platform for gram‐scale oxidative dearomatization of phenols

“…Recently, significant effort has been dedicated to establishing efficient biocatalytic reaction platforms for a wide variety of transformations. A number of approaches have been developed for conducting preparative‐scale biocatalytic reactions, including reactions with purified enzymes, crude cell lysates (Rudroff et al., ; Sheldon & Woodley, ) immobilized enzymes (Liang, Li, & Yang, ; Sheldon & van Pelt, ; Soares et al., ) lyophilized lysates, and wet whole cells (de Carvalho, ; Lin & Tao, ; Wachtmeister, Mennicken, Hunold, & Rother, ). Enzymes in various forms can be employed in batch or flow reactors (Britton, Majumdar, & Weiss, ; Tamborini, Fernandes, Paradisi, & Molinari, ).…”