Flow Process for Ketone Reduction Using a Superabsorber-Immobilized Alcohol Dehydrogenase from Lactobacillus brevis in a Packed-Bed Reactor

Abstract

Flow processes and enzyme immobilization have gained much attention over the past few years in the field of biocatalytic process design. Downstream processes and enzyme stability can be immensely simplified and improved. In this work, we report the utilization of polymer network-entrapped enzymes and their applicability in flow processes. We focused on the superabsorber-based immobilization of an alcohol dehydrogenase (ADH) from Lactobacillus brevis and its application for a reduction of acetophenone. The applicability of this immobilization technique for a biotransformation running in a packed bed reactor was then demonstrated. Towards this end, the immobilized system was intensively studied, first in a batch mode, leading to >90% conversion within 24 h under optimized conditions. A subsequent transfer of this method into a flow process was conducted, resulting in very high initial conversions of up to 67% in such a continuously running process.were described by further groups using carrier-bound ADH from Lactobacillus brevis (LB-ADH) in combination with a mobile aqueous phase [30], or magnetic nanoparticle-bound ADH [31]. In addition, Buehler et al. investigated intensively the use of ADH in aqueous/organic segmented flow systems. It was shown that emulsion formation could be eliminated, and enzymatic mass transfer-limited reactions can be enhanced [32]. Furthermore, different means of stabilization of this reaction system were investigated [33]. Very recently, our group published an improved downstream process by means of such a segmented flow process, illustrated using two different ADHs and substrates [34].In continuous processes using packed bed reactors (PBR), catalyst immobilization is a prerequisite. Also, downstream processes benefit from immobilization, as separation of the reaction mixture from the catalyst is easier, and the catalyst reusability is improved [35]. Moreover, both, operational, as well as storage stability of the catalyst can potentially be significantly increased. However, often a significant loss of enzymatic activity during the immobilization process represents a drawback, and the reproducible production of immobilized catalyst may be a further challenge [36]. Numerous techniques have been developed over the past years, which can be classified in the following three major methods: (a) binding to a solid support (carrier), (b) entrapment (encapsulation) in polymer networks, or (c) cross-linking in the form of cross-linked enzyme aggregates (CLEAs) or cross-linked enzyme crystals (CLECs) [35]. Recently, much progress in the field of enzyme immobilization has been made [37][38][39][40][41].In this work, the option of entrapment of enzymes in polymer networks has been chosen. Often for this purpose, organic polymers containing cavities, sol-gel-processed particles, or membranes are used. A special case is the use of superabsorbent polymers to immobilize the entire aqueous phase. [42,43]. In this case, the enzyme is immobilized in its native aqueous environment, whereby undesired interact...

Characterization and evaluation of immobilized enzymes for applications in flow reactors

“…Among the different strategies to engineer surface-protein binding (Figure 2A), reversible interactions (i.e. physical adsorptions) often preserve the activity of the immobilized enzymes [10,11], yet the protein orientation is hardly controlled and the enzyme leaching is plausible. To strengthen the binding and direct the immobilization, enzymes have been genetically fused to both binding modules and peptide tags.…”

Heterogeneous Catalysts “on the Move”: Flow Chemistry with Fluid Immobilised (Bio)Catalysts

“…After these successful initial experiments regarding general behavior of both phases, the first reaction system, namely the reduction of acetophenone ( 1 ) to ( R )‐1‐phenylethanol ( 2 ) catalyzed by an alcohol dehydrogenase from Lactobacillus brevis (LB‐ADH), was investigated. Over the past years, in general, alcohol dehydrogenases (ADHs) have turned out to be an efficient tool for the synthesis of chiral alcohols via ketone reduction .…”

“…We herein introduce the realization of this flow concept exemplified for two (bio‐)catalytic model processes (shown in Figure ). Firstly, an enzyme‐catalyzed reduction of acetophenone ( 1 ) to ( R )‐1‐phenylethanol ( 2 ) in the presence of an alcohol dehydrogenase (ADH), and, secondly, an enzyme‐catalysed dehydration of octanal oxime ( 3 ) to octanenitrile ( 4 ) in the presence of aldoxime dehydratase (Oxd) containing whole‐cells were studied . Both types of reactions gained broad interest in biocatalysis and led, in case of the biotransformations with alcohol dehydrogenases, already to industrial applications ,…”

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

“…to investigate a superabsorber-based immobilization strategy for application in a PBR and applicability in flow processes and observed initial conversions of up to 67% in such a continuously running process. 96 Hanefeld and co-workers used manganese dependent Granulicella tundricola hydroxynitrile lyase (GtHNL) in batch and continuous flow reactions in organic solvents for the synthesis of enantiopure cyanohydrin (Scheme 2). 97 The continuous reaction was performed in acetate buffered methyl MTBE with flow rates from 0.1 to 1.0 mL min −1 (τ = 240 s to 24 s, respectively) and at optimal flow conditions conversions of 97% in 4 min at 0.1 mL min −1 were achieved.…”