Process intensification in continuous flow biocatalysis by up and downstream processing strategies.

In this review, we focus on the holistic continuous enzymatic production and put special emphasis on process intensification by up- and downstream processing in continuous flow biocatalysis. After a brief introduction, we provide an overview of current examples of enzyme immobilization as an upstream process for flow biocatalysis. Thereafter, we provide an overview of unit operations as downstream processing strategies, namely continuous (i) liquid-liquid extraction, (ii) adsorptive downstream processing, and (iii) crystallization and precipitation. Eventually, we present our perspectives on future trends in this research field.

A holistic carrier-bound immobilization approach for unspecific peroxygenase.

Unspecific peroxygenases (UPOs) are among the most studied enzymes in the last decade and their well-deserved fame owes to the enzyme's ability of catalyzing the regio- and stereospecific hydroxylation of non-activated C-H bonds at the only expense of H2O2. This leads to more direct routes for the synthesis of different chiral compounds as well as to easier oxyfunctionalization of complex molecules. Unfortunately, due to the high sensitivity towards the process conditions, UPOs' application at industrial level has been hampered until now. However, this challenge can be overcome by enzyme immobilization, a valid strategy that has been proven to give several benefits. Within this article, we present three different immobilization procedures suitable for UPOs and two of them led to very promising results. The immobilized enzyme, indeed, shows longer stability and increased robustness to reaction conditions. The immobilized enzyme half-life time is 15-fold higher than for the free AaeUPO PaDa-I and no enzyme deactivation occurred when incubated in organic media for 120 h. Moreover, AaeUPO PaDa-I is proved to be recycled and reused up to 7 times when immobilized.

A Deep Eutectic Solvent Thermomorphic Multiphasic System for Biocatalytic Applications.

The applicability of a thermomorphic multiphasic system (TMS) composed of a hydrophobic deep eutectic solvent (DES) and an aqueous potassium phosphate buffer with a lower critical solution temperature (LCST) phase change for homogeneous biocatalysis was investigated. A lidocaine-based DES with the fatty acid oleic acid as a hydrogen-bond donor was studied. Phase diagrams were determined and presented within this study. We tested different additional components to the solvent system and observed a decrease in the cloud point of approximately 0.026 °C per concentration unit. Distribution studies revealed a clear distribution of the protein in the aqueous buffer phase (>95 %), whereas the hydrophobic substrate and educt accumulated (>95 %) in the DES-enriched layer. Finally, a reduction catalyzed by horse liver alcohol dehydrogenase was performed in a larger-scale experiment, and the biocatalyst could be recycled by simply removing the DES phase for three recycling runs.

Enzyme immobilization in hydrogels: A perfect liaison for efficient and sustainable biocatalysis.

Biocatalysis is an established chemical synthesis technology that has by no means been restricted to research laboratories. The use of enzymes for organic synthesis has evolved greatly from early development to proof-of-concept - from small batch production to industrial scale. Different enzyme immobilization strategies contributed to this success story. Recently, the use of hydrogel materials for the immobilization of enzymes has been attracting great interest. Within this review, we pay special attention to recent developments in this key emerging field of research. Firstly, we will briefly introduce the concepts of both biocatalysis and hydrogel worlds. Then, we list recent interesting publications that link both concepts. Finally, we provide an outlook and comment on future perspectives of further exploration of enzyme immobilization strategies in hydrogels.

Integration of ion exchange resin materials for a downstream-processing approach of an imine reductase-catalyzed reaction.

In this study, an ion exchange resin-based downstream-processing concept for imine reductase (IRED)-catalyzed reactions was investigated. As a model reaction, 2-methylpyrroline was converted to its corresponding product (S)-2-methylpyrrolidine with >99% of conversion by the (S)-selective IRED from Paenibacillus elgii B69. Under optimized reaction conditions full conversion was achieved using a substrate concentration of 150 and 500 mmol/L of d-glucose. Seven commercially available cation- and anion-exchange resins were studied with respect to their ability to recover the product from the reaction solution. Without any pretreatment, cation-exchange resins Amberlite IR-120(H), IRN-150, Dowex Monosphere 650C, and Dowex Marathon MSC showed high recovery capacities (up to >90%). A 150-ml preparative scale reaction was performed yielding ~1 g hydrochloride salt product with >99% purity. Any further purification steps, for example, by column chromatography or recrystallization, were not required.

Development of Ionic Liquid-Water-Based Thermomorphic Solvent (TMS)-Systems for Biocatalytic Reactions.

The applicability of ionic liquid-water-based thermomorphic solvent (TMS)-systems with an upper critical solution temperature for homogeneous biocatalysis is investigated. Cholinium- and imidazolium-based ionic liquids are used to facilitate a temperature-dependent phase change, which can be easily fine-tuned by adding salts or polar organic solvents. Within the TMS-system, a high enzymatic activity and subsequent full conversion is achieved in the intermittent monophasic reaction system of the TMS-system. Therefore, the biocatalyst can be easily recycled after separating the phases at lower temperatures.

Application of In Situ Product Crystallization and Related Techniques in Biocatalytic Processes.

This Minireview highlights the application of crystallization as a very powerful in situ product removal (ISPR) technique in biocatalytic process design. Special emphasis is placed on its use for in situ product crystallization (ISPC) to overcome unfavorable thermodynamic reaction equilibria, inhibition, and undesired reactions. The combination of these unit operations requires an interdisciplinary perspective to find a holistic solution for the underlying bioprocess intensification approach. Representative examples of successful integrated process options are selected, presented, and assessed regarding their overall productivity and applicability. In addition, parallels to the use of adsorption as a very similar technique are drawn and similarities discussed.

Recent developments in biocatalysis in multiphasic ionic liquid reaction systems.

Ionic liquids are well known and frequently used 'designer solvents' for biocatalytic reactions. This review highlights recent achievements in the field of multiphasic ionic liquid-based reaction concepts. It covers classical biphasic systems including supported ionic liquid phases, thermo-regulated multi-component solvent systems (TMS) and polymerized ionic liquids. These powerful concepts combine unique reaction conditions with a high potential for future applications on a laboratory and industrial scale. The presence of a multiphasic system simplifies downstream processing due to the distribution of the catalyst and reactants in different phases.