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.

Internal Illumination to Overcome the Cell Density Limitation in the Scale-up of Whole-Cell Photobiocatalysis.

Cyanobacteria have the capacity to use photosynthesis to fuel their metabolism, which makes them highly promising production systems for the sustainable production of chemicals. Yet, their dependency on visible light limits the cell-density, which is a challenge for the scale-up. Here, it was shown with the example of a light-dependent biotransformation that internal illumination in a bubble column reactor equipped with wireless light emitters (WLEs) could overcome this limitation. Cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene-reductase YqjM were used for the reduction of 2-methylmaleimide to (R)-2-methylsuccinimide with high optical purity (>99 % ee). Compared to external source of light, illumination by floating wireless light emitters allowed a more than two-fold rate increase. Under optimized conditions, product formation rates up to 3.7 mm h-1 and specific activities of up to 65.5 U gDCW -1 were obtained, allowing the reduction of 40 mm 2-methylmaleimide with 650 mg isolated enantiopure product (73 % yield). The results demonstrate the principle of internal illumination as a means to overcome the intrinsic cell density limitation of cyanobacterial biotransformations, obtaining high reaction rates in a scalable photobioreactor.

Recent developments in the use of peroxygenases - Exploring their high potential in selective oxyfunctionalisations.

Peroxygenases are an emerging new class of enzymes allowing selective oxyfunctionalisation reactions in a cofactor-independent way different from well-known P450 monooxygenases. Herein, we focused on recent developments from organic synthesis, molecular biotechnology and reaction engineering viewpoints that are devoted to bring these enzymes in industrial applications. This covers natural diversity from different sources, protein engineering strategies for expression, substrate scope, activity and selectivity, stabilisation of enzymes via immobilisation, and the use of peroxygenases in low water media. We believe that peroxygenases have much to offer for selective oxyfunctionalisations and we have much to study to explore the full potential of these versatile biocatalysts in organic synthesis.