Search

Additional file 1: 1. GC analyses. 2. SDS-PAGE.

The integration of enzyme-catalyzed reactions in flow systems has been boosted during the last few years. Nowadays, biocatalysis is officially recognized as a tool to increase reaction specificity and sustainability, however applications are sometimes characterized by low productivity. A logical step to improve the performance of biocatalytic reactions is represented by the combination of enzymes and flow facilities. This tutorial review aims at introducing the key concepts of flow biocatalysis, guiding the reader through its advantages and highlighting the current trends in the field to encourage innovative applications of enzymes in flow reactors. Experimental cases are also presented and discussed as a troubleshooting guide for development of flow biocatalytic processes.

We expanded the application of self-sufficient heterogeneous biocatalysts containing coimmobilized w-transaminases and pyridoxal 5´-phosphate (PLP) to efficiently operate packed-bed reactors in continuous flow. Using a w-transaminase from Halomonas elongata co-immobilized with PLP onto porous methacrylate-based carriers coated with polyethyleneimine, we operated a packed-bed reactor continuously for up to 50 column volumes at 1.45 mL x min-1 in the enantioselective deamination of model amines (α-methylbenzyl amine), yielding > 90% conversion in all cycles without exogenous addition of cofactor. In this work, we expanded the concept of self-sufficient heterogeneous biocatalysts to other w-transaminases such as the ones from Chromobacterium violaceum and Pseudomonas fluorescens. We found that enzymes with lower affinities towards PLP present lower operational stabilities in flow, even when coimmobilizing PLP. Furthermore, w-transaminases co-immobilized with PLP were successfully implemented for the continuous synthesis of amines and the sustainable metrics were assessed. These results give some clues to exploit PLP-dependent w-transaminases under industrially relevant continuous operations in a more cost-effective and environmentally friendly process.

The synthesis of alcohols from amines starting material is an excellent, yet challenging, strategy for the preparation of pharmaceuticals and polymers. Here, we developed a versatile, self-sustaining closed-loop multienzymatic platform for the biocatalytic synthesis of a large range of non-commercially available products in continuous flow with excellent yields (80>99%), reaction times and optical purity of secondary alcohols (>99 e.e.). This process was also extended to the conversion of biogenic amines into high value alcohols, such as the powerful anti-oxidant hydroxytyrosol, and the synthesis of enantiopure 2-arylpropanols via dynamic kinetic resolution of commercially affordable racemic amines. The system exploits the in situ immobilisation of transaminases and redox enzymes which were combined to cater for a fully automated, ultra-efficient synthetic platform with cofactor recycling, in-line recovery of benign by-products and recirculation of the aqueous media containing the recycled cofactors in catalytic amount, which increases the efficiency of the system by over 20-fold.

A new mild and efficient process for the preparation of aldehydes in water employed as flavour and fragrance components in food, beverage, cosmetics, as well as in pharmaceuticals was developed using a continuous-flow approach based on an immobilized pure transaminase-packed bed reactor. HEWT, an ?-transaminase from the haloadapted bacterium Halomonas elongata, has been selected for its excellent stability and substrate scope. Sixteen different amines were rapidly (3-15 min) oxidized into the corresponding aldehydes (90 to 99 %) with only 1 to 5 equivalents of sodium pyruvate. The process was fully automated allowing for the in-line recovery of the pure aldehydes (chemical purity >99% and isolated yields above 80%), without any further work-up procedure.

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a “lid” domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG.