SummaryThis review highlights the state of the art in the field of coupled chemo(enzymatic) reactions in continuous flow. Three different approaches to such reaction systems are presented herein and discussed in view of their advantages and disadvantages as well as trends for their future development.

A solvent-free, chemoenzymatic reaction sequence for the enantioselective synthesis of β-amino acid esters has been kinetically and thermodynamically characterized. The coupled sequence comprises a thermal aza-Michael addition of cheap starting materials and a lipase catalyzed aminolysis for the kinetic resolution of the racemic ester. Excellent ee values of >99% were obtained for the β-amino acid ester at 60% conversion. Kinetic constants for the aza-Michael addition were obtained by straightforward numerical integration of second-order rate equations and nonlinear fitting of the progress curves. A different strategy had to be devised for the biocatalytic reaction. Initially, a simplified Michaelis-Menten model including product inhibition was developed for the reaction running in THF as an organic solvent. Activity based parameters were used instead of concentrations in order to facilitate the transfer of the kinetic model to the solvent-free system. Observed solvent effects not accounted for by the use of thermodynamic activities were incorporated into the kinetic model. Enzyme deactivation was observed to depend on the ratio of the applied substrates and also included in the kinetic model. The developed simple model is in very good agreement with the experimental data and allows the simulation and optimization of the solvent-free process.

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.

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A sequential, chemoenzymatic process for a continuously operating production of the chiral b-amino acid ester ethyl (S)-3-(benzylamino)-butanoate was developed. The reactor set-up combined a plug-flow reactor for the thermal aza-Michael addition of benzylamine to trans-ethyl crotonate coupled to a subsequent packed-bed reactor for the lipase (Novozym 435)-catalyzed kinetic resolution of the racemic intermediate product, which was formed in the initial step. The coupled reactors were operated continuously for a time period of 4 days without significant loss of enzyme activity. The target b-amino acid ester was obtained with 92% conversion in the plug-flow reactor and 59% conversion in the packed bed reactor at high enantiomeric excess of > 98%. A space-time yield of 0.4 kg L À1 d À1 was calculated for the total reactor system and 1.8 kg L À1 d À1 based solely on the volume of the packed bed reactor. A total turnover number of 158,000 was calculated for the biocatalyst assuming the same deactivation rate as observed in batch experiments. The continuously operating, solvent-free process thus represents an efficient method for the enantioselective production of a value added (S)-b-amino acid ester starting from cheap substrates.Abbreviations: CALB = Candida antarctica lipase B; CSTR = continuously stirred tank reactor; d i = inner diameter [mm]; ee = enantiomeric excess; e = porosity; k cat,obs = apparent turnover number [h ].

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Monitoring the dispersed phase of an oil-in-water (O-W) emulsion by means of Fourier transform infrared (FTIR) spectroscopy is a challenging task, restricted to the continuous phase that is in contact with the FTIR probe. Nonetheless, real-time measurement and kinetic analysis by FTIR, including analysis of the dispersed, often non-polar phase containing substrates and/or products, is desirable. Enzymatic hydrolysis of sunflower oil was performed in an O-W emulsion. After separation of the oil phase by use of a newly developed μ-membrane module, infrared spectra were collected using an attenuated total reflectance (ATR) cell. Different chemometric models were calibrated using the partial least squares (PLS) algorithm. Online application of a chemometric model based on the FTIR spectra enabled real-time monitoring of free fatty acid concentrations in the oil phase.

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