Artificial cascade reactions involving biocatalysts have demonstrated a tremendous potential during the recent years. This review just focuses on selected examples of the last year and putting them into context to a previously published suggestion for classification. Subdividing the cascades according to the number of catalysts in the linear sequence, and classifying whether the steps are performed simultaneous or in a sequential fashion as well as whether the reaction sequence is performed in vitro or in vivo allows to organise the concepts. The last year showed, that combinations of in vivo as well as in vitro are possible. Incompatible reaction steps may be run in a sequential fashion or by compartmentalisation of the incompatible steps either by using special reactors (membrane), polymersomes or flow techniques.

The functionalization of bio‐based chemicals is essential to allow valorization of natural carbon sources. An atom‐efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α‐ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α‐hydroxylation of fatty acids combined with enantioselective oxidation by α‐hydroxyacid oxidase(s) resulted in internal recycling of the oxidant H₂O₂, thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O₂‐dependent cascade relies on catalytic amounts of H₂O₂ and releases water as sole by‐product. Octanoic acid was converted under mild conditions in aqueous buffer to 2‐oxooctanoic acid in a simultaneous one‐pot two‐step cascade in up to >99 % conversion without accumulation of hydroxyacid intermediate. Scale‐up allowed isolation of final product in 91 % yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

This paper presents a novel strategy for the simple immobilization of biomolecules on epoxy-functionalized supports via a one-pot three component reaction. Lipase B from Candida antarctica (CALB) and Rhizomucor miehei lipase (RML) as model enzymes were immobilized on epoxy-functionalized silica and mesoporous silica nanoparticles (SBA-15). Investigation of the mechanism of this reaction confirmed the participation of three functional groups including carboxylic acid groups from the enzyme surface, epoxy groups from the support and isocyanide from the reaction medium. The results revealed very rapid immobilization of 10 and 40 mg of RML on 1 gr of the supports shortly after 30 minutes of incubation. The loading capacity of the supports was also dramatically improved with the maximum loading of 158 mg of CALB and 77 mg of RML on SBA-15 and silica, respectively. Silica-epoxy-RML showed an enantiomeric excess (ee) of 92% and an E-value of 29.9 in the enantioselective esterification of (R,S)-ibuprofen.

Chemical and biocatalytic synthesis of non‐canonical α‐amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three‐step, scalable and modular one‐pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l‐α‐amino acids. In module 1, selective α‐hydroxylation of FAs is catalyzed by the P450 peroxygenase P450_CLA. By using an automated H₂O₂ supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable α‐hydroxy acids (α‐HAs; >90% α‐selective) is shown on preparative scale (up to 2.3 g L⁻¹ isolated product). In module 2, a redox‐neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of α‐HAs into l‐α‐AAs (20 to 99%). Enantiopure l‐α‐AAs (e.e. >99%) including the pharma synthon l‐homo‐phenylalanine can be obtained at product titers of up to 2.5 g L⁻¹. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs. magnified image

The oxidation of allylic alcohols is challenging to perform in a chemo‐ as well as stereo‐selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin‐dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from Arabidopsis thaliana (AtBBE15) and the 5‐(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of sec‐allylic alcohols. While primary and benzylic alcohols as well as certain sugars are well known to be transformed by flavin‐dependent oxidases, sec‐allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E‐value of up to >200. For instance HMFO V465S/T oxidized the (S)‐enantiomer of (E)‐oct‐3‐en‐2‐ol (1 a) and (E)‐4‐phenylbut‐3‐en‐2‐ol with E>200 giving the remaining (R)‐alcohol with ee>99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e. g. dimethyl sulfoxide). magnified image