Juan Mangas-Sanchez

A reductive aminase from Aspergillus oryzae

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73u2005gu2005l−1u2005d−1. An enzyme (AspRedAm) capable of coupling carbonyls with a variety of amines in a reductive amination has now been discovered. Kinetic studies revealed that the enzyme catalysed both the imine formation step, as well as the reduction step. Structure and mutagenesis studies have highlighted essential catalytic residues and preparative scale examples have demonstrated total turnover numbers of up to 32,000.

Imine reductases (IREDs)

Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field.

Highly efficient enzymatic biodiesel production promoted by particle-induced emulsification.

BackgroundAt present, the conversion of oils to biodiesel is predominantly carried out using chemical catalysts. However, the corresponding lipase-catalysed process has important advantages, which include mild reaction conditions and the possibility of using cheap, low quality feedstocks with a high free fatty acid content. Further increases in the efficiency of the enzymatic process are desired to make it even more attractive and suitable for large-scale applications.ResultsHerein, we present a simple and efficient two-phase lipase-catalysed system for the preparation of biodiesel in which different parameters (biocatalyst composition, ethanol concentration and the presence of additives) were optimised in order to obtain the maximum productivity starting from triolein with a high free oleic acid content. In the two-phase system, the enzyme tolerated high-ethanol concentrations, which made it possible to reach high conversions. The addition of silica particles increased the reaction rate substantially. It was suggested that such particles can catalyse acyl migration as a step to the full conversion to glycerol and biodiesel. However, in the system studied here, the effect of the particles was shown to be due to the formation of smaller and more uniform emulsion droplets leading to better mass transfer between the two phases. Particles of widely different size had positive effects, and the highest rate was obtained with silica particles derivatised with phenyl groups. The optimal conditions were applied to the solvent-free ethanolysis of rapeseed oil, and a yield of 96% was reached in 5xa0h. Under the mild conditions used, chemical catalysts were inefficient.ConclusionsTriacylglycerol oils with a high free fatty acid content can be efficiently converted to ethyl esters using Thermomyces lanuginosus lipase as the catalyst in an aqueous/organic two-phase system. Fast mass transfer can be achieved using silica particles, which helped to decrease the size of the emulsion droplets and thus led to a more efficient process. The high-ethanol concentration tolerated by the lipase in this system made it possible to reach almost quantitative yields.

Kinetic Resolution and Deracemization of Racemic Amines Using a Reductive Aminase

The NADP(H)‐dependent reductive aminase from Aspergillus oryzae (AspRedAm) was combined with an NADPH oxidase (NOX) to develop a redox system that recycles the co‐factor. The AspRedAm‐NOX system was applied initially for the kinetic resolution of a variety of racemic secondary and primary amines to yield S‐configured amines with enantiomeric excess (ee) values up to 99u2009%. The addition of ammonia borane to this system enabled the efficient deracemization of racemic amines, including the pharmaceutical drug rasagiline and the natural product salsolidine, with conversions up to >98u2009% and >99u2009%u2009ee Furthermore, by using the AspRedAm W210A variant it was possible to generate the opposite R enantiomers with efficiency comparable to, or even better than, the wildtype AspRedAm.

Direct Alkylation of Amines with Primary and Secondary Alcohols through Biocatalytic Hydrogen Borrowing

The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99u2009% conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97u2009% ee, releasing water as the only byproduct.

NAD(P)H-Dependent Dehydrogenases for the Asymmetric Reductive Amination of Ketones: Structure, Mechanism, Evolution and Application

Abstract Asymmetric reductive aminations are some of the most important reactions in the preparation of active pharmaceuticals, as chiral amines feature in many of the worlds most important drugs. Although many enzymes have been applied to the synthesis of chiral amines, the development of reductive amination reactions that use enzymes is attractive, as it would permit the one‐step transformation of readily available prochiral ketones into chiral amines of high optical purity. However, as most natural “reductive aminase” activities operate on keto acids, and many are able to use only ammonia as the amine donor, there is considerable scope for the engineering of natural enzymes for the reductive amination of ketones, and also for the preparation of secondary amines using alkylamines as donors. This review summarises research into the development of NAD(P)H‐dependent dehydrogenases for the reductive amination of ketones, including amino acid dehydrogenases (AADHs), natural amine dehydrogenases (AmDHs), opine dehydrogenases (OpDHs) and imine reductases (IREDs). In each case knowledge of the structure and mechanism of the enzyme class is addressed, with a further description of the engineering of those enzymes for the reductive amination of ketones towards primary and also secondary amine products.

Identification of Novel Bacterial Members of the Imine Reductase Enzyme Family that Perform Reductive Amination

Reductive amination of carbonyl compounds constitutes one of the most efficient ways to rapidly construct chiral and achiral amine frameworks. Imine reductase (IRED) biocatalysts represent a versatile family of enzymes for amine synthesis through NADPH‐mediated imine reduction. The reductive aminases (RedAms) are a subfamily of IREDs that were recently shown to catalyze imine formation as well as imine reduction. Herein, a diverse library of novel enzymes were expressed and screened as cell‐free lysates for their ability to facilitate reductive amination to expand the known suite of biocatalysts for this transformation and to identify more enzymes with potential industrial applications. A range of ketones and amines were examined, and enzymes were identified that were capable of accepting benzylamine, pyrrolidine, ammonia, and aniline. Amine equivalents as low as 2.5 were employed to afford up to >99u2009% conversion, and for chiral products, up to >98u2009%u2009ee could be achieved. Preparative‐scale reactions were conducted with low amine equivalents (1.5 or 2.0) of methylamine, allylamine, and pyrrolidine, achieving up to >99u2009% conversion and 76u2009% yield.

Biocatalytic Routes to Enantiomerically Enriched Dibenz[c,e]azepines.

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of (R)- and (S)-5-methyl-6,7-dihydro-5H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework.

Chemoenzymatic Deracemization of Secondary Alcohols by using a TEMPO–Iodine–Alcohol Dehydrogenase System

A deracemization system for secondary alcohols was established after the analysis of individual steps and their compatibility in one pot. The chemical oxidation and bioreduction occurred in a sequential manner to yield 1‐arylethanols and lineal aliphatic alcohols with excellent conversions and enantiomeric excess values. The oxidation step was performed by using 2,2,6,6‐tetramethylpiperidin‐1‐oxyl and iodine. This chemical process was extremely favored by sonication, which allowed quantitative formation of the corresponding ketone intermediates after just 1u2005h. Simple destruction of iodine in the same pot allowed sequential bioreduction of the ketones by using either Prelog or antiPrelog enzymes, which led to the preparation of the enantiopure alcohols in excellent yields.

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii

ABSTRACT Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO2) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO2 for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO2 and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO2 into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO2 reduction. Structural analysis of the enzyme through homology modeling is also presented.