Sustainable synthesis of L-phenylalanine derivatives in continuous flow by immobilized phenylalanine ammonia lyase

The application of phenyl ammonia lyases for the amination of a variety of cinnamic acids has been shown to be a cost-efficient method to produce a variety of phenylalanine analogues. Nonetheless, as many other biocatalytic tools, the process intensification, especially due to the high equivalents of ammonia needed, and the cost-efficiency of the catalyst production and use have been key points to further prove their usefulness. Here, we investigated the use of previously characterized PALs (AvPAL and PbPAL) for the amination of a series of substituted cinnamic acids. To enhance the process scalability and the reusability of the catalyst, we investigated the use of covalent immobilization onto commercially available supports, creating a heterogeneous catalyst with good recovered activity (50%) and excellent stability. The immobilized enzyme was also incorporated in continuous flow for the synthesis of 3-methoxy-phenyl alanine and 4-nitro-phenylalanine, which allowed for shorter reaction times (20 mins of contact time) and excellent conversions (88 ± 4% and 89 ± 5%) respectively, which could be maintained over extended periods of time, up to 24h. This work exemplifies the advantages that the combination of enzyme catalysis with flow technologies can have not only in the reaction kinetics, but also in the productivity, catalyst reusability and downstream processing

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts (article)

This is the final version. Available on open access from Wiley via the DOI in this recordThe dataset associated with this article is located in ORE at: https://doi.org/10.24378/exe.1623Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.Biotechnology & Biological Sciences Research Council (BBSRC)GlaxoSmithKlin

Evolution of Biocatalysis at Novartis over the last 40 Years

The fortieth anniversary of biocatalysis started at Ciba-Geigy and later at Novartis is a great time to pause and reflect on development of science and technology in this field. Enzyme-based synthesis became a highly valued enabling tool for pharmaceutical research and development over the last decades. In this perspective we aim to discuss how the scientific approaches and trends evolved over the time and present future challenges and opportunities

Nitrile biotransformationby aspergillus niger

A nitrile-converting enzyme activity was induced in Aspergillus niger K10 by 3-cyanopyridine. The whole cell biocatalyst was active at pH 3–11 and hydrolyzed the cyano group into acid and/or amide functions in benzonitrile as well as in its meta- and para-substituted derivatives, cyanopyridines, 2-phenylacetonitrile and thiophen-2-acetonitrile. Amides constituted a significant part of the total biotransformation products of 2- and 4-cyanopyridine, 4-chlorobenzonitrile, 4-tolunitrile and 1,4-dicyanobenzene, while α-substituted acrylonitriles gave amides as the sole product

Enzymatic Baeyer-Villiger Oxidation of Benzo-Fused Ketones:Formation of Regiocomplementary Lactones

Baeyer-Villiger monooxygenases (BVMOs) are enzymes that are known to catalyse the Baeyer-Villiger oxidation of ketones in aqueous media using O(2) as oxidant. Herein, we describe the oxidation of a set of diverse benzo-fused ketones by three different BVMOs in both aqueous and non-conventional reaction media. Most of the tested ketones, for example, 1-tetralone and 1- and 2-indanone, were converted by one of the employed biocatalysts. The catalytic efficiency could be improved by performing the oxidation reactions at a relatively high pH and by adding organic cosolvents. One striking observation is that absolute and complementary regioselectivities were obtained when oxidizing a range of 1-indanones using two different BVMOs. The conversion of 1-indanone by 4-hydroxyacetophenone monooxygenase (HAPMO) results in the formation of the expected lactone, 3,4-dihydrocoumarin. In contrast, by using a phenylacetone monooxygenase mutein (M-PAMO), conversion of 1-indanone leads to the formation of only the unexpected lactone, 1-isochromanone. This illustrates that by the appropriate choice of BVMO as biocatalyst, the effective and regioselective conversion of a wide range of benzo-fused ketones is feasible. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009