Enzymatic Photometric Assays for the Selective Detection of Halides.

Halides are substrates and products of a number of biotechnologically important enzymes like dehalogenases, halide methyltransferases, and halogenases. Therefore, the determination of halide concentrations in samples is important. The classical methods based on mercuric thiocyanate are very dangerous, produce hazardous waste, and do not discriminate between chloride, bromide, and iodide. In this chapter, we describe a detailed protocol for the determination of halide concentrations based on the haloperoxidase-catalyzed oxidation of halides. The resulting hypohalous acids are detected using commercially available colorimetric or fluorimetric probes. The biocatalytic nature of the assays allows them to be implemented in one-pot cascade reactions with halide-generating enzymes. Since chloride is ubiquitous in biological systems, we also describe convenient photometric assays for the selective detection of bromide and iodide in the presence of chloride, obviating the need for laborious dialyses to obtain halide-free enzymes and reagents.

Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation.

Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium-dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 µM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well-established GC-MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide-producing enzymes.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades.

Plant-derived carbohydrates are an abundant renewable resource. Transformation of carbohydrates into new products, including amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, biocatalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step biocatalysis was performed to functionalize d-galactose-containing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from Agaricus bisporus followed by the ω-transaminase from Chromobacterium violaceum (Cvi-ω-TA). Formation of 6-amino-6-deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino-2-deoxy-6-aldo-d-galactose was confirmed by mass spectrometry. The activity of Cvi-ω-TA was highest towards 6-aldo-d-galactose, for which the highest yield of 6-amino-6-deoxy-d-galactose (67 %) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6-aldo-d-galactose.