Gábor Tasnádi

Asymmetric bioreduction of activated alkenes to industrially relevant optically active compounds.

Highlights ► Activated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds bearing electron-withdrawing groups are efficiently reduced by flavoproteins from the OYE family. ► The application of ene-reductases for the pharma- and perfumery industry has been demonstrated. ► Access to both stereoisomeric products is feasible by choice of stereo-complementary enzymes or via proper substrate engineering.

Reductive dehalogenation of β-haloacrylic ester derivatives mediated by ene-reductases

The enzymatic bioreduction of β-halo-α,β-unsaturated carboxylic esters proceeded via sequential enzymatic CC reduction—β-elimination to afford saturated carboxylic esters. This novel biodegradation pathway combines the reductive activity of ene-reductases with the spontaneous β-elimination of hydrohalous acid from the unstable (saturated) intermediates. Both enantiomers of methyl 2-chloro-, 2-bromo- and 2-iodopropionate were obtained in good to excellent enantiopurity via enzyme-based stereocontrol using various members of the ‘Old Yellow Enzyme’ family of flavoproteins. Overall, this pathway resembles a reductive dehalogenation of β-halogenated acrylic esters.

Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

Abstract A set of phosphatases was evaluated for their potential to catalyze the regio‐ and stereoselective phosphorylation of alcohols using a high‐energy inorganic phosphate donor, such as di‐, tri‐ and polyphosphate. Parameters such as type and amount of phosphate donor and pH of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester. Diols were monophosphorylated with high selectivities. This biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale (6.86 g) in a flow setup with immobilized phosphatase.

Biocatalytic functionalization of hydroxyalkyl acrylates and phenoxyethanol via phosphorylation.

The enzymatic phosphorylation of phenoxyethanol, 2-hydroxyethyl acrylate and 4-hydroxybutyl acrylate catalyzed by acid phosphatases PhoN-Sf and PiACP at the expense of inorganic di-, tri-, hexameta- or polyphosphate was applied to the preparative-scale synthesis of phosphorylated compounds. The reaction conditions were optimized with respect to enzyme immobilization, substrate concentration, pH and type of phosphate donor. The mild reaction conditions prevented undesired polymerization and hydrolysis of the acrylate ester moiety. Application of a continuous flow system allowed facile scale-up and mono-phosphates were obtained in up to 26% isolated yield with space-time yields of 0.89kgL(-1)h(-1).

Investigation of Acid Phosphatase Variants for the Synthesis of Phosphate Monoesters

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC‐Mm from Morganella morganii and NSAP‐Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC‐Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative‐scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187–2195.

Smart Nanoparticles for Selective Immobilization of Acid Phosphatases

An easy to use method combining the selectivity of metal chelate affinity binding with strong covalent linking was developed for immobilization of non‐specific acid phosphatases bearing a His‐tag from crude cell lysate. Silica nanoparticles were grafted with aminopropyl functions which were partially transformed further with EDTA dianhydride to chelators. The heterofunctionalized nanoparticles charged with Ni2+ as the most appropriate metal ion were applied as support. First, the His‐tagged phosphatases were selectively bound to the metal‐chelate functions of the support. Then, the enzyme‐charged silica nanoparticles were further stabilized by forming a covalent linkage between nucleophilic moieties at the enzyme surface and free amino groups of the support using neopentylglycol diglycidylether as the most effective bifunctional linking agent. The phosphatase biocatalysts obtained by this method exhibited better phosphate transfer activity with a range of alcohols and PPi as phosphate donor in aqueous medium applying batch and continuous‐flow modes than the ones immobilized on conventional supports. Furthermore, this novel strategy opens up novel possibility for efficient immobilization of other His‐tagged recombinant enzymes.

Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters

Abstract Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by‐product are the main obstacles associated with the use of pyrophosphate in the phosphatase‐catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl‐compounds. Among them, acetyl phosphate delivered stable product levels and high phospho‐transfer efficiency at the lower functional pH‐limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative‐scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L−1 h−1 space‐time yield and 75% reduction of barium phosphate waste.