Transaminase-Catalyzed Racemization with Potential for Dynamic Kinetic Resolutions.

Dynamic kinetic resolution (DKR) reactions in which a stereoselective enzyme and a racemization step are coupled in one pot would represent powerful tools for the production of enantiopure amines through enantioconvergence of racemates. The exploitation of DKR strategies is currently hampered by the lack of effective, enzyme-compatible and scalable racemization strategies for amines. In the present work, the proof of concept of a fully biocatalytic method for amine racemization is presented.

Cross-linked aggregates of (R)-oxynitrilase: a stable, recyclable biocatalyst for enantioselective hydrocyanation.

[Reaction: see text] The (R)-oxynitrilase from almonds was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with 1,2-dimethoxyethane and subsequent cross-linking using glutaraldehyde. The resulting preparation was a highly effective hydrocyanation catalyst under microaqueous conditions, which suppress the nonenzymatic background reaction. The beneficial effect of these latter conditions on the hydrocyanation of slow-reacting aldehydes is demonstrated.

A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates.

Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent.

Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase.

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization.

A two-step, one-pot enzymatic synthesis of cephalexin from D-phenylglycine nitrile.

A cascade of two enzymatic transformations is employed in a one-pot synthesis of cephalexin. The nitrile hydratase (from R. rhodochrous MAWE)-catalyzed hydration of D-phenylglycine nitrile to the corresponding amide was combined with the penicillin G acylase (penicillin amidohydrolase, E.

Penicillin acylase-catalyzed ampicillin synthesis using a pH gradient: a new approach to optimization.

The penicillin acylase-catalyzed synthesis of ampicillin by acyl transfer from D-(-)-phenylglycine amide (D-PGA) to 6-aminopenicillanic acid (6-APA) becomes more effective when a judiciously chosen pH gradient is applied in the course of the process. This reaction concept is based on two experimental observations: 1) The ratio of the initial synthesis and hydrolysis rates (V(S)/V(H)) is pH-dependent and exhibits a maximum at pH 6.5-7.

Evaluation of the performance of immobilized penicillin G acylase using active-site titration.

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme.