Semi-rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures.

Enzymes are industrially applied under increasingly diverse environmental conditions that are dictated by the efforts to optimize overall process efficiency. Engineering the operational stability of biocatalysts to enhance their half-lives under the desired process conditions is a widely applied strategy to reduce costs. Here, we present a simple method to enhance enzyme stability in the presence of monophasic aqueous/organic solvent mixtures based on the concept of strengthening the enzyme's surface hydrogen-bond network by exchanging surface-located amino acid residues for arginine.

In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection.

Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process.

Exploiting racemases.

Chiral resolutions of racemic mixtures are limited to a theoretical yield of 50 %. This yield can be doubled by integration of a step-wise or continuous racemization of the non-desired enantiomer. Many of the different routes along which the racemization step can be conducted require harsh treatments and are therefore often incompatible with the highly functionalized state of many compounds relevant for the life science industries.

A separation-integrated cascade reaction to overcome thermodynamic limitations in rare-sugar synthesis.

Enzyme cascades combining epimerization and isomerization steps offer an attractive route for the generic production of rare sugars starting from accessible bulk sugars but suffer from the unfavorable position of the thermodynamic equilibrium, thus reducing the yield and requiring complex work-up procedures to separate pure product from the reaction mixture. Presented herein is the integration of a multienzyme cascade reaction with continuous chromatography, realized as simulated moving bed chromatography, to overcome the intrinsic yield limitation. Efficient production of D-psicose from sucrose in a three-step cascade reaction using invertase, D-xylose isomerase, and D-tagatose epimerase, via the intermediates D-glucose and D-fructose, is described.

Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8.

Highly efficient production of rare sugars D-psicose and L-tagatose by two engineered D-tagatose epimerases.

Rare sugars are monosaccharides that do not occur in nature in large amounts. However, many of them demonstrate high potential as low-calorie sweetener, chiral building blocks or active pharmaceutical ingredients. Their production by enzymatic means from broadly abundant epimers is an attractive alternative to synthesis by traditional organic chemical means, but often suffers from low space-time yields and high enzyme costs due to rapid enzyme degradation.

Model-based identification of optimal operating conditions for amino acid simulated moving bed enantioseparation using a macrocyclic glycopeptide stationary phase.

Teicoplanin aglycone columns allow efficient separation of amino acid enantiomers in aqueous mobile phases and enable robust and predictable simulated moving bed (SMB) separation of racemic methionine despite a dependency of the adsorption behavior on the column history (memory effect). In this work we systematically investigated the influence of the mobile phase (methanol content) and temperature on SMB performance using a model-based optimization approach that accounts for methionine solubility, adsorption behavior and back pressure. Adsorption isotherms became more favorable with increasing methanol content but methionine solubility was decreased and back pressure increased.

Simulated moving bed enantioseparation of amino acids employing memory effect-constrained chromatography columns.

Teicoplanin aglycone-based chromatography columns (Chirobiotic TAG) enable amino acid enantioseparation with aqueous mobile phases, which perfectly accommodates the distinct hydrophilicity of most amino acids. Therefore, this stationary phase constitutes a promising option in particular for preparative-scale separations that require high feed concentrations for economic operation. However, detailed studies revealed a solute-related memory effect when this column is subjected to high loadings of amino acids, conditions that prevail in SMB operation.

Novel method for high-throughput colony PCR screening in nanoliter-reactors.

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents.

Towards preparative asymmetric synthesis of beta-hydroxy-alpha-amino acids: L-allo-threonine formation from glycine and acetaldehyde using recombinant GlyA.

The diastereospecific formation of L-allo-threonine, catalyzed by the serine hydroxymethyltransferase GlyA form Escherichia coli, was studied with regard to the application in continuous processes. Process design will rely on a suitable description of enzyme stability and kinetics under relevant process conditions. Therefore, the effects of addition of organic co-solvents--methanol and acetonitrile--to the reaction mixtures on activity, stability, and diastereoselectivity were investigated.

Adsorption behavior of a teicoplanin aglycone bonded stationary phase under harsh overload conditions.

Silica-bonded teicoplanin aglycone allows enantioseparation of amino acids by reversed-phase liquid chromatography with a low organic solvent content. However, a reversible change in the adsorption behavior leading to a retention time shift (RTS) was observed when a preparative scale column was treated with harsh preparative chromatography-like conditions between finite-injection HPLC runs conducted under exactly the same conditions. This behavior was observed for all five investigated aliphatic and aromatic amino acids.

Integrated operation of continuous chromatography and biotransformations for the generic high yield production of fine chemicals.

The rapid progress in biocatalysis in the identification and development of enzymes over the last decade has enormously enlarged the chemical reaction space that can be addressed not only in research applications, but also on industrial scale. This enables us to consider even those groups of reactions that are very promising from a synthetic point of view, but suffer from drawbacks on process level, such as an unfavourable position of the reaction equilibrium. Prominent examples stem from the aldolase-catalyzed enantioselective carbon-carbon bond forming reactions, reactions catalyzed by isomerising enzymes, and reactions that are kinetically controlled.

Suitability of teicoplanin-aglycone bonded stationary phase for simulated moving bed enantioseparation of racemic amino acids employing composition-constrained eluents.

The suitability of a teicoplanin-aglycone based chiral stationary phase for the simulated moving bed (SMB) enantioseparation of amino acids under enzyme-compatible conditions was shown following a procedure that is based solely on model-based simulations and HPLC experiments. A set of eight amino acids could be separated employing aqueous solvent containing only 10% (v/v) methanol, five of them with baseline resolution. The impact of type and concentration of organic modifier and pH modifier and pH on the separation characteristics of racemic methionine was investigated.