Zusammengeheftete Untereinheiten: Die begrenzte Stabilität von Multimeren (siehe Bild) wird oft mit der Dissoziation der Untereinheiten in Verbindung gesetzt. Ein neues, auf Kristallstrukturanalyse, Sequenzalignment und Sättigungsmutagenese basierendes Verfahren wurde genutzt, um systematisch Proteinreste in der Grenzfläche zwischen den Untereinheiten zu identifizieren, die zu einer höheren Stabilität führen.

No abstract

The coupling of the enantioselective reduction catalyzed by Old Yellow Enzymes (OYEs), together with the in situ substrate feeding product removal (SFPR) concept, significantly improved the productivity of the g-scale preparation of ethyl (S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP), an important precursor of several PPAR-α/γ agonists, such as Tesaglitazar. The OYEs and the glucose dehydrogenase for cofactor regeneration were cloned, overexpressed in Escherichia coli, and purified. The synthetic sequence was completed by a NaClO2 oxidation employing cheap and environmentally friendly conditions. The product was obtained in 94% yield and with an ee of 98% over the two steps.

Abstract:We exploit the functional promiscuity of an engineered thermostable variant of a promiscuous D-tagatose epimerase (DTE Var8) to morph it 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 shell around the substrate binding site of Var8 yielded 8-sites mutant IDF8 with an 9-fold improved kcat for the epimerization of D-fructose, and the 6-sites mutant ILS6 with an 14-fold improved epimerization of L-sorbose compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site that supposedly facilitates the entry of the polar substrate, whereas the improvement in variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures will inform future engineering efforts of these and other isomerizing enzymes for the production of rare.

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. On the other hand, continuous chromatography concepts such as the simulating moving bed technology have matured and are increasingly realized on industrial scale for the efficient separation of difficult compound mixtures - including enantiomers - with unprecedented efficiency. We propose that coupling of enzyme reactor and continuous chromatography is a very suitable and potentially generic process concept to address the thermodynamic limitations of a host of promising biotransformations. This way, it should be possible to establish novel in situ product recovery processes of unprecedented efficiency and selectivity that represent a feasible way to recruit novel biocatalysts to the industrial portfolio.

Integrated operation of biotransformation and simulated moving bed (SMB) separation is an attractive option for high-yield manufacturing of commercially relevant compounds such as rare sugars and sialic acids from equilibrium-limited isomerase- or aldolase-catalyzed reactions. Here, we present the first lab-scale implementation of such a process using the production of d-psicose, which is currently under consideration as low calorie sweetener, by d-tagatose epimerase-catalyzed epimerization from d-fructose as a model system. While a typical batchwise eprimerization of d-fructose would stop at 25%, a yield of 97% was obtained when operating the fully integrated process consisting of SMB, enzyme membrane reactor (EMR) and nanofiltration (NF) for a number of days with absolute product purities. Next to the proof of principle, important process characteristics such as startup time, stability and robustness were investigated. By pre-equilibrating the NF unit to the projected conditions, startup times could be reduced to the contributions from EMR and SMB (in this case below 5 h) which was perfectly in line with the projected range of operation time of a few days. Robustness was probed by introduction of a perturbation, specifically a 2-fold increase in process feed concentration, which did not compromise any of the set specifications. Next, long-term operation of the respective units indicated a potential process time of at least 5 days, which could be easily extended in the future by engineering a more stable enzyme variant and implementing a cleaning-in-place approach for SMB column regeneration. In summary, the principle feasibility of such process integration for fine chemical synthesis could be successfully demonstrated.

Integration of enantioseparation by simulated moving bed (SMB) and mild enzymatic racemization enables the production of single enantiomers from a racemic mixture in theoretically 100 % yield and hence overcomes the 50 % yield limitation of conventional SMB processes. We implemented such a process consisting of a Chirobiotic TAG column-SMB, an amino acid racemase-containing enzyme membrane reactor, and a nanofiltration unit for concentration of the distomer-enriched SMB raffinate prior to racemization on lab-scale for the production of enantiopure D-methionine. The integrated process scheme was operated continuously for over 30 hours without significant variations in product concentration and purity and with a yield of 93.5 %, demonstrating the feasibility of this integrated process concept. Furthermore, a rational analysis of the integrated process on the basis of a shortcut model was conducted. The process model consists of a true moving bed equilibrium stage model to represent the SMB, a continuous stirred tank reactor model with reversible Michaelis-Menten kinetics to represent the enzyme membrane reactor, a nanofiltration model and feed node mass balances, and enabled the identification of optimal operating points (flow rate ratios, enzyme concentration) at a variety of process specifications and objectives. Optimal operating points were calculated for different cost distributions between the applied materials such as stationary phase, enzyme, solvent, and nanofiltration membrane. By assigning plausible pricing data and lifetimes to the respective materials, variable costs for the specific process considered in this work were estimated.

No abstract