Aminotransferases (ATs) received much attention as biocatalyst in the last decades and are utilized for the synthesis of chiral amines, amino alcohols and amino acids. More recently, enzymatic synthesis of enantiopure β‐amino acids is of increasing interest, for their application in peptidomimetics or other drug related compounds. For economic process management it is of major interest to acquire reusable biocatalysts. This is achievable by immobilization of the enzymes. A wide range of immobilization techniques for ATs exists. Two of the most common procedures are entrapment in sol‐gel or calcium‐alginate and alternatively covalent binding to chitosan support. For these traditional immobilization techniques, high amounts of purified enzyme solutions are required which is cost intensive. A major step toward faster and straightforward immobilization is the insertion of affinity tags, like a His‐tag, to the enzyme coding gene. Immobilization on non‐porous polyvinyl magnetic micro beads functionalized with IDA (iminodiacetic acid) leads to a fast and easy one‐step purification and immobilization of a β‐amino acid AT from Variovorax paradoxus. For loading of the functionalized beads, three divalent metal ions (Ni2+, Co2+ and Cu2+) were tested. The best activity was obtained with Ni2+ and almost no activity was detected after loading the beads with Cu2+. The immobilized AT was successfully tested to synthesize several enantiopure β‐amino acids via kinetic resolution. Further examination and characterization of this immobilized AT showed improved stability compared to the crude cell extract. The immobilized AT revealed enhanced pH and thermal stability compared to the crude cell extract. High activity was preserved up to 38 days of storage at 4°C. Moreover, the use of magnetic beads allows an easy and mild recycling of the immobilized enzyme after the reaction, leading to an efficient reuse of the immobilized enzyme, for at least seven cycles.

β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods—namely, the classical Z’-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening.

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Despite their key role in numerous natural compounds, β-amino acids have rarely been studied as substrates for microbial degradation. Fermentation of the newly isolated Paraburkholderia strain BS115 and the type strain P. phytofirmans PsJN with β-phenylalanine (β-PA) as sole nitrogen source revealed (S)-selective transamination of β-PA to the corresponding β-keto acid by both strains, accompanied by substantial formation of acetophenone (AP) from spontaneous decarboxylation of the emerging β-keto acid. While the PsJN culture became stationary after entire (S)-β-PA consumption, BS115 showed further growth at a considerably slower rate, consuming (R)-β-PA without generation of AP which points to a different degradation mechanism for this enantiomer. This is the first report on degradation of both enantiomers of any β-amino acid by one single bacterial strain.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0676-2) contains supplementary material, which is available to authorized users.

wasserziehenden Eigenschaften der hydrophilen Lösemittel konnten durch Bestimmung des Wassergehalts in der Lipidphase bestätigt werden. Am Beispiel der 2-Ethylhexylester, die als Schmierstoffester Verwendung finden, konnte gezeigt werden, dass sehr hohe Produktausbeuten erreicht werden und die Bildung von Fettsäuren unterdrückt werden kann.[1] U. Schörken, Eur.