Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) — a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. Key points • Database mining identified a thermostable amine transaminase in the β-alanine:pyruvate transaminase subfamily. • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C. • A tetrameric structure is not necessarily associated with a higher operational stability Graphical abstract

The efficient multifunctionalization by one-pot or cascade catalytic systems has developed as an important research field, but is often challenging due to incompatibilities or crossreactivities of the catalysts leading to side product formation. Herein we report the stereoselective preparation of cis-and trans-4-aminocyclohexanol from the potentially bio-based precursor 1,4-cyclohexanedione. We identified regio-and stereoselective enzymes catalyzing reduction and transamination of the diketone, which can be performed in a one-pot sequential or cascade mode. For this, we identified regioselective keto reductases for the selective mono reduction of the diketone to give 4-hydroxycyclohexanone. The system is modular and by choosing stereocomplementary amine transaminases, both cisand trans-4-aminocyclohexanol were synthesized with good to excellent diastereomeric ratios. Furthermore, we identified an amine transaminase that produces cis-1,4-cyclohexanediamine with diastereomeric ratios > 98 : 2. These examples highlight that the high selectivity of enzymes enable short and stereoselective cascade multifunctionalizations to generate high-value building blocks from renewable starting materials.

Amine transaminases (ATAs) are biocatalysts for the synthesis of chiral amines and can be identified in sequence databases by specific sequence motifs. This study shows that the activity level towards the model substrate 1-phenylethylamine can be predicted solely from the sequence. To demonstrate this, 15 putative ATAs with a different distribution of hydrophobic or hydrophilic amino acid side chains near the active site were characterized. Hydrophobic side chains were associated with a high activity level and were a better predictor of activity than global sequence identity to known ATAs with high or low activities. Enzyme stability investigations revealed that four out of the 15 ATAs showed a good operational stability.

Piper betel L. belongs to the family Piperaceae. It has been an important medicinal agent since ages in various traditional and folk systems of medicine. Leaves obtained from the local market were shade dried and powdered. Different solvents were used based on polarity to extract phytochemicals from this powder using a Soxhlet extractor and separated using rotary vacuum evaporator. Thin layer chromatography was run using different solvent systems in different ratios for identifying essential compounds of Piper betel and for standardizing the ratios at which better resolution of compounds taken place. Antimicrobial activities were tested on twelve bacterial and three fungal species. Also, anti fibrin activity was tested on erythrocytes by using the extracts obtained by the plant. The zone of inhibitions formed due to the anti microbial activity were measured and found that mixtures of ethyl acetate and ethanol were effective. The percentage of clot lysis was found to be appreciable for ethyl acetate extract of the Piper leaves.