The chemoenzymatic flow synthesis of enantiomerically pure captopril, a widely used antihypertensive drug, is accomplished starting from simple, inexpensive, and readily available reagents. The first step is a heterogeneous biocatalyzed regio‐ and stereoselective oxidation of cheap prochiral 2‐methyl‐1,3‐propandiol, performed in flow using immobilized whole cells of Acetobacter aceti MIM 2000/28, thus avoiding the use of aggressive and environmentally harmful chemical oxidants. The isolation of the highly hydrophilic intermediate (R)‐3‐hydroxy‐2‐methylpropanoic acid is achieved in‐line by using a catch‐and‐release strategy. Then, three sequential high‐throughput chemical steps lead to the isolation of captopril in only 75 min. In‐line quenching and liquid–liquid separation enable breaks in the workflow and other manipulations to be avoided.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

hornekiae 15A gave highly stereoselective reduction of (R,S)-1, while H. aquamarina 9B enantioselectively hydrolysed (R,S)-1; in both the cases, enantiomerically pure unreacted substrate could be easily recovered and purified at molar conversion below 57-58%, showing the potential of DHABs extremophile microbiome and marine-derived enzymes in biocatalysis.

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates.Analysis of the genome of Pichia glucozyma CBS 5766 revealed the occurrence of a sequence (called KRED1-Pglu) with high homology with known benzil reductases. The corresponding Histagged protein was successfully expressed in E. coli BL21(DE3)Star, yielding an active enzyme accounting for about 40% of the total protein content of the cell extract (Fig.1). When the series of aryl ketones is docked into enzyme active site, the orientation of the top-scoring poses changes depending on the length of aliphatic chain which defines the stereochemistry of the reductive reaction. Figure 2 shows the relative placement of acetophenone and butyrophenone, and their position with respect to the reductive hydride. Lengthening the aliphatic chain produces steric with amino acids in the enzyme catalytic site and results in an inversion of prevailing orientation during the interaction. As a result, the stereochemistry predicted for the stereocenter of the alcohol products is different in either case and identical to what is obtained in vitro. Biotransformations and modelling Protein cloning, expression and purificationMolar conversion and enantioselectivity towards different ketones and ketoesters were determined by performing biotransformations at 50 mg-scale , using an enzyme-coupled system (glucose-glucose dehydrogenase-GDH-from Bacillus megaterium). Contact: martina.contente@unimi.itTemperature and pH optima were studied conducting the benzil-reductase test at temperatures between 15°C and 50°Cand at pH values in 4-12 range (using suitable buffers).Activity measurements were performed spectrophotometrically at 340 nm by determining the consumption of NAD(P)H at 25°Cin a half-micro cuvette (total volume: 1 mL) for 5 min. KRED1-Pglu showed• highest activity between 30 °C and 35 °C• stability up to a temperature of 30 °C highest activity at pH between 7.0 and 8.0stability at pH between 6.0 and 8.015 mg/g cell of pure proteine were obtained

Hydrolytic enantioselective cleavage of different racemic non-steroidal anti-inflammatory drugs (NSAIDs) ester derivatives has been studied. An engineered esterase form Bacillus subtilis (BS2m) significantly outperformed homologous enzymes from Halomonas elongata (HeE) and Bacillus coagulants (BCE) in the enantioselective hydrolysis of naproxen esters. Structural analysis of the three active sites highlighted key differences which explained the substrate preference. Immobilization of a chimeric BS2m-T4 lysozyme fusion (BS2mT4L1) was improved by resin screening achieving twice the recovered activity (22.1 ± 5 U/g) with respect to what had been previously reported, and was utilized in a packed bed reactor. Continuous hydrolysis of α-methyl benzene acetic acid butyl ester as a model substrate was easily achieved, albeit at low concentration (1 mM). However, the high degree of insolubility of the naproxen butyl ester resulted in a slurry which could not be efficiently bioconverted, despite the addition of co-solvents and lower substrate concentration (1 mM). Addition of Triton® X-100 to the substrate mix yielded 24% molar conversion and 80% e.e. at a 5 mM scale with 5 min residence time and sufficient retention of catalytic efficiency after 6 h of use.