A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.
Soft microfluidic systems that capture, store, and perform biomarker analysis of microliter volumes of sweat, in situ, as it emerges from the surface of the skin, represent an emerging class of wearable technology with powerful capabilities that complement those of traditional biophysical sensing devices. Recent work establishes applications in the real-time characterization of sweat dynamics and sweat chemistry in the context of sports performance and healthcare diagnostics. This paper presents a collection of advances in biochemical sensors and microfluidic designs that support multimodal operation in the monitoring of physiological signatures directly correlated to physical and mental stresses. These wireless, battery-free, skin-interfaced devices combine lateral flow immunoassays for cortisol, fluorometric assays for glucose and ascorbic acid (vitamin C), and digital tracking of skin galvanic responses. Systematic benchtop evaluations and field studies on human subjects highlight the key features of this platform for the continuous, noninvasive monitoring of biochemical and biophysical correlates of the stress state.
The asymmetric synthesis of chiral amines using prochiral ketones was carried out with (S)-specific omega-transaminase (omega-TA) from Vibrio fluvialis JS17. This reaction is inhibited severely by both products, (S)-amine and deaminated ketone. In addition, thermodynamic equilibrium strongly favored the reverse reaction. L-Alanine proved to be the best amino donor based on easy removal of the products. Optimal pH of the reactions with both whole cells and cell-free extract was 7. Amino acceptor reactivities of ketone substrates and reaction profiles of the asymmetric synthesis showed that the initial rate as well as the reaction yield were lower when the resulting (S)-amine from a prochiral ketone substrate was a more reactive amino donor. The yield could be increased dramatically by removing pyruvate, which is a more inhibitory product than (S)-alpha-methylbenzylamine [(S)-alpha-MBA] when acetophenone and L-alanine are used as an amino acceptor and donor, respectively. The removal of pyruvate was carried out by incorporating lactate dehydrogenase (LDH) in cell-free extract or by using whole cells. The whole cell reaction yielded a much better result. When 25 mM benzylacetone and 30 mM acetophenone were used as an amino acceptor with 300 mM L-alanine, 90.2% and 92.1% of the reaction yields after 1 day were obtained with whole cells, respectively. Enantiomeric excesses of both (S)-alpha-MBA and (S)-1-methyl-3-phenylpropylamine [(S)-MPPA] were all above 99%.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.