A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

Simultaneous detection of ozone and nitrogen dioxide by oxygen anion chemical ionization mass spectrometry: a fast-time-response sensor suitable for eddy covariance measurements

We report on the development, characterization, and field deployment of a fast-time-response sensor for measuring ozone (O3) and nitrogen dioxide (NO2) concentrations utilizing chemical ionization time-of-flight mass spectrometry (CI-ToFMS) with oxygen anion (O2-) reagent ion chemistry. We demonstrate that the oxygen anion chemical ionization mass spectrometer (Ox-CIMS) is highly sensitive to both O3 (180 counts s−1 pptv−1) and NO2 (97 counts s−1 pptv−1), corresponding to detection limits (3σ, 1 s averages) of 13 and 9.9 pptv, respectively. In both cases, the detection threshold is limited by the magnitude and variability in the background determination. The short-term precision (1 s averages) is better than 0.3 % at 10 ppbv O3 and 4 % at 10 pptv NO2. We demonstrate that the sensitivity of the O3 measurement to fluctuations in ambient water vapor and carbon dioxide is negligible for typical conditions encountered in the troposphere. The application of the Ox-CIMS to the measurement of O3 vertical fluxes over the coastal ocean, via eddy covariance (EC), was tested during the summer of 2018 at Scripps Pier, La Jolla, CA. The observed mean ozone deposition velocity (vd(O3)) was 0.013 cm s−1 with a campaign ensemble limit of detection (LOD) of 0.0027 cm s−1 at the 95 % confidence level, from each 27 min sampling period LOD. The campaign mean and 1 standard deviation range of O3 mixing ratios was 41.2±10.1 ppbv. Several fast ozone titration events from local NO emissions were sampled where unit conversion of O3 to NO2 was observed, highlighting instrument utility as a total odd-oxygen (Ox=O3+NO2) sensor. The demonstrated precision, sensitivity, and time resolution of this instrument highlight its potential for direct measurements of O3 ocean–atmosphere and biosphere–atmosphere exchange from both stationary and mobile sampling platforms.

Технології комплексоутворення в системі краун-етер – KO2 –розчинник, як один з методів отримання стабільної форми аніон-радикалу кисню

Study of reactions of obtaining anion-radical of oxygen: one-electron oxygen reduction and complex formation in the system of crown-ether - potassium superoxide - solvent. Investigation of the possibility of regulating the activity of two different supramolecular structures containing oxygen anion radical to create new antioxidant systems. The oxygen anion radical in solution was obtained by the method of electrochemical oxygen reduction, as well as by complexation in the system of crown ether – potassium superoxide – solvent. The amount of oxygen radical anion obtained was monitored spectrophotometrically using a high-quality reaction with tetrazolium blue and the potentiometric method using an exchange reaction with butyl bromide. The activity of two different supramolecular structures containing oxygen anion radical is compared. It is shown that in the transition from the chemical method of generation of oxygen anion radical to electrochemical, the activity of the anion radical in the reaction with alkyl halide increases slightly. Spectrophotometrically and potentiometrically determined the kinetic parameters of the electrochemical oxygen reduction in the aprotic medium, as well as the process of complexation in the system of crown ether – potassium superoxide – solvent. The activity of two different supramolecular structures containing anion-radical of oxygen with the (Crown-K)+ and (tetraethylammonium)+ ions in reaction with propyl bromide in acetonitrile was compared. The conducted studies allow us to choose the optimal mode and structure of the complex - the sources of oxygen anion radical, depending on the task and goals of the experiment. The study of the complexation reaction in the system of crown ethers - potassium superoxide - solvent, along with the electrochemical method of obtaining active oxygen, is necessary not only in terms of obtaining the most stable form of oxygen anion radical, but also the creation of model systems that allow to analyze the mechanisms and patterns of the processes in biological systems. The obtained results indicate the prospect of using the investigated supramolecular complexes containing oxygen anion radical to create new antioxidant systems that can be used as new classes of antioxidants for engineering, medicine and pharmacy purposes.