Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis–Menten constants <i>K</i>_m for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine <i>K</i>_m and turnover number <i>k</i>_cat for trypsin digestion of angiotensin II (<i>K</i>_m and <i>k</i>_cat are determined to be 6.4 mM and 1.3 s^–1, respectively)

Carbon Nanodots-Based Fluorescent Turn-On Sensor Array for Biothiols

Biothiols play important roles in biological processes. In this study, a novel sensor array-based method was proposed to detect and differentiate biothiols. The sensor array was constructed using three kinds of Ag⁺–sensitive carbon nanodots (CDs). The CDs were synthesized with amino acids and urea as carbon sources via a simple microwave method. Results revealed that Ag⁺ can bind with CDs and depress the fluorescence of CDs, while the subsequently joined biothiols can take Ag⁺ away from CDs and recover the fluorescence of CDs. Due to the different binding ability between Ag⁺ and various CDs, as well as Ag⁺ and various biothiols, the CD–Ag⁺ array exhibits a unique pattern of fluorescence variations when interacting with six biothiol samples (cysteamine, dithiothreitol, mercaptosuccinic acid, glutathione, mercaptoacetic acid, and mercaptoethanol). Principal component analysis (PCA) was applied to analyze the pattern and generate a clustering map for a clearer identification of these biothiols. PCA can also be employed to simplify the established three-sensor array into a two-sensor array. Both the three- and two-sensor arrays can identify these biothiols in a wide biothiol concentration range (>10 μM)

Metal–Organic Framework Derived Magnetic Nanoporous Carbon: Novel Adsorbent for Magnetic Solid-Phase Extraction

The fabrication of a magnetic nanoporous carbon (MNPC) via one-step direct carbonization of Co-based metal–organic framework has been achieved without using any additional carbon precursors. The morphology, structure, and magnetic behavior of the as-prepared Co-MNPC were characterized by using the techniques of scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, Raman spectroscopy, N₂ adsorption, and vibrating sample magnetometer. The Co-MNPC has a high specific surface area, large pore volume, and super paramagnetism. Its performance was evaluated by the magnetic solid-phase extraction of some neonicotinoid insecticides from water and fatmelon samples followed by high-performance liquid chromatographic analysis. The effects of the main experimental parameters that could affect the extraction efficiencies were investigated. The results demonstrated that the Co-MNPC had an excellent adsorption capability for the compounds

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N‑Terminus

The GH11 xylanase XynA from Streptomyces rameus L2001 has favorable hydrolytic properties. However, its poor thermal stability hinders its widespread application in industry. In this study, mutants Mut1 and Mut2 were constructed by rationally combining the mutations 11YHDGYF16, 23AP24/23SP24, and 32GP33. The residual enzyme activity of these combinational mutants was more than 85% when incubated at 80 and 90 °C for 12 h, and thus are the most thermotolerant xylanases known to date. The reduced flexibility of the N-terminus, increased overall rigidity, as well as the surface net charge of Mut1 and Mut2 may be partially responsible for the improved thermal stability. In addition, the specific activity and catalytic efficiency of Mut1 and Mut2 were improved compared with those of wild-type XynA. The broader catalytic cleft and enhanced flexibility of the “thumb” of Mut1 and Mut2 may be partially responsible for the improved specific activity and catalytic efficiency