4 research outputs found
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><sub>m</sub> for these two reactions were
determined to be 214 ÎĽM and 172 ÎĽM, respectively, which
are in good agreement with the values of 300 ÎĽM and 230 ÎĽM
reported in literature, validating the DESI-MS approach. Furthermore,
this time-resolved DESI-MS also allowed us to determine <i>K</i><sub>m</sub> and turnover number <i>k</i><sub>cat</sub> for trypsin digestion of angiotensin II (<i>K</i><sub>m</sub> and <i>k</i><sub>cat</sub> are determined to be
6.4 mM and 1.3 s<sup>–1</sup>, 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<sup>+</sup>–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<sup>+</sup> can
bind with CDs and depress the fluorescence of CDs, while the subsequently
joined biothiols can take Ag<sup>+</sup> away from CDs and recover
the fluorescence of CDs. Due to the different binding ability between
Ag<sup>+</sup> and various CDs, as well as Ag<sup>+</sup> and various
biothiols, the CD–Ag<sup>+</sup> 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 ÎĽM)
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, N<sub>2</sub> 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