Effects of TiC, Si, and Al on Combustion Synthesis of Ti3SiC2/TiC/Ti5Si3 Composites.

The fabrication of Ti3SiC2 from TiC-containing reactant compacts was investigated by combustion synthesis in the mode of self-propagating high-temperature synthesis (SHS). The initial sample composition was formulated based on (3 - x)Ti + ySi + (2 - x)C + xTiC + zAl, with stoichiometric parameters of x from 0 to 0.7, y = 1.0 and 1.2, and z = 0 and 0.1. For all samples studied, combustion was sufficiently exothermic to sustain the reaction in the SHS manner. Due to the dilution effect of TiC, combustion wave velocity and reaction temperature substantially decreased with TiC content. When compared with the TiC-free sample, the TiC-containing sample facilitated the formation of Ti3SiC2 and the TiC content of x = 0.5 produced the highest yield. Excess Si (y = 1.2) to compensate for the evaporation loss of Si during combustion and the addition of Al (z = 0.1) to promote the phase conversion were effective in improving the evolution of Ti3SiC2. All final products were composed of Ti3SiC2, TiC, and Ti5Si3. For the TiC-containing samples of x = 0.5, the weight fraction of Ti3SiC2 increased from 67 wt.% in the sample without extra Si and Al to 72 wt.% in the Si-rich sample of y = 1.2 and further up to 85 wt.% in the Si-rich/Al-added sample of y = 1.2 and z = 0.1. As-synthesized Ti3SiC2 grains were in a thin plate-like shape with a thickness of 0.5-1.0 µm and length of about 10 µm. Ti3SiC2 platelets were closely stacked into a layered structure.

Affinity Immobilization of a Bacterial Prolidase onto Metal-Ion-Chelated Magnetic Nanoparticles for the Hydrolysis of Organophosphorus Compounds.

In this study, silica-coated magnetic nanoparticles (SiMNPs) with isocyanatopropyltriethoxysilane as a metal-chelating ligand were prepared for the immobilization of His6-tagged Escherichia coli prolidase (His6-EcPepQ). Under one-hour coupling, the enzyme-loading capacity for the Ni2+-functionalized SiMNPs (NiNTASiMNPs) was 1.5 mg/mg support, corresponding to about 58.6% recovery of the initial activity. Native and enzyme-bound NiNTASiMNPs were subsequently characterized by transmission electron microscopy (TEM), superparamagnetic analysis, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. As compared to free enzyme, His6-EcPepQ@NiNTASiMNPs had significantly higher activity at 70 °C and pH ranges of 5.5 to 10, and exhibited a greater stability during a storage period of 60 days and could be recycled 20 times with approximately 80% retention of the initial activity. The immobilized enzyme was further applied in the hydrolysis of two different organophosphorus compounds, dimethyl p-nitrophenyl phosphate (methyl paraoxon) and diethyl p-nitrophenyl phosphate (ethyl paraoxon). The experimental results showed that methyl paraoxon was a preferred substrate for His6-EcPepQ and the kinetic behavior of free and immobilized enzymes towards this substance was obviously different. Taken together, the immobilization strategy surely provides an efficient means to deposit active enzymes onto NiNTASiMNPs for His6-EcPepQ-mediated biocatalysis.

High-level expression and molecular characterization of a recombinant prolidase from Escherichia coli NovaBlue.

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene from Escherichia coli NovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-ß-D-galactoside IPTG-induced E. coli M15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass of EcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris-HCl buffer (pH 8.0) as a dimeric form. The optimal conditions for EcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ ion. Kinetic analysis with Ala-Pro as the substrate showed that the K m and k cat values of EcPepQ were 8.8 mM and 926.5 ± 2.0 s-1, respectively. The thermal unfolding of EcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-U value of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity of EcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability of EcPepQ allows to adjust the reaction conditions more suitable for EcPepQ-catalyzed bioprocess.