Notes

A risk signature of four aging-related genetics provides clinical prognostic price and is also of the tumor resistant microenvironment throughout glioma.
Microtuning the substrate-binding pocket (SBP) of EHs has emerged as an effective approach to manipulate their enantio- or regio-selectivities and activities towards target substrates. Here, the enantioselectivity (enantiomeric ratio, E) of AuEH2 towards a racemic (rac-) ortho-trifluoromethyl styrene oxide (o-TFMSO) was improved via microtuning its SBP. Based on the analysis on the crystal structure of AuEH2, its specific residues I192, Y216, R322 and L344 lining the SBP in close to the catalytic triad were identified for site-saturation mutagenesis. After screening, five single-site mutants were selected with E values elevated from 8 to 12-25 towards rac-o-TFMSO. To further improve E, four double-site mutants were constructed by combinatorial mutagenesis of AuEH2R322V separately with AuEH2I192V, AuEH2Y216F, AuEH2L344A and AuEH2L344C. Among all the mutants, AuEH2R322V/L344C possessed the largest E of 83 with activity of 67 U/g wet cell. The kinetic resolution of 200 mM rac-o-TFMSO was conducted at 0 °C for 5.5 h using 80 mg/mL wet cells of E. coli/Aueh2R322V/L344C, a transformant expressing AuEH2R322V/L344C, retaining (S)-o-TFMSO with 98.4 % ees and 49.3 % yields. Furthermore, the molecular docking simulation analysis indicated that AuEH2R322V/L344C more enantiopreferentially attacks the terminal carbon (Cβ) in the oxirane ring of (R)-o-TFMSO than AuEH2.The Δ1-dehydrogenation of 3-ketosteroid substrates is a crucial reaction in the production of steroids. Although 3-ketosteroid Δ1-dehydrogenase (KsdD) catalyzes this reaction with high efficiency and selectivity, the low stability and high cost of the purified enzyme catalyst have limited its industrial application. In this study, an epoxy support was used to evaluate the covalent immobilization of KsdD from Pimelobacter simplex, and the best androsta-1,4-diene-317-dione (ADD) production was achieved after optimized immobilization of KsdD enzyme in 1.5 M NaH2PO4- Na2HPO4 buffer (pH 6.5) for 12 h at 25 °C. The immobilized KsdD exhibited higher tolerance toward 20 % methanol. The dehydrogenation reaction reached a conversion efficiency of up to 90.0 % in 2 h when using 0.6 mg/mL of 4-androstene-317-dione (AD). The W299A and W299 G mutants of KsdD were also immobilized, and both showed the better catalytic performance with higher kcat/KM values compared with the wild type (WT). The immobilized W299A, W299 G and WT KsdD respectively maintained 70.5, 65.7 and 38.7 % of their initial activity at the end of 15 reaction cycles. Furthermore, the W299A retained 66.3 % of the initial activity after 30 days of incubation at 4 °C, and was more stable than free KsdD, Thus, the immobilized W299A is a promising biocatalyst for steroid dehydrogenation. In this study, we investigated the application of immobilized enzymes for the dehydrogenation of steroids, which will be of great importance for improving the development of green technology and sustainable use of biocatalysts in the steroid manufacturing industry.A simple, convenient and efficient enzyme immobilization method through phytic acid (PA) modified α-Glucosidase (α-Glu)/Cu3(PO4)2·3H2O hybrid nanoflower was developed. The structural properties of the materials were studied by several characterization techniques. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were optimized, and the enzyme kinetics and inhibition parameters were determined. The PA modified α-Glu/Cu3(PO4)2·3H2O hybrid nanoflower had better enzymatic activity under a wide pH range and high temperature than the free one. ABC294640 clinical trial After seven successive cycles, the PA modified α-Glu/Cu3(PO4)2·3H2O hybrid nanoflower could still maintain approximately 63.0 % of its initial immobilized enzyme activity. The Michaelis-Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of acarbose were determined as 0.77 mM and 15.01 μM, respectively. In addition, the material was applied to evaluate the inhibitory activity of ten phenolic compounds on α-Glu, and epicatechin gallate, gallocatechin gallate, epigallocatechin gallate and rosmarinic acid showed good inhibitory activity with % of inhibition of (53.42 ± 2.39)%, (37.28 ± 1.32)%, (37.08 ± 0.63)% and (35.53 ± 0.23)%, respectively. These results indicate that the PA modified hybrid nanoflower is an efficient method of α-Glu immobilization.Phosphotriesterase (PTE) is considered to be a good biodegradation agent for organophosphorus pesticides. However, the instability of the free PTE limits its application. In this study, the free PTE was hybridized with copper ions (Cu2+) to enhance its catalytic stability and activity. The acquired particles were freeze-dried after precipitation with PO43- at 4 °C for 72 h. Scanning electron microscopy showed that the Cu-PTE complexes formed flower-like nanoparticles after hybridization. The characteristic peaks of both the enzyme and metal material were revealed by Fourier transform-infrared spectroscopy. X-ray diffraction analysis indicated that PTE was encapsulated in the Cu3(PO4)2·3H2O based hybrid nanoflowers. Compared with free PTE, the catalytic activity of Cu-PTE hybrid nanoflowers was significantly increased about 2.2 fold. The catalytic efficiency (kcat/Vmax) of Cu-PTE hybrid nanoflowers was 1.76 fold than that of free PTE. The stability of the immobilized PTE under thermal and pH conditions was improved and the tolerance of it to organic solvents was also enhanced. Moreover, the Cu-PTE hybrid nanoflowers still exhibited 72.3 % relative activity after ten consecutive reactions. In general, this is the first time to use copper based hybrid nanoflowers to immobilize PTE, and the immobilized enzyme shows excellent performance on OPs degradation. The Cu-PTE hybrid nanoflowers may have great potential in the biodegradation of organophosphorus compounds in future.L-amino acid ligases (Lals) are promising biocatalysts for the synthesis of dipeptides with special biological properties. However, their poor (or broad) substrate specificity limits their industrial applications. To address this problem, a molecular engineering method for Lals was developed to enhance their catalytic performance. Based on substrate channeling, entrances to the active site for different substrates were identified, and the "gate" located around the active site pocket, which plays an essential role in substrate recognition, was then engineered to facilitate acceptance of L-Gln. Two mutants (L110Y and N108F/L110Y) were discovered to display significantly increased catalytic activity toward L-Ala and L-Gln in the biosynthesis of Ala-Gln. The catalytic efficiency (kcat/ Km) of the L110Y and N108F/L110Y mutants was improved by 2.64-fold and 4.06-fold, respectively, compared with that of the wild type. N108F/L110Y was then further applied for batch production of Ala-Gln, which showed that the released Pi yield was 694.
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