Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

The Golgi-localized transporter OsPML4 contributes to manganese homeostasis in rice.

Manganese (Mn), an indispensable plant micronutrient, functions as a vital enzyme co-factor in numerous biochemical reactions. In rice, the Golgi-localized PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), a member of the UNCHARACTERIZED PROTEIN FAMILY (UPF0016), plays a pivotal role in Mn homeostasis, particularly in rapidly developing tissues. This study focused on the functional characterization of another UPF0016 family member in rice, OsPML4, to elucidate its involvement in Mn homeostasis. OsPML4 had a 73% sequence identity with OsPML3 and exhibited expression in both shoots and roots, albeit at a lower transcriptional level than OsPML3. Furthermore, subcellular localization studies confirmed that OsPML4 localizes in the Golgi apparatus. Notably, heterologous expression of OsPML4 restored growth in the Mn uptake-deficient yeast strain Δsmf1 under Mn-limited conditions. Under Mn-deficient conditions, OsPML4 knockout exacerbated the decline in shoot dry weight and intensified necrosis in young leaves of OsPML3 knockout lines, which displayed stunted growth. The Mn concentration in OsPML3PML4 double knockout lines was lower than in wild-type (WT) and OsPML3 knockout lines. At the reproductive phase, OsPML3PML4 double knockout lines exhibited reduced fertility and grain yield compared to WT and OsPML3 knockout lines. Notably, reductions were observed in the deposition of cell wall polysaccharides and the content of Lea (Lewis A structure)-containing N-glycans in the young leaves of OsPML3PML4 double knockout lines, surpassing the reductions in WT and OsPML3 knockout lines. These findings underscore the significance of OsPML4 in Mn homeostasis in the Golgi apparatus, where it co-functions with OsPML3 to regulate cell wall polysaccharide deposition and late-stage Golgi N-glycosylation.

A portable ascorbic acid in sweat analysis system based on highly crystalline conductive nickel-based metal-organic framework (Ni-MOF).

Conductive metal-organic frameworks can provide unique porous structures, large pore volumes, many catalytically active sites and high crystallinity, and so are becoming increasingly important and attractive as electrocatalytic materials. The present work synthesized nanorods of the conductive compound Ni3(2,3,6,7,10,11-hexaiminotriphenylene)2 (Ni3(HITP)2) with a high degree of crystallinity from HITP ligands and Ni2+ ions. Screen-printed electrodes made with this material were employed to fabricate an enzyme-free sensor for the detection of ascorbic acid (AA). The sensor exhibited good catalytic activity during the electrocatalytic analysis of AA in alkaline media, attributed to the synergistic effect of highly active Ni-N4 catalytic sites in the nanorods, the two-dimensional superimposed honeycomb lattice of the Ni3(HITP)2, and the large specific surface area of this material. The latter property facilitated efficient electron transfer during catalytic oxidation. A portable electrochemical AA detection system was developed using Ni3(HITP)2 as the electrode material together with application-specific integrated circuits and a smartphone application with App. Good sensing performance was obtained, including a wide linear range (2-200 µM) with high sensitivity (0.814 µA µM-1 cm-2), and low detection limit (1 µM). This system can be used to monitor AA levels and trends in sweat to assess vitamin C intake as a part of personal health management.

Selective oxidation of cyclohexane on a novel catalyst Mg-Cu/SBA-15 by molecular oxygen.

The novel catalysts xMg-2.3Cu/SBA-15 with copper and magnesium oxide co-supported on mesoporous silica were synthesized by an impregnation method. The newly synthesized catalysts were characterized using a series of techniques such as BET, XRD, H2-TPR, UV-vis, XPS, EDS and TEM. The catalytic performance was evaluated by using selective oxidation of cyclohexane with molecular oxygen as the oxidant in a solvent free system. The incorporation of magnesium improved the dispersion of copper oxide and prevented the deep oxidation of cyclohexanol and cyclohexanone. The selectivity of K/A oil was up to 99.3% with 12% conversion of cyclohexane over the 1.2Mg-2.3Cu/SBA-15 catalyst. To our knowledge, this is the best result for the heterogeneous oxidation of cyclohexane by O2.