Nanozeolite NaX ion exchanged with different transition metals (Mn 2+ ,Cu 2+ , Co 2+ , Zn 2+ , Ni 2+ ) was used as a solid support for the immobilization of the lipases of Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML). The nanozeolite-enzyme complexes were used as heterogeneous catalysts for the transesterification reaction of palm oil to fatty acid ethyl esters (FAEEs). The most relevant results were obtained with the Thermomyces lanuginosus enzyme immobilized on nanozeolitic supports ion exchanged with Ni 2+ . Although these zeolitic supports were able to immobilize a relatively small amount of the enzyme (43.7%) in comparison with the other nanozeolitic supports, the FAEE yields obtained with Nano-X/Ni/0.5M-TLL complexes were above 94%. These results revealed an unusual synergistic effect between the Thermomyces lanuginosus enzyme and the nickel ion-exchanged nanozeolitic support; this effect was not observed for the complexes prepared with the Rhizomucor miehei enzyme. Bioinformatics calculations were performed for both enzymes by taking into consideration the crystallographic structures of the enzymes and the zeta potential of the surface of the nanozeolitic supports. By combining calculations of the protein electrostatic potential surface and normal mode analyses in a model, we were able to propose an explanation for the synergistic effect between the lipases and the nanozeolitic supports. The synergistic effect could be explained through an allosteric mechanism describing the interaction between aspartic acid residues 102 and 158 of the Thermomyces lanuginosus lipase and the positively charged zeolitic support surface. This interaction results in the stabilization of the opening of the enzyme lid and leaves its catalytic triad permanently exposed to the reaction medium.
Nanozeolites with different crystallographic structures (Nano/TS1, Nano/GIS, Nano/LTA, Nano/BEA, Nano/X, and Nano-X/Ni), functionalized with (3-aminopropyl)trimethoxysilane (APTMS) and crosslinked with glutaraldehyde (GA), were studied as solid supports for Thermomyces lanuginosus lipase (TLL) immobilization. Physicochemical characterizations of the surface-functionalized nanozeolites and nanozeolite-enzyme complexes were performed using XRD, SEM, AFM, ATR-FTIR, and zeta potential measurements. The experimental enzymatic activity results indicated that the nanozeolitic supports functionalized with APTMS and GA immobilized larger amounts of enzymes and provided higher enzymatic activities, compared to unfunctionalized supports. Correlations were observed among the nanozeolite surface charges, the enzyme immobilization efficiencies, and the biocatalyst activities. The catalytic performance and reusability of these enzyme-nanozeolite complexes were evaluated in the ethanolysis transesterification of microalgae oil to fatty acid ethyl esters (FAEEs). TLL immobilized on the nanozeolite supports functionalized with APTMS and GA provided the most efficient biocatalysis, with FAEEs yields above 93% and stability during five reaction cycles. Lower FAEEs yields and poorer catalytic stability were found for nanozeolite-enzyme complexes prepared only by physical adsorption. The findings indicated the viability of designing highly efficient biocatalysts for biofuel production by means of chemical modulation of nanozeolite surfaces. The high biocatalyst catalytic efficiency observed in ethanolysis reactions using a lipid feedstock that does not compete with food production is an advantage that should encourage the industrial application of these biocatalysts.
The cytotoxic response, cellular uptake, and metabolomic profile of HeLa and HaCaT cell lines treated with cobalt ferrite nanoparticles (CoFeO NPs) were investigated in this study. Cell viability assays showed low cytotoxicity caused by the uptake of the nanoparticles at 2 mg/mL. However, metabolomics revealed that these nanoparticles impacted cell metabolism even when tested at a concentration that presented low cytotoxicity according to the cell viability assay. The two cell lines shared stress-related metabolic changes such as increase in alanine and creatine levels. A reduced level of fumarate was also observed in HeLa cells after treatment with the nanoparticles, and this alteration can inhibit tumorigenesis. Fumarate is considered to be an oncometabolite that can inhibit prolyl hydroxylase, and this inhibition stabilizes HIF1α, one of the master regulators of tumorigenesis that promotes tumor growth and development. In summary, this study showed that nanoparticle-treated HeLa cells demonstrated decreased concentrations of metabolites associated with cell proliferation and tumor growth. The results clearly indicated that treatment with these nanoparticles might cause a perturbation in cellular metabolism.
Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.
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