Purification and biochemical characterization of a novel carbonic anhydrase II from erythrocytes of camel (Camelusdromedarius).

A novel carbonic anhydrase II (CA II) from erythrocytes of camel (Camelus dromedarius) was purified to homogeneity using affinity chromatography and biochemically characterized. Specific activity of 140.88 U/mg was obtained with 745.17-fold purification and 25.37% yield. The enzyme was a monomer with a lower molecular weight (25 kDa) and lower Zn content (0.50 mol of Zn per mol of protein). The enzyme showed higher optimum temperature (70 °C) and pH (pH 9.0), moreover, it was stable at higher temperatures and strongly alkaline pH as judged by thermodynamic parameters (Ea, kd, Ed, t1/2, D-value, Z-value, ΔH, ΔG and ΔS). The enzyme was inhibited by cations (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Ni2+, Mg2+ and Zn2+) as well as by anions (Br‾, CH3COO‾, ClO4‾, CN‾, F‾, HCO3‾, I‾, N3‾, NO3‾ and SCN‾), some anions (C6H5O73-, CO32-, SeO3‾ and SO42-) does not affect enzyme activity. Effect of various chemicals on enzyme activity was also investigated. Km, Vmax, kcat and kcat/Km values for 4-NPA were found to be 1.74 mM, 0.0093 U/mL, 0,0039 s-1 and 0,0023 s-1 mM-1, respectively. With these interesting biochemical properties, camel CA II represents promising candidate for harsh industrial applications, in particular, for a successful biomimetic CO2 sequestration process.

Eco-friendly degradation of reactive red 195, reactive blue 214, and reactive yellow 145 by Klebsiella pneumoniae MW815592 isolated from textile waste.

The water is used in many textile manufacturing steps beyond cleaning. The quantity and the significant chemical load of the effluents generated constitute the primary challenge of the textile industry. In order to discover new sustainable methods to overcome this problem, the aim of this research was to study the potential for degradation of Reactive Blue 214, Reactive Red 195, and Reactive Yellow 145 using a dye degrading bacterium. Sequencing analysis reveals it to be Klebsiella pneumoniae MW815592. This strain completely decolorized artificial effluent (200 mg/L) after 42 h at pH 9 and 46 °C. The decolorization rate increased in the presence of glucose and yeast extract (2 g). In addition, our finding revealed that the decolorization is due to biodegradation rather than adsorption on the bacterial surface.

Purification and biochemical characterization of catalase that confers protection against hydrogen peroxide induced by stressful desert environment: the Camelus Dromedarius kidney catalase.

Camel is continually exposed to stressful desert environment that enhances generation of reactive oxygen species, including hydrogen peroxide (H2O2). Catalase plays an important role in detoxification of H2O2. A highly active catalase from camel kidney was purified to homogeneity, with a specific activity of 1,774,392 U/mg protein, using ion exchange and metal chelate affinity chromatography. The molecular weight of the enzyme was 268 kDa consisting of four identical subunits of 63 kDa. The enzyme showed higher optimum temperature (45 °C) and higher activation energy (4.37 kJ mol-1). The thermodynamic parameters, ΔH, ΔG and ΔS, were determined. The effect of various metal ions and chemicals on enzyme activity was investigated. Km, Vmax, kcat and kcat/Km values for H2O2 were found to be 46 mM, 10,715,045 U/mg, 48,265,968 s-1 and 2,966,562 s-1 mM-1, respectively. Camel kidney catalase displayed higher affinity efficiency for H2O2 and can protect reduced glutathione (GSH) from oxidation by H2O2. Sodium azide was found to be a noncompetitive inhibitor of enzyme with Ki and IC50 of 17.88 µM and 20.94 µM, respectively. Camel catalase showed unique biochemical properties. Interestingly, camel catalase can protect molecules (GSH) and organ functions (kidney) from the toxic effects of H2O2 induced by stressful desert environment.

Synthesis, Characterization, Biological Activity and Molecular Docking Studies of Novel Organotin(IV) Carboxylates.

Four new carboxylates complexes with general formula R2SnL2 and R3SnL, where R = n-butyl (1, 3), methyl (2, 4) and L = 4-Chlorophenoxyacetate, were synthesized in significant yields. FT-IR analysis revealed a chelating (1 and 2) and a bridging bidentate (3 and 4) coordination modes for the carboxylate ligand in solid state which was further confirmed by the single crystal X-ray analysis of complex 4. The NMR data (1H, 13C and 119Sn) revealed a higher coordination number around the tin center in R2SnL2 (1 and 2) compared to R3SnL (3 and 4). A close matching was observed between the experimental and calculated structures (obtained at B3LYP/6-31G* + LANL2DZ basis set). Quantum chemical analysis indicates that the carboxylate moiety has the major contribution in the formation of filled and unfilled orbitals as well as in ligand to ligand intramolecular charge transfer during the electronic transitions. The cytotoxicity data of the screened compounds evaluated against lung cancer cell line (A549) and normal lung fibroblast cell line (MRC-5) revealed that 1, 3 and 4 have shown dose dependent cytotoxic effects while HL and 2 have shown steady and low cytotoxic activities. The antibacterial activity of complexes 1-4 is higher than that of HL. Molecular docking study showed an intercalation binding mode for complex 3 with DNA (docking score = -3.6005) involving four polar interactions. Complex 3 docking with tubulin (PDB ID 1SA0) with colchicine as a target protein resulted in three polar interactions (docking score -5.2957). Further, the docking analysis of the HL and 1-4 has shown an adequate interactions with the coronavirus SARS-CoV-2 spike protein, nucleocapsid protein and human angiotensin converting enzyme (ACE2).

A novel acid phosphatase from cactus (Opuntia megacantha Salm-Dyck) cladodes: Purification and biochemical characterization of the enzyme.

Acid phosphatase (ACP) plays an important role in regulating phosphate nutrition in plants. Herein, for the first time, a novel ACP from Opuntia megacantha Salm-Dyck cladodes was purified to homogeneity and biochemically characterized. Specific activity of 8.78 U/mg was obtained with 11.29-fold purification and 15% yield. ACP was purified as monomer with molecular weight of 44 kDa as determined by SDS-PAGE under denaturing and nondenaturing conditions. Optimum pH and temperature for ACP activity was 5.5 and 60 °C, respectively. Thermodynamic parameters (Ea, ΔH, ΔG and ΔS) were also determined. ACP activity was stimulated by Ca2+, strongly inhibited by Cu2+ and Fe3+, and moderately inhibited by Mg2+ and Zn2+. Br-, CN-, F-, I- and N3- weakly inhibited ACP activity, where more than 70% of enzyme activity was remained at 5 mM. In addition, effect of ß-ME, Cys, DTT, EDTA, H2O2, PMSF, SDS and TX-100 on ACP activity was investigated. km, Vmax, kcat and kcat/km of ACP for p-NPP were found to be 0.09 mM, 2.75 U/mL, 9.60 s-1 and 106.67 s-1 mM-1, respectively. The biochemical properties of ACP from Opuntia megacantha Salm-Dyck cladodes provide novel features with other plant ACPs and basic knowledge of ACP in Opuntia species.

Purification and biochemical characterization of a novel copper, zinc superoxide dismutase from liver of camel (Camelus dromedarius): An antioxidant enzyme with unique properties.

A novel copper, zinc superoxide dismutase (CuZnSOD) was purified to homogeneity from the liver of an animal well adapted to the stressful living conditions of the desert, the camel (Camelus dromedarius). The biochemical properties of camel liver CuZnSOD were examined. The purified enzyme had a native molecular weight of 28 kDa, as judged by gel filtration chromatography, and showed a single band at 27 kDa on SDS-PAGE, indicating that it is a monomeric protein. Optimal activity of the purified enzyme occurred at 43 °C and pH 6.0, and the activation energy was 1.42 kJ/mol. CuZnSOD activity was strongly inhibited by ß-ME, DTT, H2O2 and SDS and slightly inhibited by EDTA, NaN3 and PMSF. Al3+, Ca2+, Cd2+, Mg2+ and Zn2+ stimulated CuZnSOD activity, whereas Ba2+, Co2+, Fe2+ and Ni2+ inhibited it. The purified enzyme contained 0.010 µg of Cu and 0.69 µg of Zn per mg of protein. Km, Vmax, kcat and kcat/Km values for NBT and riboflavin were 16.27 and 0.16 µM, 20.85 and 21.54 U/mg, 9.65 and 9.97 s-1, and 0.59 and 62.33 s-1 µM-1, respectively. Camel liver CuZnSOD exhibited unique biochemical properties compared to those of other CuZnSODs, including lower molecular weight with a monomeric structure, higher optimum temperature, very low Ea, very low optimum pH, very low contents of Cu and Zn, and higher affinity, turnover number and catalytic efficiency for riboflavin. These unique properties of camel liver CuZnSOD might be related to the ability of this animal to inhabit stressful desert conditions.

Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, ß-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al3+, Cd2+ and Mg2+, whereas Ca2+, Co2+ and Ni2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The Km and Vmax of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with Ki value of 14.43µM, the IC50 was found to be 16.71µM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert.