In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis.

BACKGROUND: Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. OBJECTIVE: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. METHODS: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. RESULTS: It was determined that doses of the recombinant Omp25 protein greater than 0.1 µg/mL are toxic to RAW cells. Doses of 1 µg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 µg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. CONCLUSION: Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.

Effect of Met/Leu substitutions on the stability of NAD+-dependent formate dehydrogenases from Gossypium hirsutum.

NAD+-dependent formate dehydrogenases (FDHs) are extensively used in the regeneration of NAD(P)H and the reduction of CO2 to formate. In addition to their industrial importance, FDHs also play a crucial role in the maintenance of a reducing environment to combat oxidative stress in plants. Therefore, it is important to investigate the response of NAD+-dependent FDH against both temperature and H2O2, to understand the defense mechanisms, and to increase its stability under oxidative stress conditions. In the present study, we characterized the oxidative and thermal stability of NAD+-dependent FDH isolated from cotton, Gossypium hirsutum (GhFDH), by investigating the effect of Met/Leu substitutions in the positions of 225, 234, and 243. Results showed that the single mutant, M234L (0.72 s-1 mM-1), and the triple mutant, M225L/M234L/M243L (0.55 s-1 mM-1), have higher catalytic efficiency than the native enzyme. Substitution of methionine by leucine on the position of 243 increased the free energy gain by 670 J mol-1. The most remarkable results in chemical stability were seen for double and triple mutants, cumulatively. Double and triple substitution of Met to Leu (M225L/M243L and M225L/M243L/M234L) reduce the kefin by a factor of 2 (12.3×10-5 and 12.8×10-5 s-1, respectively.Key points• The closer the residue to NAD+, in which we substituted methionine to leucine, the lower the stability against H2O2 we observed.• The significant gain in the Tm value for the M243L mutant was observed as +5°C.• Residue 234 occupies a critical position for oxidation defense mechanisms. Graphical abstract (a) Methionine amino acids on the protein surface are susceptible to oxidative stress and can be converted to methionine sulfoxide by reactive oxygen derivatives (such as hydrogen peroxide). Therefore, they are critical regions in the change of protein conformation and loss of activity. (b) Replacing the amino acid methionine, which is susceptible to oxidation due to the sulfur group, with the oxidation-resistant leucine amino acid is an important strategy in increasing oxidative stability.

Microbiota profiling and screening of the lipase active halotolerant yeasts of the olive brine.

Searching for novel enzymes that could be active in organic solvents has become an area of interest in recent years. Olive brine naturally provides a suitable environment for the survival of halophilic and acidophilic microorganisms and the resulting genome is thought to be a gene source for determining the halophilic and acidophilic proteins that are active in a non-aqueous organic solvent medium, and so it has been used in several biotechnological and industrial applications. In this study, microbial analysis of natural, cracked green olive brine from the southern region of Turkey has been made by next-generation sequencing of the brine metagenome for the first time in the literature. The number of reads assigned to fungal operational taxonomic units was the highest percentage (73.04%) with the dominant representation of Ascomycota phylum (99% of fungi). Bacterial OTU was 3.56% of the reads and Proteobacteria phylum was 65% of the reads. The lipase production capacity of the yeasts that were grown on the media containing elevated concentrations of NaCl (1-3 M) was determined on a Rhodamine B-including medium. Molecular identification of the selected yeasts was performed and 90% of sequenced yeasts had a high level of similarity with Candida diddensiae, whereas 10% showed similarity to Candida boidinii. The hydrolytic lipase activities using olive oil were analyzed and both yeasts showed cell-bound lipase activity at pH 3.0.

Stress response of NAD+-dependent formate dehydrogenase in Gossypium hirsutum L. grown under copper toxicity.

Cotton (Gossypium hirsutum L.), which is not directly involved in the food chain, appears to be a suitable candidate to remove heavy metals from the food chain and to be a commercial plant which could be planted in contaminated soils. The key point of this approach is selection of the right genotype, which has heavy metal resistance or hyperaccumulation properties. Therefore, in the present study, two G. hirsutum genotypes, Ersan-92 and N-84S, were grown under copper stress and investigated to obtain further insights about the heavy metal tolerance mechanisms of plants by focusing on the expression of NAD+-dependent formate dehydrogenase (FDH). In accordance with the results, which were obtained from RT-PCR analysis and activity measurements, in the Ersan-92 root tissue, FDH activity increased significantly with increasing metal concentrations and a 6.35-fold higher FDH activity was observed in the presence of 100-µM Cu. As opposed to Ersan-92, the maximum FDH activity in the roots of N-84S, which were untreated with copper as the control plants, was measured as 0.0141-U mg-1 g-1 FW, and the activity decreased significantly with the increasing metal concentrations. The metallothionein (GhMT3a) transcript level of the plants grown in a medium containing different Cu concentrations showed nearly the same pattern as that of the FDH gene transcription. It was observed that while the tolerance of N-84S in the lower Cu concentration reduces remarkably, Ersan-92 continues to struggle up to 100-µM Cu. The results of the SOD analysis also confirm this activity of Ersan-92 against the Cu stress.

Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.).

NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2), providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO2 reduction. Here, the truncated form of the Gossypium hirsutum fdh1 cDNA was cloned into pQE-2 vector, and overexpressed in Escherichia coli DH5α-T1 cells. Recombinant GhFDH1 was purified 26.3-fold with a yield of 87.3%. Optimum activity was observed at pH 7.0, when substrate is formate. Kinetic analyses suggest that GhFDH1 has considerably high affinity to formate (0.76 ± 0.07 mM) and NAD+ (0.06 ± 0.01 mM). At the same time, the affinity (1.98 ± 0.4 mM) and catalytic efficiency (0.0041) values of the enzyme for NADP+ show that GhFDH1 is a valuable enzyme for protein engineering studies that is trying to change the coenzyme preference from NAD to NADP which has a much higher cost than that of NAD. Improving the NADP specificity is important for NADPH regeneration which is an important coenzyme used in many biotechnological production processes. The Tm value of GhFDH1 is 53.3 °C and the highest enzyme activity is measured at 30 °C with a half-life of 61 h. Whilst further improvements are still required, the obtained results show that GhFDH1 is a promising enzyme for NAD(P)H regeneration for its prominent thermostability and NADP+ specificity.

Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase.

In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6 × 104-fold and 5 × 104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating "smarter libraries" for improving the properties of cmFDH.

Improved Candida methylica formate dehydrogenase fermentation through statistical optimization of low-cost culture media.

NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is of use in the regeneration of NAD(P)H coenzymes, and therefore has strong potential for practical application in chemical and medical industries. A low-cost production of recombinant Escherichia coli (E. coli) containing FDH from Candida methylica (cmFDH) was optimized in molasses-based medium by using response surface methodology (RSM) based on central composite design (CCD). The beet molasses as a sole carbon source, (NH4)2HPO4 as a nitrogen and phosphorus source, KH2PO4 as a buffer agent, and Mg2SO4 · 7H2O as a magnesium and sulfur source were used as variables in the medium. The optimum medium composition was found to be 34.694 g L⁻¹ of reducing sugar (equivalent to molasses solution), 8.536 g L⁻¹ of (NH4)2HPO4, 3.073 g L⁻¹ of KH2PO4, and 1.707 g L⁻¹ of Mg2SO4 · 7H2O. Molasses-based culture medium increased the yield of cmFDH about three times compared to LB medium. The currently developed media has the potential to be used in industrial bioprocesses with low-cost production.

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase.

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10(-13)M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k(1)) of about 2x10(-3)s(-1) (by deduction k(-1) is about 10(-4)s(-1)) and assembles into the active dimeric state with a bimolecular rate constant (k(2)) of about 2x10(4)M(-1)s(-1). The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k(-2) approximately 3x10(-7)s(-1)).

Improving the purification of NAD+-dependent formate dehydrogenase from Candida methylica.

The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.