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.

The novel gene yvfI in Bacillus subtilis is essential for bacilysin biosynthesis.

Using transposon mutagenesis in Bacillus subtilis PY79, three independent mutants defective in production of bacilysin were isolated. To identify the genes in these mutant loci affecting bacilysin biosynthesis, the inserted transposon and its flanking regions were cloned and sequenced from each mutant. Transposon insertions in these three mutants were found to be in the yvfI gene which encodes an unknown protein similar to GntR family transcriptional regulators. For further confirmation, deletion mutants were constructed in which nucleotides 196-314 of the yvfI gene were removed. All resulting yvfI (Delta196-314)::spc deletion mutants exhibited bacilysin-negative phenotypes, as in the case of the yvfI::Tn10::spc insertional mutants. The lacR gene, encoding a transcriptional regulator, resides immediately downstream from the yvfI gene. Therefore, an insertion mutation was created in the lacR gene to demonstrate that the bacilysin negative phenotype is actually due to the mutation in the yvfI gene and not a polar effect of yvfI mutation on the downstream gene. As expected, all resulting lacR mutant derivatives of PY79 still produced bacilysin.