Biochemical characterization of a novel thermostable feruloyl esterase from Geobacillus thermoglucosidasius DSM 2542T.

The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542T was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11 kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50 °C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50 °C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). Km, Vmax, kcat and kcat/Km values for p-nitrophenyl caprylate were determined as 0.035 mM, 11,735 µmol/min/mg protein, 5491 (1/s) and 156,885 s-1 mM-1 respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry.

Cloning, characterization and paper pulp applications of a newly isolated DyP type peroxidase from Rhodococcus sp. T1.

A newly identified ligninolytic Rhodococcus strain (Rhodococcus sp. T1) was isolated from forestry wastes (Trabzon/Turkey). The DyP type peroxidase of Rhodococcus sp. T1 (DyPT1) was cloned, characterized and paper treated for industrial applications. Molecular weight of the protein was about 38 kDa. The kinetic parameters were 0.94 mM and 1417.53 µmol/min/mg for Km and Vmax, respectively. The enzyme was active at the temperature range of 25-65 °C and optimum temperature was 35 °C, enzyme was stable up to 6 days at room temperature. Optimum pH of the DyPT1 was 4.0 and it was stable between pH 4.0-6.0 up to 8 days at room temperature. Effects of some metal ions, Hemin, and some chemical agents on DyPT1 were determined. Hemin has implemented protective effects on the stability and the activity of the enzyme in long time periods when added into growing medium. DyPT1 was applied to eucalyptus kraft pulp for analyzing the bleaching efficiency, physical and optical tests of the manufuctared paper were carried out. Application of lignin peroxidase to kraft pulp caused a decrease of 5.2 units for kappa number and an increase from 52.05 to 64.18% in the delignification rate.

Generation of acid mine drainage around the Karaerik copper mine (Espiye, Giresun, NE Turkey): implications from the bacterial population in the Acisu effluent.

The Karaerik Cu mine is a worked-out deposit with large volumes of tailings and slags which were left around the mine site without any protection. Natural feeding of these material and run-off water from the mineralised zones into the Acisu effluent causes a serious environmental degradation and creation of acid mine drainage (AMD) along its entire length. This research aims at modelling the formation of AMD with a specific attempt on the characterisation of the bacterial population in association with AMD and their role on its occurrence. Based on 16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community was determined to consist of Acidithiobacillus ferrivorans, Ferrovum myxofaciens, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans as iron-oxidising bacteria, Acidocella facilis, Acidocella aluminiidurans, Acidiphilium cryptum and Acidiphilium multivorum as iron-reducing bacteria, and Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidiphilium cryptum as sulphur-oxidising bacteria. This association of bacteria with varying roles was interpreted as evidence of a concomitant occurrence of sulphur and iron cycles during the generation of AMD along the Acisu effluent draining the Karaerik mine.

Sulfonamide inhibition studies of the ß-carbonic anhydrase from the newly discovered bacterium Enterobacter sp. B13.

The genome of the newly identified bacterium Enterobacter sp. B13 encodes for a ß-class carbonic anhydrases (CAs, EC 4.2.1.1), EspCA. This enzyme was recently cloned, and characterized kinetically by this group (J. Enzyme Inhib. Med. Chem. 2016, 31). Here we report an inhibition study with sulfonamides and sulfamates of this enzyme. The best EspCA inhibitors were some sulfanylated sulfonamides with elongated molecules, metanilamide, 4-aminoalkyl-benzenesulfonamides, acetazolamide, and deacetylated methazolamide (KIs in the range of 58.7-96.5nM). Clinically used agents such as methazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, zonisamide, sulthiame, sulpiride, topiramate and valdecoxib were slightly less effective inhibitors (KIs in the range of 103-138nM). Saccharin, celecoxib, dichlorophenamide and many simple benzenesulfonamides were even less effective as EspCA inhibitors, with KIs in the range of 384-938nM. Identification of effective inhibitors of this bacterial enzyme may lead to pharmacological tools useful for understanding the physiological role(s) of the ß-class CAs in bacterial pathogenicity/virulence.

Cloning, expression and biochemical characterization of a ß-carbonic anhydrase from the soil bacterium Enterobacter sp. B13.

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co(2+) affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the ß-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 °C and pH of 8.3: kcat of 4.8 × 10(5) s(-1) and kcat/Km of 5.6 × 10(7) M(-1) × s(-1). This activity was potently inhibited by acetazolamide which showed a KI of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO3 biomineralization processes.

Molecular analysis of the genus Anoxybacillus based on sequence similarity of the genes recN, flaA, and ftsY.

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550-600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3-42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.