Pharmacokinetics following intravenous administration and pharmacodynamics of cefquinome in buffalo calves.

Disposition following single intravenous injection (2 mg/kg) and pharmacodynamics of cefquinome were investigated in buffalo calves 6-8 months of age. Drug levels in plasma were estimated by high-performance liquid chromatography. The plasma concentration-time profile following intravenous administration was best described by a two-compartment open model. Rapid distribution of cefquinome was evident from the short distribution half-life (t ½ α = 0.36 ± 0.01 h), and small apparent volume of distribution (Vd area = 0.31 ± 0.008 L/kg) indicated limited drug distribution in buffalo calves. The values of area under plasma concentration-time curve, elimination half-life (t ½ ß ), total body clearance (ClB), and mean residence time were 32.9 ± 0.56 µg · h/mL, 3.56 ± 0.05 h, 60.9 ± 1.09 mL/h/kg, and 4.24 ± 0.09 h, respectively. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration of cefquinome were 0.035-0.07 and 0.05-0.09 µg/mL, respectively. A single intravenous injection of 2 mg/kg may be effective to maintain the MIC up to 12 h in buffalo calves against the pathogens for which cefquinome is indicated.

Enhancing the expression of recombinant small laccase in Pichia pastoris by a double promoter system and application in antibiotics degradation.

Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5 mg/L ciprofloxacin (CIP) and 99% of 100 mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.

Crystal structure of a thermophilic fungal cyanase and its implications on the catalytic mechanism for bioremediation.

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.

Enzymatic treatment of phenolic pollutants by a small laccase immobilized on APTES-functionalised magnetic nanoparticles.

In this study, we have successfully synthesized magnetic nanoparticles (MNPs), functionalised them by silanization and used them for the covalent immobilization of a recombinant small laccase (rSLAC) from Streptomyces coelicolor. The immobilized recombinant laccase (MNP-rSLAC) was subsequently used for the treatment of phenol, 4-chlorophenol (4-CP) and 4-fluorophenol (4-FP). The enzyme completely degraded 80 µg/mL of the selected phenolic compounds within 2 h in the presence of a natural mediator, acetosyringone. The MNP-rSLAC retained > 73% of initial activity (2,6-dimethoxyphenol as substrate) after 10 catalytic cycles and could be easily recovered from the reaction mixture by the application of magnetic field. Furthermore, immobilised rSLAC exhibited better storage stability than its free counterpart. The Michaelis constant (Km) value for the immobilised rSLAC was higher than free rSLAC, however the maximum velocity (Vmax) of the immobilised SLAC was similar to that of the free rSLAC. Growth inhibition studies using Escherichia coli showed that rSLAC-mediated treatment of phenolic compounds reduced the toxicity of phenol, 4-CP and 4-FP by 90, 60 and 55%, respectively. Interestingly, the presence of selected metal ions (Co2+, Cu2+, Mn2+) greatly enhanced the catalytic activity of rSLAC and MNP-rSLAC. This study indicates that immobilized small laccase (MNP-rSLAC) has potential for treating wastewater contaminated with phenolic compounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02854-0.

Simultaneous removal of heavy metals and cyanate in a wastewater sample using immobilized cyanate hydratase on magnetic-multiwall carbon nanotubes.

Global environmental problems allied with waste management require novel approaches for the simultaneous removal of heavy metals and other associated compounds including cyanate. In this study, iron-oxide filled multi-walled carbon nanotubes (m-MWCNTs) were successfully synthesized and characterized by field emission gun scanning electron microscopy (FEGSEM), high-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD). The m-MWCNTs were amino-functionalized for the covalent immobilization of a recombinant cyanate hydratase (rTl-Cyn), and were characterized by fourier transform infrared (FTIR) spectroscopy. The immobilized rTl-Cyn on the m-MWCNTs (m-MWCNT-rTl-Cyn) had long term storage stability and showed great potential towards cyanate biodegradability. We found that m-MWCNT-rTl-Cyn retained >94% of the initial activity even after 10 repeated cycles of bio-catalysis. Strikingly, the m-MWCNT-rTl-Cyn simultaneously reduced the concentration of chromium (Cr), iron (Fe), lead (Pb) and copper (Cu) by 39.31, 35.53, 34.48 and 29.63%, respectively as well as the concentration of cyanate by ≥84%, in a synthetic wastewater sample.

A novel strategy for the efficient removal of toxic cyanate by the combinatorial use of recombinant enzymes immobilized on aminosilane modified magnetic nanoparticles.

Cyanase detoxifies cyanate by transforming it to ammonia and carbon dioxide in a bicarbonate-dependent reaction, however, dependence on bicarbonate limits its utilization in large-scale applications. A novel strategy was therefore developed for overcoming this bottleneck by the combined application of cyanase (rTl-Cyn) and carbonic anhydrase (rTl-CA). The synergistic effect of rTl-Cyn and rTl-CA could reduce the dependence of bicarbonate by 80%, compared to using rTl-Cyn alone. Complete degradation of cyanate (4 mM) was achieved with buffered conditions and 85 ±â€¯5% degradation with industrial wastewater sample, when 20 U of rTl-Cyn was applied. Furthermore, a similar percentage of degradation was achieved using 80% less bicarbonate, when rTl-Cyn and rTl-CA were used together under identical conditions. In addition, rTl-Cyn and rTl-CA were immobilized onto the magnetic nanoparticles and their catalytic activity, stability and reusability were also evaluated. This is the first report on the synergistic biocatalysis by rTl-Cyn and rTl-CA, for cyanate detoxification.

Synthesis, molecular docking, antimicrobial, antioxidant and toxicity assessment of quinoline peptides.

A series of quinoline based peptides were synthesized by a one-pot reaction through Ugi-four component condensation of lipoic acid, cyclohexyl isocyanide, aniline derivatives and 2-methoxy quinoline-3-carbaldehyde derivatives under microwave irradiation. The products were obtained in excellent yields and high purity. Solvent optimization and the effect of microwave irradiation with various powers were also observed. All the synthesized compounds were characterized by FTIR, NMR spectral data and elemental analysis. A total of eight peptides were subjected to antimicrobial, antioxidant and toxicity evaluation. Among them, four peptides showed potential towards antibacterial screening with Bacillus cereus, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Candida albicans, Candida utilis and three peptides showed antioxidant test positive (DPPH). Besides, toxicity of all the peptides were evaluated by using brine shrimp and it was observed that four peptides showed mortality rate less than 50% up to 48h. Molecular docking studies revealed that the higher binding affinity of the two peptides toward DNA gyrase than ciprofloxacin based on Libdock score. The described chemistry represents a facile tool to synthesize complex heterocycles of pharmaceutical relevance in a highly efficient and one-pot fashion. The advantages of this method are its green approach, inexpensive solvent, shorter reaction times and excellent yields.

Secretory expression of recombinant small laccase from Streptomyces coelicolor A3(2) in Pichia pastoris.

This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ±â€¯10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142.85 µM, 10 µM and 54.55 µM and the corresponding kcat values were 60.6 s-1, 25.36 s-1 and 27.84 s-1, respectively. The t1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.

Expression of a novel recombinant cyanate hydratase (rTl-Cyn) in Pichia pastoris, characteristics and applicability in the detoxification of cyanate.

A recombinant Pichia pastoris harbouring the cyanate hydratase gene (rTl-Cyn) from the thermophilic fungus Thermomyces lanuginosus SSBP yielded a high titre of extracellular cyanate hydratase (100±13UmL-1) which was ∼10-fold higher than the native fungal strain. The purified rTl-Cyn had a molecular mass of ∼20kDa on SDS-PAGE, with Km, Vmax, kcat and kcat/Km values of 0.34mM, 2857.14µmolesmg-1min-1, 2.14×104s-1 and 6.3 ×107M-1s-1, respectively. Its properties of thermostability, pH stability, and heavy metals insensitivity, make it a suitable candidate for bioremediation in extreme environments. The rTl-Cyn was able to degrade toxic cyanate completely with the liberation of ammonia, which was confirmed by FTIR analysis. This is the first report of any known cyanate hydratase that has been expressed in P. pastoris, characterized and effectively evaluated for cyanate detoxification.

Recombinant HAP Phytase of the Thermophilic Mold Sporotrichum thermophile: Expression of the Codon-Optimized Phytase Gene in Pichia pastoris and Applications.

The codon-optimized phytase gene of the thermophilic mold Sporotrichum thermophile (St-Phy) was expressed in Pichia pastoris. The recombinant P. pastoris harboring the phytase gene (rSt-Phy) yielded a high titer of extracellular phytase (480 ± 23 U/mL) on induction with methanol. The recombinant phytase production was ~40-fold higher than that of the native fungal strain. The purified recombinant phytase (rSt-Phy) has the molecular mass of 70 kDa on SDS-PAGE, with K m and V max (calcium phytate), k cat and k cat/K m values of 0.147 mM and 183 nmol/mg s, 1.3 × 10(3)/s and 8.84 × 10(6)/M s, respectively. Mg(2+) and Ba(2+) display a slight stimulatory effect, while other cations tested exert inhibitory action on phytase. The enzyme is inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K and 2,3-bunatedione, but resistant to both pepsin and trypsin. The rSt-Phy is useful in the dephytinization of broiler feeds efficiently in simulated gut conditions of chick leading to the liberation of soluble inorganic phosphate with concomitant mitigation in antinutrient effects of phytates. The addition of vanadate makes it a potential candidate for generating haloperoxidase, which has several applications.

Characteristics of Recombinant Phytase (rSt-Phy) of the Thermophilic mold Sporotrichum thermophile and its applicability in dephytinizing foods.

Sporotrichum thermophile produces very low titres of phytase (St-Phy) extracellularly, which is acidstable, thermostable, and protease insensitive with broad substrate specificity, and therefore, the gene encoding phytase (St-Phy) has been cloned and expressed in E. coli. The purified recombinant phytase (rSt-Phy) has the molecular mass of 55 kDa with Km and Vmax (calcium phytate), kcat and kcat/Km of 0.143 mM, 185.05 nmoles mg(-1)  s(-1), 5.1 × 10(3) s(-1), and 3.5 × 10(7) M(-1) s(-1), respectively. Mg(2+) and Ba(2+) display slight stimulatory effect on the enzyme, while it is inhibited by other ions to a varied extent. The enzyme is also inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K, and 2,3-butanedione but resistant to both pepsin and trypsin. The rSt-Phy is useful in dephytinization of tandoori and naan (unleavened flat Indian breads), and bread, liberating soluble inorganic phosphate that mitigates anti-nutrient effects of phytic acid.

Green rapid biogenic synthesis of bioactive silver nanoparticles (AgNPs) using Pseudomonas aeruginosa.

The present work was focused on isolating a bacterial strain of Pseudomonas sp. with the ability to synthesise AgNPs rapidly. A strain of Pseudomonas aeruginosa designated JO was found to be a potential candidate for rapid synthesis of AgNPs with a synthesis time of 4h in light, at room temperature which is a shorter time period noticed for the synthesis when compared to the previous reports Biosynthesis of AgNPs was achieved by addition of culture supernatant with aqueous silver nitrate solution (1 mM). The reaction mixture exhibits change in colour from green to brown with a peak at 420 nm corresponding to the plasmon absorbance of AgNPs by UV-vis spectroscopy. The nanoparticles were characterised by X-ray diffraction (XRD), energy-dispersive X-ray analysis, Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), Zetasizer and transmission electron microscopy (TEM). The XRD spectrum exhibited 2θ values corresponding to the silver nanocrystals. TEM and SEM micrographs revealed the extracellular formation of polydispersed elongated nanoparticles with an average size of 27.5 nm. Synthesised nanoparticles showed antibacterial property against both gram-positive and gram-negative microorganisms, but more effective towards gram-negative.

Effects of novel brominated flame retardants on steroidogenesis in primary porcine testicular cells.

Brominated flame retardants are chemicals with fire quenching properties which are extensively used in manufacturing. Historically, less regulated use of legacy brominated flame retardants (BFRs) for a number of years has resulted in ubiquitous contamination of the environment. As a result, some of the more persistent BFRs have been phased out and are being replaced by a next generation of brominated compounds for which there is little toxicological data. The study investigated effects of 2-ethylhexyl tetrabromobenzoate (TBB), 1,2,5,6-tetrabromocyclooctane (TBCO), and bis-(2-ethylhexyl) tetrabromophthalate (TBPH)on steroidogenesis in a porcine primary testicular cell model. TBB did not affect sex-steroid production in this cell model; rather the data suggest a flux towards synthesis of aldosterone and cortisol via up-regulation of CYP21A2. At the greatest concentrations of TBCO and TBPH tested greater production of sex hormones testosterone (T) and estradiol (E2) was observed. Effects were mediated by regulation of multiple molecular targets in the steroidogenesis pathway; CYP11A in the case of TBPH and CYP17A1 in the case of TBCO. This investigation is the first of its kind to use a testicular mixed population cell model to investigate mechanism(s) of action of three chemically diverse compounds currently used in commercial fire retardants.

Pharmacokinetics, urinary excretion and plasma protein binding of ofloxacin in water buffalo calves (Bubalus bubalis).

Pharmacokinetics and urinary excretion of an intravenous dose of 5 mg.kg-1 ofloxacin were investigated in water buffalo calves. Plasma concentrations of ofloxacin were determined by high-performance liquid chromatography. Ofloxacin was rapidly distributed from the central to the peripheral compartment as evidenced by a short distribution half-life (0.09 h ± 0.003 h) and high K12 (4.7 h(-1) ± 0.1 h(-1)), and was detected in plasma for 8 h. The large volume of distribution (2.48 L.kg(-1) ± 0.18 L.kg(-1)) obtained in this study indicated high distribution of ofloxacin in water buffalo calves. The elimination half-life, the area under the plasma drug concentration-time curve and total body clearance were 2.11 h ± 0.13 h, 6.20 µg.mL(-1) ± 0.23 µg.mL(-1).h and 0.81 mL.kg(-1).h(-1) ± 0.03 mL.kg(-1).h(-1), respectively. About 18.7% of administered drug was bound to plasma proteins and approximately 32.5% of the administered dose was recovered in urine within 48 h. The results of the study indicated a favourable pharmacokinetic profile of ofloxacin in water buffalo calves, which suggests that ofloxacin may be effective against urinary pathogens in this species.