Determination of lansoprazole in pharmaceuticals using flow injection with rhodamine 6G-diperiodatoargentate(III) chemiluminescence detection.

A chemiluminescence (CL) method based on rhodamine 6G (R6G)-diperiodatoargentate(III) (silver(III) complex) reaction in acid solution is reported for the determination of lansoprazole (LNP) combined with a flow injection (FI) technique. The most likely mechanism for CL reaction was elucidated considering reported data, spectrophotometric and spectrofluorimetric studies. The weak CL reaction between R6G and silver(III) complex could be magnanimously increased in the presence of LNP with a limit of detection (LOD) of 0.002 mg L-1 (S/N = 3), a linear range of 0.01 to 10 mg L-1 (R2 = 0.9997, n = 7), a relative standard deviation (RSD) of 1.2 to 3.2% (n = 4) and an injection throughput of 140 h-1 . No interference activity of commonly found excipients in LNP was detected. After LNP extraction from pharmaceutical samples, the recovery rate ranging from 93 to 110% (RSD, 1.4-3.3%, n = 4) was calculated. The results of the proposed flow CL method were assessed with a spectrophotometric approach applying paired Student's t-test and the calculated value (0.178) was lower than the distributed value (2.20) at a 95% confidence limit.

Effect of Y50H and S187G substitutions on thermostability and exonuclease activity of TK1646 from Thermococcus kodakarensis.

TK1646 is a highly thermostable single strand specific 3'-5' exonuclease. Exonucleases play important role in maintaining the genome integrity at elevated temperatures. Therefore, it is important to examine the factors contributing to thermostability of these exonucleases. In this study we report on production, purification and characterization of S187G and Y50H mutants of TK1646, focusing on the factors leading to thermostability of TK1646. Characterization of the recombinant proteins indicated that these substitutions did not drastically affect the catalysis of single stranded DNA. However, both of these substitutions reduced the thermostability of the recombinant proteins. Half-lives of Y50H and S187G mutants were 95 and 155 min, respectively, at 100 °C in comparison to 180 min of the wild type. Bioinformatics analysis indicated an increase in solvent accessibility of the mutated residues and disruption of hydrogens bonds. Molecular modelling and superimposition of the 3D structures of the mutants and the wild type demonstrated that one of the active site residues, Glu145, was shifted away from the metal ion in both the mutants which may be responsible for the decrease in catalytic activity. Compact secondary structure, hydrophobicity and hydrogen bonding might be the major factors contributing to the thermostability of TK1646.

Flow-injection determination of manganese (II) using surfactant enhanced diperiodatonickelate (IV)-rhodamine 6G chemiluminescence.

Chemiluminescence (CL) of the rhodamine 6-G-diperiodatonickelate (IV) (Rh6-G-Ni(IV) complex) in the presence of Brij-35 was examined in an alkaline medium and implemented using flow-injection analysis to analyze Mn(II) in natural waters. Brij-35 was identified as the surfactant of choice that enhanced CL intensity by about 62% of the reaction. The calibration curves were linear in the range 1.7 × 10-3 - 0.2 (0.9990, n = 7) and 8.0 × 10-4 - 0.1 µg ml-1 (0.9990, n = 7) with limits of detection (LODs) (S:N = 3) of 5.0 × 10-4 and 2.4 × 10-4 µg ml-1 without and with using an in-line 8-hydroxyquinoline (8-HQ) resin mini-column, respectively. The sample throughput and relative standard deviation were 200 h-1 and 1.7-2.2% in the range studied respectively. Mn(II) concentrations in certified reference materials and natural water samples was successfully determined. A brief discussion about the possible CL reaction mechanism is also given. In addition, analysis of V(III), Cr(III) and Fe(II) was also performed without and with using an in-line 8-HQ column and selective elution of each metal ion was achieved by adjusting the pH of the sample carrier stream with aqueous HCl solution.

Pcal_0632, a phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Pyrobaculum calidifontis.

Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.

Identification of a novel copper-activated and halide-tolerant laccase in Geobacillus thermopakistaniensis.

Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (ESU71923) annotated as laccase. However, the gene product did not display any laccase-like activity against the substrates examined. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 was prepared in Escherichia coli. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7-7.5. The purified enzyme was highly tolerant to various halides and organic solvents, thus having a potential for various industrial applications. To the best of our knowledge, this is the first characterization of a laccase from genus Geobacillus which identifies a gene responsible for functional laccase in this genus.

Draft Genome Sequence of Geobacillus thermopakistaniensis Strain MAS1.

Geobacillus thermopakistaniensis strain MAS1 was isolated from a hot spring located in the Northern Areas of Pakistan. The draft genome sequence was 3.5 Mb and identified a number of genes of potential industrial importance, including genes encoding glycoside hydrolases, pullulanase, amylopullulanase, glycosidase, and alcohol dehydrogenases.

Truncated Type II isopentenyl diphosphate isomerase from hyperthermophilic Achaeon Thermococcus kodakaraensis implicates the necessity of its N-terminal amino acid residues in protein thermostability.

The enzyme isopentenyl diphosphate isomerase (IDI, EC 5.3.3.2) interconverts isopentenyl diphosphate and dimethylallyl diphosphate. We had previously cloned Tk-idi gene encoding the thermostable Tk-IDI enzyme from Thermococcus kodakaraensis KOD1. Four putative start codons were found on Tk-idi gene at 123, 213, 297 and 321 positions downstream of the first start codon. In the present work four mutants were obtained by deleting 123, 213, 297 and 321 nucleotides from the 5'-end of Tk-idi gene to obtain Tk-idim, Tk-idim1, Tk-idim2, and Tk-idim3, respectively. When we tried to express these truncated genes in Escherichia coli only Tk-idim was expressed in the active form. The product, Tk-IDIM, was purified and characterized. The molecular mass of the enzyme, estimated by gel filtration chromatography, was 300 kDa which indicated that the truncated enzyme retained the octameric form. The removal of 41 N-terminal amino acids did not exhibit a significant effect on the enzyme activity however, the thermostability of the enzyme decreased. The decrease in thermostability of Tk-IDIM correlated well with the results of circular dichroism (CD) analysis and structural modeling.

Indolepyruvate ferredoxin oxidoreductase: An oxygen-sensitive iron-sulfur enzyme from the hyperthermophilic archaeon Thermococcus profundus.

Thermococcus profundus is a strictly anaerobic sulfur-dependent archaeon that grows optimally at 80°C by peptide fermentation. Indolepyruvate ferredoxin oxidoreductase (IOR), an enzyme involved in the peptide fermentation pathway, was purified to homogeneity from the archaeon under strictly anaerobic conditions. The maximal activity was obtained above the boiling temperature of water (105°C), with a half-life of 62min at 100°C and 20min at 105°C. IOR was oxygen-sensitive with a half-life of 7h at 25°C under aerobic conditions. The specific activity of T. profundus IOR was found to be dependent on the number of [4Fe-4S] clusters in the enzyme.

Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis.

Enzymatic and thermodynamic characteristics of type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (Tk-IDI) from Thermococcus kodakaraensis, which catalyzes the interconversion of IPP and DMAPP, were examined. FMN was tightly bound to Tk-IDI, and the enzyme required NADPH and Mg2+ for the isomerization in both directions. The melting temperature (Tm), the change of enthalpy (deltaH(m)), and the heat capacity change (deltaC(p)) of Tk-IDI were 88.0 degrees C, 444 kJ mol(-1), and 13.2 kJ mol(-1) K(-1), respectively, indicating that Tk-IDI is fairly thermostable. Kinetic parameters dramatically changed when the temperature crossed 80 degrees C even though its native overall structure was stably maintained up to 90 degrees C, suggesting that local conformational change would occur around 80 degrees C. This speculation was supported by the result of the circular dichroism analysis that showed the shift of the alpha-helical content occurred at 80 degrees C.

An instant measurement of oxidoreductase activity above 100 degrees C by monitoring the absorbance change.

A conventional absorbance monitoring method using a cuvette covered with a tight rubber cap was found to be applicable for measuring oxidoreductase activity at temperatures up to 115 degrees C. Using this method, the optimal temperatures of the enzymes, including oxygen-sensitive enzymes from a hyperthermophilic archaeon Thermococcus profundus, were determined.