Cloning,Sequence Analysis and Three-dimensional Structure Prediction of DNA Pol I from Thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. <Emphasis Type="Italic">MKK</Emphasis> Isolated from an Iranian Hot Spring

Molecular phylogenetic analysis of a novel thermophilic eubacterium isolated from an Iranian hot spring using 16S rDNA sequence showed that the new isolate belongs to genera Geobacillus. DNA pol I gene from this isolate was amplified, cloned, sequenced, and the three-dimensional (3D) structure of deduced amino acid sequence was predicted. Sequence analysis revealed the gene is 2,631 bp long, encodes a protein of 876 amino acids with a calculated molecular mass of 99 kDa, and belongs to family A DNA polymerases. Comparison of 3′–5′exonuclease domain of Klenow fragment (KF) with corresponding region of newly identified DNA pol I (MF), the large fragment of Bacillus stearothermophilus DNA pol I (BF) and Klentaq1, revealed not only deletions in three regions compared to KF, but that three of the four critical metal-binding residues in KF (Asp355, Glu357, Asp424, and Asp501) are altered in MF as well. Predicted 3D structure and sequence alignments between MF and BF showed that all critical residues in the polymerase active site are conserved.     

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors, and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H₂O₂ and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t _1/2 from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H₂O₂ (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state k _cat and K _m values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.     

Modified nanoalumina sorbent for sensitive trace cobalt determination in environmental and food samples by flame atomic absorption spectrometry

1-(2-pryidylazo)-2-naphthol (PAN) immobilized on sodium dodecyl sulfate-coated nano alumina was developed for the preconcentration and determination of metal cations Co (II) from environmental and food samples. The research results displayed that adsorbent has the highest adsorption capacity for Co (II) in this system. Desorption by elution of the adsorbent with 2.0?ml of a mixture of nitric acid and ethanol was carried out. After phase separation, the enriched analyte in the final solution is determined by flame atomic absorption spectrometry (FAAS) by using a micro sample introduction system. Analytical influencing parameters including pH value, amount of sorbent, equilibrium time, sample volume, volume and concentration of eluent were examined. The effect of common matrix ions has also been investigated and it was found that they had no influence on cobalt preconcentration. Under the optimum experimental conditions, the maximum capacity of sorbent was obtained as 20?mg?g^?1. The preconcentration factor and limit of detection were found to be 250 and 0.15?µg?L^?1, respectively. This method showed good precision with the relative standard deviation (RSD) of 2.4% and 2.1% in concentrations of 20 and 50?µg?L^?1, respectively. The accuracy of the method was evaluated by comparison of results with those obtained by electrothermal atomic absorption spectrometry. This method was successfully applied for preconcentration and determination of Co (II) in environmental and food samples.     

Monophosphoramide derivatives: synthesis and crystal structure,theoretical and experimental studies of their biological effects

Molecular Diversity - Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. In this investigation, we synthesized and...     

Remarkable Improvement of Methylglyoxal Synthase Thermostability by His–His Interaction

Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine–histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HH^O), and another two histidines (TMGS-HHH^O) were inserted between Arg22 and His23 (H^O). In comparison with the wild type, TMGS-HH^O thermostability increased remarkably, whereas TMGS-HHH^O was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHH^O was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHH^O. These results demonstrated that histidine–histidine interactions at position 23 in TMGS-HH^O probably have the main role in TMGS thermostability.