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Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X‐ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three‐dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik‐Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half‐life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild‐type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase. [ABSTRACT FROM AUTHOR]

Azo dyes are being extensively used in textile industries, so finding a proper solution to decolorize them is of high importance. In order to find azo dye decolorizing strains among haloarchaea, which are well known for their tolerance to harsh environmental conditions, fifteen haloarchaeal strains were screened. Halogeometricum sp. strain A and Haloferax sp. strain B with the highest decolorization ability (95% and 91% for Remazol black B; both about 60% for Acid blue 161, respectively) were selected for further studies. It was shown that both strains were able to grow and decolorize the dye in a medium containing up to 5 M NaCl, with optimum decolorization activity at 2.5-3.4 M, pH 7 and a wide temperature range between 30 to 45 °C. Moreover, both strains were able to tolerate and decolorize up to 1,000 mg l-1 Remazol black B. Also, they were able to survive in 5,000 mg l-1 of the dye after 20 days'incubation. Glucose and yeast extract were found to be the best carbon and nitrogen sources in the decolorization medium for both strains. This is the first report studying decolorization of azo dyes using halophilic archaea. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses. [ABSTRACT FROM AUTHOR]

Abstract: Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H2O2 exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris–HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35min at 80°C) and T m (from 73 to 77.5°C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H2O2 stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H2O2 tolerance in concentrated buffer. [Copyright &y& Elsevier]