Tailor-made biocatalysts based on scarcely studied acidic horseradish peroxidase for biodegradation of reactive dyes.

Peroxidases (EC 1.11.1.7) have enormous biotechnological applications. Usage of more abundant, basic isoforms of peroxidases in diagnostic kits and/or in immunochemistry has led to under exploitation and disregard of horseradish peroxidase (HRP) acidic isoforms. Therefore, acidic horseradish peroxidase (HRP-A) isoenzyme was used for the preparation of a biocatalyst with improved ability in dye decolorization. Ten biocatalysts were prepared by covalent binding of enzyme to chitosan and alginate, adsorption followed by cross-linking on inorganic support (aluminum oxide), and encapsulation in spherical calcium alginate beads via polyethylene glycol. Model dyes of 50 to 175 mg l-1 were removed by the biocatalysts. Among the tested biocatalysts, the three with the highest specific activity and biodegradation rate were further studied (Chitosan-HRP, Al-Gel-HRP and Al-HRP-Gel). The impact of hydrogen peroxide concentration on dye decolorization was examined on the Chitosan-HRP biocatalyst, since the HRP is susceptible to inhibition/inactivation by high H2O2. On the other hand, H2O2 is needed as a co-substrate for the HRP, and the H2O2/dye ratio can greatly influence decolorization efficiency. Concentrations of H2O2 ranging from 0.22 to 4.4 mM showed no difference in terms of impact on the biocatalyst decolorization efficiency. The high decolorization efficiency of the biocatalysts was validated by the removal of 25 and 100 mg l-1 anthraquinone (Remazol Brilliant Blue R (RBBR)), triphenylmethane (Coomassie Brilliant Blue (CBB)), acridine (Acridine Orange (AO)), and formazan metal complex dye (Reactive Blue 52 (RB52)). After the seven consecutive decolorization cycles, the decolorization was still 53, 78, and 67% of the initial dye for the Al-HRP-Gel, Al-Gel-HRP, and Chitosan-HRP immobilizate, respectively. The results obtained showed potential of otherwise neglected acidic HRP isoforms as a cost-effective biocatalyst with significant potential in wastewater dyestuff treatment.

Acidic horseradish peroxidase activity abolishes genotoxicity of common dyes.

The aim of this study was to investigate the impact of dyes on DNA before and after enzymatic decolorization by acidic horseradish peroxidase (HRP-A). The comet assay is easy and feasible method widely used to measure DNA damage and repair. The medium-throughput comet assay was employed for assessment of genotoxic effects of 8 dyes in BEAS-2B cells. We have incorporated a digestion with bacterial endonuclease (formamidopyrimidine DNA glycosylase, FPG) to detect oxidized bases in the case of single and double azo dyes, Orange II (OR2) and Amido Black 10B (AB), respectively. This allowed detection 8-oxo-7,8-dihydroguanine, one of most abundant oxidized bases in nuclear DNA. In the case of AB there was no indication of DNA damage, either strand brakes or FPG-sensitive sites before and after decolorization. The OR2 induced DNA damage (in terms of percentage of DNA in comet tails). Also, the frequency of FPG-sensitive sites increased with OR2 concentration. After decolorization no DNA damaging effects was seen at all. The interaction studies of OR2 and AB, before and after decolorization, with calf thymus DNA has been investigated by absorption and fluorescence spectroscopy. The results provide support for the idea that in some cases enzymatic decolorization contributes to lower genotoxicity potential.