Date Published: February 12, 2013
Author(s): Thitikorn Chanwun, Nisaporn Muhamad, Nion Chirapongsatonkul, Nunta Churngchow.
Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0–10.0. The enzyme was heat stable over a wide range of temperatures (0–60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by β-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications.
Peroxidases (EC 188.8.131.52) are enzymes that oxidize various hydrogen donors in the presence of H2O2 or organic hydroperoxides. They catalyse many different and important biochemical and physiological reactions in most living organisms. Plant peroxidases are involved in diverse physiological functions such as lignin biosynthesis (Gross 2008), suberization (Bernards et al. 1999), wound healing (Kumar et al. 2007), fruit ripening (Huang et al. 2007), auxin metabolism and disease resistance (Veitch 2004). The peroxidase superfamily can be divided into three classes according to their origin, amino acid homology and metal-binding capability. Class I includes the plant intracellular peroxidases as well as prokaryotic enzymes from mitochondria and chloroplasts (Passardi et al. 2007). Class II comprises extracellular fungal peroxidases: lignin-degrading peroxidase (LiP), and manganese peroxidase (MnP), and includes monomeric glycoproteins involved in the degradation of lignin. Fungal LiP and MnP belonging to this class of peroxidases have been most commonly studied for dye decolorization. Class III consists of secretory plant peroxidases, with multiple tissue specific functions; e.g. removal of H2O2 from chloroplasts and cytosol, oxidation of toxic compounds, biosynthesis of cell walls, defence responses towards wounding, indole-3-acetic acid catabolism, ethylene biosynthesis, etc. Some of the most well known peroxidases in this class are the horseradish peroxidase (HRP), turnip peroxidase (TP), bitter gourd peroxidase (BGP) and soybean peroxidase (SBP). Class III peroxidases are also monomeric glycoproteins containing four conserved disulphide bridges, and require calcium ions for their activities (Schuller et al. 1996). Peroxidases from plant tissues are able to oxidize a wide range of phenolic compounds, such as o-dianisidine, guaiacol, pyrogallol, chlorogenic acid, catechin and catechol (Passardi et al. 2005).
A peroxidase enzyme from H. brasiliensis cell suspension was extracted, purified and characterized by determination of some of its biochemical properties and application in the dye decolorization. The results showed that the peroxidase was relatively easily purified through three steps, anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) then followed by preparative SDS-PAGE. The binding of the enzyme to the Con A-agarose column indicated that the peroxidase was a glycoprotein compatible with the previous studies reporting that most of the plant peroxidases were glycoproteins (Johansson et al. 1992; Kvaratskhelia et al. 1997; Passardi et al. 2005). Our purified peroxidase had a molecular weight of 70 kDa as determined by SDS-PAGE while the molecular weights of various peroxidases have been reported to be in the range of 30–150 kDa (Regalado et al. 2004). A similar molecular weight for the purified peroxidase has been previously reported from Pseudomonas sp. SUK1 (86 kDa), Leptogium saturninum (79 kDa) and Kocuria rosea MTCC 1532 (66 kDa), (Kalyani et al. 2011; Liers et al. 2011; Parshetti et al. 2012). The molecular weight of this obtained peroxidase was equal to those of the polyphenol oxidase previously characterized in our laboratory (Muhamad et al. 2012); however, at that time the peroxidase staining was not attempted. Now, the duplicated gels were stained with substrates of each enzyme and elucidated that it possessed the activities of both enzymes. By the mentioned purification procedure, the obtained protein also exhibited high activity of polyphenol oxidase inferring that it is a bifunctional enzyme. The bifunctional activity (peroxidase-polyphenol oxidase) also appeared in Satsuma mandarin, turnip and Brassica oleracea L. (Fujita et al. 1980a, 1980b ; Rahman et al. 2012). However, the fact that whether our purified protein is a single protein chain exhibiting both activities of peroxidase and polyphenol oxidase is being pursued to elucidate. In this report the enzyme was studied in term of peroxidase since our previous work has reported the biochemical characteristics of polyphenol oxidase enzyme (Muhamad et al. 2012). The optimal pH of the purified enzyme was 5.0 which were close to those reported from Jatropha curcas, Streptomyces sp. and L. leucocephala (Cai et al. 2012; Fodil et al. 2012; Pandey and Dwivedi 2011). For pH stability, its activity was retained at pH values between 5.0 and 10.0, and it had no activity at a pH < 4.0. Previous studies have reported that peroxidases lose their activities at low pH due to the instability of the heme molecule bound to the enzyme (Adams and Gorg 2002). Our purified peroxidase likewise contains heme groups since a peak of its spectrum was present at 302 nm (data not shown) which similar to the spectrum of other purified peroxidases containing heme groups (Gold et al. 1984; Kalyani et al. 2011; Tien and Kent Kirk et al. 1984). Moreover, the enzyme was heat stable over a wide range of temperatures (0–60°C), and about 40% of its activity was lost at 70°C within 30 min. The heat stability of this enzyme was similar to the peroxidases isolated from Jatropha curcas, artichoke and Euphobia cotinifolia (Cai et al. 2012; Cardinali et al. 2011; Kumar et al. 2011). The effect of various compounds on this peroxidase showed β-mercaptoethanol strongly inhibited its activity. This results indicated that at least one disulfide bond within the structure was important for its activity. Schuller et al. (1996) has reported that Class III peroxidases are monomeric glycoproteins; containing four conserved disulphide bridges. Our peroxidase was also the glycoprotein which contains disulphide bonds; therefore our enzyme may belong to class III peroxidase. The NaN3 could strongly inhibit this peroxidase. This chemical substance has been reported to be an inhibitor for all peroxidases (Pandey and Dwivedi 2011) as it can coordinate with the metal ion of a metal enzyme causing toxicity (Schwartz et al. 2001), for example, NaN3 acts as an inhibitor on a peroxidase from Jatropha curcas and Viscum angulatum (Cai et al. 2012; Das et al. 2011). Our purified enzyme was slightly inhibited by EDTA, a chelating agent, like those from Jatropha curcas and Viscum angulatum (Cai et al. 2012; Das et al. 2011). In addition, SDS which is a strong anionic detergent slightly inhibited its activity probably due to a conformational change of the enzyme. The enzymatic decolorization of dyes by this purified enzyme was examined using a UV–vis spectrophotometer. The results showed that the enzymatic activity could decolorize the triphenylmethane dye group. Aniline blue, malachite green, methyl green and water blue were rapidly decolorized (83–97%) within 24 h. This enzyme also decolorized brilliant green, bromocresol purple, crystal violet, fuchsin and methyl violet (49–68%) within 24 h, and the residuals were then cleared within 72 h. Recently, it has been reported that many aromatics dyes can be decolorized by peroxidase through the precipitation or breaking of the aromatic ring structure (Husain 2010). Many previous studies have also reported that bacterial and fungal peroxidases from, for example, Phanerochaete chrysosporium and Bjerkandera adusta could decolorize dyes (Bumpus and Brock 1988; Mohorčič et al. 2006). However using microorganisms to decolorize dyes involves high costs, alternative sources such as plants are now being considered. Our purified peroxidase could react on extreme condition, so it may be used as an alternative enzyme to treat water pollutants. Our study provided a new perspective for the use of this enzyme or a related system in environmental biotechnology. Further studies will focus on the purification and characterization of peroxidase enzymes from other tissues of H. brasiliensis such as leaves and on the identification of the products obtained from the decolorization process by this enzyme. The authors declare that they have no competing interests. Source: http://doi.org/10.1186/2191-0855-3-14