Defense-Related Polyphenol Oxidase from Hevea brasiliensis Cell Suspension: Purification and Characterization

Abstract

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V(max)/K(m) ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH 6.0. The PPO activity was retained at pH 4.0-10.0 and temperature 10-60 °C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and β-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.

Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

“…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.…”

“…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.…”

“…The cell suspension of H. brasiliensis was prepared according to Muhamad et al (2012) and Te-chato et al (2002). First, calli were cultivated from the integument of H. brasiliensis seeds on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 1 mg/mL of 2,4-dichlorophenoxy acetic acid (2,4-D) and 1 mg/mL of 6-benzylaminopurine (BA), pH 5.7 under dark conditions at 25±2°C.…”

“…Protein extraction was performed following some modifications of the procedure of Muhamad et al (2012). Briefly, collected cells were ground with liquid N 2 and extracted in 0.2 M phosphate buffer, pH 6.5, containing 0.25% (v/v) Triton X-100 and 3% (w/v) polyvinylpolypyrrolidone (PVPP).…”

“…Unexpectedly, the purified peroxidase also possessed polyphenol oxidase activity inferring that it is a bifunctional enzyme. Since the properties of polyphenol oxidase was studied in the previous article (Muhamad et al 2012), the purified enzyme was therefore analyzed based on the characteristics of peroxidase. The molecular weight of this peroxidase and some properties such as the effects of temperature, pH, some inhibitors and its activity on the dye decolorization were also reported.…”