EXTRACTION OF ACTIVE POLYPHENOL OXIDASE AND PEROXIDASE FROM A PLANT RICH IN PHENOLICS: BACKHOUSIA MYRTIFOLIA
Cut Backhousia myrtifolia, an Australian native ornament suffers from a severe postharvest browning syndrome. Leaf and floral tissues of this plant contain high amounts of phenolics. This together with the latent stage of the oxidative enzymes polyphenol oxidase (PPO) and peroxidase (POD) makes the study of enzymatic properties difficult. Therefore, extraction and/or purification of these enzymes are important to overcome this problem. Samples were extracted with different phenol removing agents in sodium phosphate buffer (pH 6.8). PPO was activated by the addition of detergents (e.g., TritonX-100) and POD was extracted with mild treatments (e.g., sucrose solution). For partial enzyme purification, high temperature induced phase separation was used. Results showed that the inclusion of 5% polyvinylpolypyrrolidone (PVPP) in the extraction buffer was effective in removing polyphenols. Latent PPO in crude extract from B. myrtifolia leaves was activated using 2% v/v TTX-100, resulting in an almost 30-fold increase in activity. The same optimised extraction protocol also improved POD activity. While the phase separation step improved enzyme activity, it failed to maintain total protein content. Fresh extract of B. myrtifolia leaf tissue was, therefore, initially concentrated by filtering with an Amicon® PL-10 unit. However, concentrated endogenous phenolics interfered with enzyme activity. The results indicated that in studying PPO and POD in phenolic rich B. myrtifolia tissues, PVPP and high temperature-induced phase separation effectively improved enzymatic activity.
Sommano, S. (2015). EXTRACTION OF ACTIVE POLYPHENOL OXIDASE AND PEROXIDASE FROM A PLANT RICH IN PHENOLICS: BACKHOUSIA MYRTIFOLIA. Acta Hortic. 1088, 547-551
Australian native flower, latent PPO, peripheral membrane POD, total phenol