Recovery of Active Polyphenol Oxidase and Peroxidase from Plant Tissues with High Phenolics and Chlorophylls

The present protocol described extraction of active polyphenol oxidase and peroxidase from a plant rich in phenolics and chlorophylls in the post-harvest browning syndrome of B. myrtifolia. Initially, general optimisation using conventional enzyme extractions was performed. However, along with membrane-bound proteins, chlorophylls and phenols were also released with Triton X (TTX). With a view to obtaining high enzymatic activity, removal of the released chlorophylls and phenols by formation of TTX-114 micelles in the detergent rich phase after hightemperature induced phase separation was tested.


Experimental Design
This protocol was conducted towards characterising PPO and POD in the post-harvest browning syndrome of Backhousia myrtifolia, an Australian native ornamental plant cultivated for cut flower production. Leaf and floral tissues of this particular species are known for high phenolic and chlorophyll contents which is the interferences for enzymatic analyses.
Initially, general optimisation using conventional enzyme extractions was performed. However, along with membrane-bound proteins, chlorophylls and phenols were also released with TTX. With a view to obtaining high enzymatic activity, removal of the released chlorophylls and phenols by formation of TTX-114 micelles in the detergent rich phase after high-temperature induced phase separation was tested. 2. The solution was kept at 4°C for 15 min and then transferred to a water bath at 30°C for 10 min. The solution became cloudy due to the formation of large mixed micelles of detergent, hydrophobic proteins, and chlorophylls [15,16].

Materials
3. This solution was centrifuged at 5,000 × g for 15 min at room temperature. The clear supernatant was used to measure both PPO and POD enzyme activity.

Protein extraction for electrophoresis studies
To improve the yield of protein with a view to maximising enzymatic activity of the extracts, the extracts were filtered through an Amicon ® ultra unit. Ten millilitres of the extracts was pipetted into a filter unit chamber, capped and then centrifuged at 4°C and 5,000 × g for 10 min. The retentate (~ 5 mL) remaining in the filter chamber and the eluate (~ 5 mL) in the receiving chamber were tested for enzymatic activity and protein content. Protein content was also determined spectrophotometrically using the Bio-Rad protein assay kit as a standard [17]. Enzymatic activity assays were carried as described herein. 2. After mixing, the cuvette was immediately transferred into the spectrophotometer and absorbance at 410 nm was recorded every 30 s for 3 min at room temperature.
3. An increase in absorbance indicated the formation of brown pigment (o-quinone). PPO activity was calculated as the slope of absorbance against time [19]. 5. The increase in absorbance was recorded at 470 nm at room temperature for 3 min. 6. The reaction assay with 200 µL deionised water instead of H2O2 was also analysed. 7. POD activity was calculated by subtracting the activity without H2O2 (PPO activity) from the activity with H2O2 [21]. 8. One unit of activity was defined as the amount of enzyme that caused a change in absorbance of 0.001 units per min.
3. The gels were run at 150 V constantly for 115 min at room temperature. 4. The native polyacrylamide gels were stained for PPO activity by immersion in 0.1 M catechol solution containing 1.5 g CaCl2, 0.2 g EDTA, 0.15 g boric acid and 2.0 g Tris for 5. The same gels were then incubated in 100 mM H2O2 for a further 15 min for POD activity [22][23].

Expected Results
PPO and POD from B. myrtifolia leaf and floral tissues were recoveries during the extraction with different phenol removing agents in sodium phosphate buffer (pH 6.8). PPO was activated by the addition of detergents (e.g. TritonX-114) and POD was extracted with mild treatments (e.g. sucrose solution). For partial enzyme purification, high temperature induced phase separation was used. The inclusion of 5% polyvinylpolypyrrolidone (PVPP) in the extraction buffer was effective in removing polyphenols (Table 1). As presented in Sommano [9] a 0.1 M sodium phosphate buffer pH 6.8.
Latent PPO in crude extract from B. myrtifolia leaves was activated using 2% v / v TTX-114, resulting in an almost 30-fold increase in activity. The same optimised extraction protocol also improved POD activity (Table 2). 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 (Table 3). As presented in Sommano [9] n/d = not detected.
Values are means (n = 3) ± SE. Upon staining in 0.1 M catechol solution for PPO activity, a single activity band was found in each lane for crude leaf, crude freeze-dried leaf and crude flower extracts (Figure 1. a). However, no activity was present for the freeze-dried extract of floral tissue. No POD activity band was detected in the same gel post-stained in a 100 mM H2O2 solution (Figure 1. b). Phenols and chlorophylls were evident in the lanes as greenish-brown pigments. Enzyme activities of crude and concentrated protein extracts (AFU retentate) were also compared on Native PAGE (Figure 2.). No POD activity band was detected in any extracts, even with concentration through the AFU (data not shown).

5.Conclusion
In studying PPO and POD in plant phenolic rich like B. myrtifolia tissues, a combination of PVPP and high temperature-induced phase separation effectively improved enzymatic activity, including activity bands on native electrophoresis.