## 3 Results and Discussion  
Table 1 shows the sequential purification of PPO isolated from stored material. The procedure started with preparing a crude extract from stored iceberg lettuce, precipitation with (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) (80% salt saturation), followed by anion exchange chromatography and gel filtration chromatography. This procedure gave partially purified iceberg lettuce PPO. Ammonium sulfate fractionation as the first step of iceberg lettuce PPO purification proved convenient and effective in removing large amounts of non-targeted proteins and brown pigments, by reducing the total proteins by almost 54% and 61% and yielding 50% and 48% in fresh and stored samples, respectively. Further purification by loading the dialyzed enzyme on Sephadex G-75 columns (1.6 cm \(\times\) 80 cm) with 0.01 M phosphate buffer (pH 5) containing 0.02% sodium azide eliminated much of the remaining non-targeted proteins. As  
a result, the PPO from material was purified over 12-fold, with a 13.35% activity recovery; in turn, PPO from stored was purified over 14-fold, with a 14.21% yield. Similar results were obtained by [PERSON] et al. (2011), who reported the purification fold for PPO from fennel seeds was 20-fold. Based on the obtained results (Figure 1A), it can be concluded that PPO exhibits activity over a relatively wide pH range (from 4 to 9), with the optimum pH identified in the range of 5-7 (using pyrocatechol as a substrate). The highest activity was observed at pH 5. The enzyme very quickly lost its activity in an alkaline condition. Similar results were obtained by [PERSON] (2010), who reported that the optimum pH for butter lettuce is 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. Moreover, [PERSON] and [PERSON] (2011) showed that PPO activity is the highest in pH 4.5 using catechol as the substrate. In turn, [PERSON], [PERSON], and [PERSON] (2012) have reported that the optimal pH value for blackberry fruits was 6.5 using catechol as the substrate, and 5.5 and 8.0 for raspberry fruits using catechol as the substrate ([PERSON] et al., 2013). Generally, most vegetables and fruits show the highest activity at or near pH 7 in the literature ([PERSON] et al., 2013; [PERSON] et al., 2014), but these values may be different for PPO obtained from various sources of the enzyme and may be dependent on the substrate used in the reaction. In most cases, the pH optima have been reported to be between 4.0 and 8.5; it should be noted that they are dependent not only on the vegetable raw material but also on the purity of the enzyme being tested. Figure 1B shows the influence of temperature on PPO activities in the assay conditions (pH 5 and 0.05 M pyrocatechol as a substrate). The optimum temperature for PPO is 50 \({}^{\circ}\)C. Above this temperature, a rapid loss of enzyme activity was observed. What is surprising is that the enzyme was still active at 80 \({}^{\circ}\)C; however, the activity was relatively low (20% relative activity). The optimal value of the temperature for PPO is dependent on the source of the enzyme and also the kind of substrate used for the reaction. According to the literature data, the optimum temperature for PPO is in the range of 40-60 \({}^{\circ}\)C depending on the raw material. Similar results concerning the same enzyme, but isolated from a different substrate, were presented by [PERSON], [PERSON], [PERSON], and [PERSON] (2016); [PERSON], [PERSON], and [PERSON] (2017); and also [PERSON], [PERSON], [PERSON], and [PERSON] (2018). They studied the optimum temperature for PPO isolated from strawberries (50 \({}^{\circ}\)C), Shona cabbage (60 \({}^{\circ}\)C), and persimmons (55 \({}^{\circ}\)C), respectively.