Purification and characterization of polyphenol oxidase from pepper seed

Enzymes, because of taking the duty of regulation at reactions for living organisms and catalysing with high-level and large-scale selectivity, sometimes cause distruptions by affecting the organisms negatively in undesirable situations. To prevent these, it will be only possible that the enzymes should be characterised by identifying the mechanisms and identifiying their appropriate inhibitors. In recent years, beneficial studies on enzymes have increased in vitro. Novadays, a lot of enzymes known for their structures and mechanisms are used different areas such as medical science, biotechnology and nutrition. Enzymatic browning affects the agriculture negatively not only by deteriozing the yield but also economically. So, the studies on these enzymes which cause browning will provide benefit in that area. The aim of this study is to refine and characterise the PPO enzyme in the seed of green pepper grown in The Southeastern Anatolia. For this purpose, the enzyme insulated from the seed of pepper was partially purified later with ammonium sulfate sedimentation, dialysis, ultrafiltration and gel filtration chromatography. Features of enzyme, including optimum pH and temperature, temperature stability, the effects of inhibitors and carbonhydrates were determined. The results obtained showed that among the tested substrates, 4-methylcatechol had the highest relation with the enzyme which displayed a higher thermal stability, and sodium azide, EDTA, SDS and asorbic acid inhibited the enzyme by 64,4%, 60,10%, 36,6% and 65,7%, respectively. In addition, among the carbonhydrates investigated, ribose has activated the enzyme at %34, glucose and sucrose have inhibited the enzyme. Using 4-methylcatechol as substrate the optimum temperature of PFO enzyme was found as 40'C, when catechol used the optimum temperature was found as 30'C. The optimum pH values were 5.0 and 7.0 in turn by using catechol and 4-methylcatechol substrates, respectively. Besides these, PPO's specificity and the temperature stability were studied. Km and Vmax values for PPO enzyme at optimum pH and temperature for catechol and 4-methylcatechol have been found by the Linewear-Burk Approach. According to Vmax/Km values, the most appropriate substrate was 4-methylcatechol. In addition, it has been observed that when the temperature inactivation time increases the PFO activity decreases dramatically in parallel.