Purification and Characterization of Polyphenoloxidase from Guava Infected with Fruit-rot Disease

Abstract

Polyphenoloxidase (PPO), an important oxidative enzyme which played a vital role in the brewing of guava after infection with fruit-rot disease was purified from disease-affected guava. The method was accomplished by ion exchange chromatography of crude enzyme extract on DEAE-cellulose followed by CM-cellulose. The purified enzyme was found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. Molecular weight of the enzyme was estimated to be 178 kDa and 180.5 kDa by gel filtration and SDS-PAGE respectively. The enzyme appeared to be a single polypeptide chain as revealed by SDS-PAGE either in presence or in absence of reducing agent. Optimum activity of the enzyme was at 48EC and pH around 8. Activity was destroyed completely in presence of EDTA, a metal chellator. Purified PPO showed an estimated k m value of 4.4 mM for catechol as substrate.

Partial purification and characterization of the quality deteriorating enzymes from Indian pink guava (Psidium guajava L.), var. Lalit

“…The molecular weight of guava PPO (* 70 kDa) was similar to wounding-induced PPO from cowpea (Vigna unguiculata) seedlings (73 kDa), however, guava infected with fruit rot disease was found to have a molecular weight of 180 kDa (Razzaque et al 2000;Pinto et al 2008). In nature, various other PPO comparable to guava PPO includes 76 kDa for litchi fruit PPO, 72 kDa for Indian tea leaf PPO, 70 kDa for plantain PPO and 69 kDa for potato PPO (Mdluli 2005).…”

“…However, optimum pH for activity of guava PME and PPO was observed in the alkaline range (8.5 and 8.0, respectively). Optimum pH for guava POD and AO has not been reported in the literature; however, optimum pH for guava PPO infected with the fruit rot disease and guava PME has been reported to be 8 and 8-9, respectively (Razzaque et al 2000;da Silva Cerqueira Leite et al 2006). POD from other fruit sources such as papaya and sweet gourd reported having optimum activity at neutral pH (7.0 and 7.2, respectively) (Koksal et al 2012;Pandey et al 2012).…”

“…Though POD and PPO were active in the temperature range of 5-95 °C, at extreme temperatures their activities were curtailed. PPO purified from guava infected with fruit rot disease was found to have optimum activity at a temperature of 48 °C (Razzaque et al 2000). The optimum temperature for enzyme activity varies with species, cultivar and with the environmental conditions at which fruit has been grown.…”

Pectinase hydrolysis of guava pulp: effect on the physicochemical characteristics of its juice

“…The enzymatic treatment was done at a temperature of 43 ± 2 °C. This temperature is substantially equal to the optimum activity temperature of the guava polyphenol oxidase which is 48 °C ( Razzaque et al., 2000 ).…”

“…To these aerobic conditions coupled with the presence of ascorbic acid could be added the temperature and the reaction time. The enzymatic treatment was done at a temperature of 43 AE 2 C. This temperature is substantially equal to the optimum activity temperature of the guava polyphenol oxidase which is 48 C (Razzaque et al, 2000). Also, during processing, the color value increases for a given enzyme concentration.…”