PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF IONICALLY UNBOUND POLYPHENOL OXIDASE FROMMusa paradisiacaLEAF

Abstract

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol(-1). It showed higher specificity with catechol (K(m) = 8 mM) as compared to 4-methylcatechol (K(m) = 10 mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.

Purification and characterization of polyphenol oxidase from purslane

“…The previous studies also raported that optimum pH values were 7.0 for Amasya apple (Dktay et al, 1995), parsley (Lin et al, 2016), Yali pear (Zhou & Feng, 1991), cocoa (Lee et al, 1991), artichoke (Dogan et al, 2005), Jackfruit (Tao et al, 2013), pH 7.2 for Barbados cherry (Kumar et al, 2008), pH 5.5 for ispir sugar bean (Sakiroglu et al, 2013) and pH 5.4 for Ataulfo mango (Cheema & Sommerhalter, 2015), using catechol as a substrate. Diwakar (2015) also found a similar result with Cinnamomum tamala obtaining the optimum pH and temperature values of 7.0 and 50 °C, respectively. The optimum temperature for cucumber was also found as 50 °C (Miller et al, 1990).…”

“…However, this loss was accompanied by a rise in specific enzyme activity. Moreover, the overall degree of PPD purification was reasonably good compared with that from pear (13.3-fold; Ziyan & Pekyardimci, 2004), Chinese parsley (6.5-fold; Lin et al, 2016) and Cinnamomum tamala (2.63-fold; Diwakar, 2015). The recovery percentage for purslane PPD obtained in the present study was also better than those in most studies reported previously.…”

Assessment of fruit flesh browning in ‘Amasya’ apple cultivar and its two selected sibs

“…The previous studies reported that the leading causes of enzymatic browning are phenolic compounds, PPO, temperature , and pH (MARTINEZ and WHITAKER, 1995;SINGH et al, 2018). Optimal activity of browning enzymes is determined at pH 5.0-7.0 in fruit species and vegetables (OKTAY et al, 1995;TAO et al, 2013;DIWAKAR, 2015;GÜL GÜVEN et al, 2017), and it is prevented when the presence of acids (SINGH et al, 2018). In this study, the genotype '109' with the lowest pH value was already indicated the lowest flesh browning incidence.…”

Biochemical characterization and thermal inactivation of polyphenol oxidase from elephant foot yam (Amorphophallus paeoniifolius)

“…The native gel was kept on dry catechol paper along with the glassplate and dark brown activity bands appeared after 5 min of contact Molecular weight of the enzyme can also be estimated very accurately by this blot zymography studies. Enzymes isolated from various plant sources suggested that the PPO enzyme can exist as monomer as reported in Musa paradisiaca Leaf and Lactarius piperatus PPO 40 kDa (Diwakar and Mishra 2011;Oz et al 2013), Butter lettuce 60 kDa (Gawlik-Dziki et al 2008) and Wheat bran 37 kDa (Yoshiki et al 2008).…”