Purification and Biochemical Characterization of Polyphenol Oxidase from Alanya Banana (Musa carevendishi)

Abstract

Polyphenol oxidase was isolated from Alanya banana (Musa
carevendishi) fruit grown in Southern Turkey with ammonium sulfate
precipitation and dialysis method. One protein fraction was obtained
with gel filtration chromatography. Optimum temperature for banana
polyphenol oxidase was 25 °C with catechol substrate. Polyphenol
oxidase showed maximum activity at pH 7.0 with catechol, caffeic acid,
pyrogallol, 7.2 with 4-methylcatechol, 7.4 with L-tyrosine, 7.8 with
p-cresol, 8.0 with gallic acid. K_m and V_max values in the case of 20 mM
catechol as substrate were 15.95 mM and 1776 ΔA min^-1, respectively.
Six different inhibitor were tested in this study and the most effective
inhibitors for banana polyphenol oxidase were found to be L-cysteine,
β-mercaptoethanol and sodium diethyldithiocarbamate. Inhibition
constants (K_i) were calculated for every inhibitor at optimum pH of polyphenol
oxidase activity. Polyphenol oxidase isoforms were determined
by using the partially purified banana polyphenol oxidase. Polyphenol
oxidase isoforms migrated as 4 active bands with catechol substrate,
4 bands with Coomassie Brillant R-250 during native polyacrylamide
gel electrophoresis (N-PAGE). On the SDS-PAGE, banana polyphenol
oxidase produced 5 bands of ca. 105 kDa, 52 kDa, 26 kDa, 18 kDa and
15 kDa molecular weights. The activation energy, inactivation rate constant
and half-lives (t_1/2) of polyphenol oxidase were also calculated.