title: Purification and characterization of membrane-bound polyphenoloxidase (mPPO) from Snake fruit [Salacca zalacca (Gaertn.) Voss].
abstract: Membrane-bound polyphenoloxidase (mPPO) an oxidative enzyme which is responsible for the undesirable browning reaction in Snake fruit (Salacca zalacca (Gaertn.) Voss) was investigated. The enzyme was extracted using a non-ionic detergent (Triton X-114), followed by temperature-induced phase partitioning technique which resulted in two separate layers (detergent-poor phase at the upper layer and detergent-rich phase at the lower layer). The upper detergent-poor phase extract was subsequently fractionated by 40-80% ammonium sulfate and chromatographed on HiTrap Phenyl Sepharose and Superdex 200 HR 10/30. The mPPO was purified to 14.1 folds with a recovery of 12.35%. A single prominent protein band appeared on native-PAGE and SDS-PAGE implying that the mPPO is a monomeric protein with estimated molecular weight of 38kDa. Characterization study showed that mPPO from Snake fruit was optimally active at pH 6.5, temperature 30°C and active towards diphenols as substrates. The K(m) and V(max) values were calculated to be 5.46 mM and 0.98 U/ml/min, respectively, when catechol was used as substrate. Among the chemical inhibitors tested, l-cysteine showed the best inhibitory effect, with an IC(50) of 1.3 ± 0.002 mM followed by ascorbic acid (1.5 ± 0.06 mM), glutathione (1.5 ± 0.07 mM), EDTA (100 ± 0.02 mM) and citric acid (186 ± 0.16 mM).
authors.affiliation: Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Nurul Aqilah Mohd
authors.initials: NA
authors.lastname: Zaini
authors.forename: Azizah
authors.initials: A
authors.lastname: Osman
authors.forename: Azizah Abdul
authors.initials: AA
authors.lastname: Hamid
authors.forename: Afshin
authors.initials: A
authors.lastname: Ebrahimpour
authors.forename: Nazamid
authors.initials: N
authors.lastname: Saari
mesh_terms: D028023:Arecaceae / Q000737:chemistry / Q000201:enzymology*; D004156:Catechol Oxidase / Q000737:chemistry* / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D004795:Enzyme Stability; D005638:Fruit / Q000737:chemistry / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010940:Plant Proteins; D004156:Catechol Oxidase