Purification And Characters Analysis Of Membrane-Bound Polyphenol Oxidase From Apples(Malus Domestica)

Membrane-bound polyphenoloxidase (mPPO) accounted for about85%of the total PPO and can be stimulated under certain conditions. However, few studies have focused on mPPO in Fuji apple. mPPO has been purified from Fuji apple (Malus domestica Borkh. cv. Red Fuji). The enzymology properties and structural were determined. Mass identification and bioinformation prediction of the mPPO were also carried out. The main results were as follows:(1) This study compared the properties of mPPO from three different kinds of apple (Fuji, Golden Delicious, Granny Smith). The properties of mPPO from them were significantly differentThe mPPO of red Fuji had the highest enzyme activity at pH8.00. The optimize reaction pH of mPPO from Golden Delicious was4.50, while the activity of mPPO from Granny Smith was highest between pH7.50-8.00. The optimal temperature for Fuji mPPO was55℃and the activity of mPPO from Golden Delicious was highest between35and55℃with no significantly difference. The optimal temperature for Granny Smith mPPO was within70～75℃. In high temperature, mPPO from Golden Delicious was more stable than that from red Fuji and Granny Smith. When using catechol as substrate, mPPO from Granny Smith was strongest affinity to the substrate, but the Vmax of mPPO from Fuji was the largest. The crude extract of mPPO from Golden Delicious and red Fuji had similar enzymic bands.(2) The best conditions for isolation and purification of mPPO in Fuji apple were:ultrasonic extraction for10min,50%～80%saturation of ammonium sulfate precipitation, gradient elution with0.1M NaCl. The mPPO was purified64.30folds with a high specific activity (387032.97U.mg-1). The Native-PAGE and SDS-PAGE are single bands for mPPO and the purified mPPO is a dimmer of a subunit with a molecular weight of67.3kDa. The results of mass spectrometry were analyzed and compared in the protein database showed that the purified protein was a polyphenol oxidase.(3) We compared membrane-bound with soluble polyphenol oxidase (mPPO and sPPO, respectively) from Fuji apple. The specific activity of mPPO was34.12higher than that of sPPO. mPPO was more stable than sPPO at pH5.00～8.50. Although mPPO was more easily inactivated at25-55℃, it is still more active than sPPO in this temperature range. The optimum substrate of mPPO was4-methyl catechol, followed by catechol. L-cysteine had the highest inhibitory effects on mPPO followed by ascorbic acid and glutathione. Surprisingly, EDTA increased mPPO activity.(4) The calculation of sub-cellular localization indicated that mPPO from Fuji was a hydrophobic membrane protein in chloroplast with a39amino acid residues signal peptide and two extracellular membrane peptides (82-97for N-,548-567for C-). The three-dimensional model and binding site of mPPO to4-methyl catechol were studied using molecular docking. The three-dimensional structure of mPPO consisted of sixa-helices, two shortb-strands, and ten random coils. The putative substrate-binding pocket contained six polar or charged amino acids, His191, His221, Trp224, Trp228, Phe227, and Val190. Trp224and Trp228formed hydrogen bonds with4-methyl-catechol.(5) The effect of different inhibitors on mPPO from Fuji was studied. The inhibition effect, type and mechanism were different when using different substrates, such as catechol and4-methyl catechol. Glutathione expressed as a effective competitive inhibition on mPPO when using4-methyl catechol as a substrate. When using catechol and4-methyl catechol as substrates, L-cysteine, glutathione, ascorbic acid all made a lag time on the activity of mPPO, but with different lag trend. The fluorescence emission wavelength of mPPO from Fuji is303.84nm which expected that the Trp residues within the protein were buried inside mPPO. Inhibitors used in our study were all expressed a quenching on fluorescence emission of mPPO with different degree. The effect of ascorbic acid on fluorescence quenching of mPPO was the most significant. The synchronous fluorescence emission peak (AX.=15nm) of mPPO was at280nm, while there were two fluorescence emission peak (230nm for the main peak and270nm for secondary peak) at Δλ=60nm. EDTA gave the strongest quenching effect on main peaks and ascorbic acid on secondary peak.