Isolation,Purification,Biochemical And Structural Properties Of Polyphenol Oxidase From Lily Bulb(Lilium Lancifolium Thunb)

Lily bulb is one of the first medicinal and edible plants approved by the Ministry of Health of China.It is rich in nutrients and has many biological active substances in addition to basic nutrients.However,lily bulb is prone to browning during processing and storage,which is mainly caused by polyphenol oxidase（PPO）.In view of enzymatic browning,the properties of PPO in lily bulb were studied.The main results were as follows:1.Biochemical properties of three kinds of lily bulb polyphenol oxidases were studied.Among the three lily varieties,Lilium lancifolium Thunb（JD）PPO was the most prone to browning,followed by Lilium davidii var.unicolor cotton（LZ）and Lilium brownie var.viridulum（LY）.Their optimal p H values are 4.0 and 6.5～7.0,and the optimal temperature is 15℃.In terms of thermal inactivation,LZ PPO showed good resistance to thermal inactivation above 75℃,while JD PPO showed the best resistance at 45℃.The thermal inactivation of PPO in the three lily species all followed the first-order kinetic model,and the E_a values of JD,LY and LZ were144.28,138.00 and 107.12 k J mol^-1,respectively.In terms of substrate specificity,all the three lily PPO species showed the best affinity for 4-methylcatechins,while all the three lily PPO species showed no monopholase activity.In terms of inhibitors,thiourea and L-cysteine had the highest inhibitory effects on PPO of the three lily bulb.2.The separation and purification method of polyphenol oxidase from Lilium lancifolium Thunb was studied.The optimal crude extraction conditions were as follows:p H 6.5 phosphate buffer,solid-liquid ratio of 1:3,extraction time of 4 h,PVPP content of 2%.After 30～50%ammonium sulfate precipitation,DEAE-Sepharose Fast Flow and Sephadex G-100 chromatography-chromatography,the purified product was 14.64 times with a specific enzyme activity of 153900 U/mg.The purified protein was identified by SDS-PAGE and Natvie-PAGE electrophoresis to obtain a single band with molecular weight of about 63 k U and 135 k U,which was considered to be a dimer protein containing the same subunit.The results of mass spectrometry showed that the peptide similarity of PPO3 was 23.8%with the sequence number ANG57909.1（A0A173DSG7）in the universal protein database of Lilium genus,which provided the credibility for the purified protein to be a PPO family protein.3.The structural properties of polyphenol oxidase in lily bulb was analyzed.Based on the amino acid sequence obtained by mass spectrometry analysis,the following results were obtained from the perspective of bioinformatics:PPO（ANG57909.1）was composed of 563 amino acids with a theoretical molecular weight of 62.9 k U and an isoelectric point of 6.95,close to neutral p H.The PPO is a plastid protein.The secondary structure prediction showed that 58.79%of the amino acids were randomly curled,20.78%wereα-helical and 17.41%wereβ-folded.Using apple PPO（PDB numbered 6ELS＿A）as the template,the three-dimensional structure model of PPO was obtained by homology modeling.The Cu atoms in the active center of PPO were linked to His 151,His 172,His 181（Cu A）and His 307,His 311,His 341（Cu B）,respectively.Molecular docking results showed that 4-methylcatechol was more likely to bind to PPO than catechol,and residues Phe 337 and Tyr 312 were the catalytic cavities of the two,respectively.