Purification,Identification,Homologous Modeling And Enzymatic Browning Of PPO From Lotus Seed

In this thesis,the structure of polyphenol oxidase,the category and content of phenolic compounds as well as the interaction process between PPO and substrate were intensely studied in a bid to tackle the problem of lotus browning which led to limits and difficulties on enriching lotus products,transportation and resources utilization.The high purified lotus PPO,obtained through optimized separation and purification method,were identified by mass spectrometry and compared with spectrometry database,which unveiled the structural information of it.The phenolic compound in lotus seed were extracted by ultrasonic with ethanol and analysis of qualitative and quantitative were performed by HPLC and Q-TOF LC/MS.There were specific phenolic substrates identified from the disparity of color and reaction speed when the lotus PPO reacts with several known phenolic acids.With the homogenous modeling,the three-dimensional structure of lotus PPO was constructed.The molecular docking software had simulated the interaction between PPO and specific substrate.The main research results are as follows:1.The comparation between four methods of extracting lotus seed PPO was made,homogenization post-acetone extraction method was better in PPO speciffic acivity(289.04±6.25U/mg)and maintaining the total acivity.The results of optimzing homogenization post-acetone extraction as follows: the ratio of solid-to-solvent 1:5,extract time 2 h,phosphate buffer containing 0.8 % TritonX-100 and 2 % PVPP at pH=7.The rude extraction was purified by DEAE Sepharose Fast Flow ion exchange column chromatography and Sephadex G-75 gel filtration chromatography.The specific activity of purified PPO was 7926.68 U/mg,a purification factor of 26.92,yeild was 38.48 %.The moecular weights of this enzyme was about 58 kDa.2.It was the first time to evaluate 14 kinds of phenolic compounds in lotus through HPLC and Q-TOF LC/MS:Chlorogenic acid,Caffeic acid,p-coumaric acid,Ferulaic acid,Hyperin,Quercetin-3-rutinoside,Quercetin,Kaempferol,Apigenin,Gallic acid,Catechin,L-Epicatechin,Sinapic acid glucoside,Apigein-8-C-xyloside-6-C-glucoside.We found the Hyperin has the highest proportion which was 13.71±0.23 mg/100 g and the rank of content from high to low is Chlorogenic acid,p-coumaric acid,Ferulaic acid,Quercetin,Sinapic acid glucoside,Catechin,Caffeic acid,Apigenin,Apigein-8-C-xyloside-6-C-glucoside,Kaempferol,L-Epicatechin,Gallic acid.3.With the comparison of lotus in 2014 and 2015,the different variation of content of total phenol was discovered.The content of total phenol in 2015's lotus was increased from 284.61 mg/100 g to 362.78 mg/100 g in 12 days.On the contrary,the situation in 2014's lotus was reversed.The peak was in the zero day which is 501.08 mg/100 g and declined to 419.30 mg/100 g within 5 days.4.The purified PPO of lotus seed was performed by tapping,enzymolysis,chromatography,mass spectrometry analysis to get the results which searched the swissprot protein database and Nelumbo nucifera Gaertn database.Finally,PPO in lotus seed had a compatible Polyphenol oxidase-Nelumbo nucifera that the serial number is E5L9E4,matching degree score reaches 203 points,the sequence similarity is 300,the molecular weight is 56.8kDa,amino acid sequence and Cu active center sequence has also been learned.5.The seven identified phenolic acids had a test with lotus seed PPO respectively.It was found that Catechin ?max of 425 nm,the measured enzyme activity was 1126.00±20.22 U/mL,and L-Epicatechin ?max of 430 nm,the enzyme activity was 1435.33±65.68 U/mL;Gallic Acid ?max of 385 nm,the enzyme activity was 1247.67±23.16 U/mL,and there is no characteristic absorption peak in the visible light wavelength for chlorogenic acid.So the enzyme activity was only 522.00±9.00 U/mL for chlorogenic acid at 420 nm.Meanwhile,the browning products in these four kinds of phenolic acid substrate were not the same.When it came to catechin,the measured enzyme activity was the highest among all the candidates.As to color,oxidation and browning products of gallic acid is the most likely to browning color.So the L-Epicatechin and gallic acid both the important substrates.6.The three-dimensional of PPO from lotus seed has sixteen ? helixs,five ? folds and some random coils,which using the 2P3X_A and 1BT3_A protein as template by homology method.To find the active site of the PPO by predicting active site software.then,the interaction process between L-epicatechin Gallic acid with PPO was simulated docking by the CDOCKER of Discovery Studio 2.1.L-epicatechin combinated the PPO through Hydrogen-bond,hydrophobic,Van der Waals interactions and ?-? conjugation.Gallic acid combinated the PPO through ydrogen-bond,hydrophobic and Van der Waals interactions.Those interactions would changing the confromation of PPO in order to make the cavity was suitable for the combination with phenolic substrates.When L-epicatechin compared with galiic acid,the former has ?-? conjugation effect and more hydrogen bonding interactions between amino acids,which leads the docking system to more stable.So it is flexible to combine and more suitable as a phenolic substrate of PPO from lotus seedr.