Purification,Identification And Characterization Of Soluble Peroxidase Isoforms In Litchi Pericarp

Litchi（Litchi chinensis Sonn cv.Wuye）pericarp was utilized as material.The soluble peroxidase（POD）isoforms were purified and the biochemical properties for individual isoperoxidase were characterized,respectively.The in vitro degradation of anthocyanin in litchi pericarp by soluble POD was investigated.The sequences of peptide fragments for individual isoperoxidase digested by trypsin were analyzed by tandem mass spectrometry,respectively.The POD cDNAs encoding the soluble isoforms were obtained by molecular cloning,respectively.Finally,the membrane/wall-bound PODs in litchi pericarp were studied preliminarily.The main results were as follows:Pericarp of mature fruit was homogenized in 0.1 M Tris-HCI buffer（pH8.0）with additional of 30%PVPP（w/w）.After filtration and centrifugation,the POD crude extract was fractioned with 40%saturation of solid ammonium sulfate.Four soluble isoperoxidases named A1,A2,B1 and B2 were purified through column chromatography of Streamline Phenyl,DEAE-52,Phenyl Sepharose and Superdex-200,respectively.The apparent molecular weights of them were 55.1KD,43.3KD,48.9KD and 43.3KD,respectively estimated by SDS-PAGE and gel filtration.A1,A2 showed maximum activity at 35 and 30℃,respectively.B1 and B2 were in the range of 25-50℃.The optimum pH of Alwas 5.6 in citric acid-phosphate buffer,A2 was 6.0 in phosphate buffer;B1 and B2 were all 6.5 in phosphate buffer.The substrate specificity results illustrated that,guaiacol and ABTS were the most suitable substrates;the four isoperoxidases showed different activities for 4-methoxyphenol,chlorogenic acid,gallic acid,catechol,epicatechin,4-methylcatechol,hydroquinone and ascorbic acid and,no activity for pyrogallic acid and benzylalcohol.The metal ions tested exhibited little effect on the activities of the POD isoforms.The activities of the four isoperoxidases were inhibited completely by DTT at a final concentration of lmmol/L,20%-50%of the activities were inhibited by AsA at O.lmmol/L,when the concentration reached lmmol/L,the isoperoxidases were completely inactivated,Cysteine showed a specific inhibition on the activity of Al.The four POD isoforms showed the lowest Km values for（-）-epicatechin among the phenolic substances tested,suggesting that（-）-epicatechin is the most suitable endogenous substrate for soluble POD in litchi pericarp.In addition,the isoperoxidases also exhibited catechol oxidase（PPO）activities.The PPO properties of them were also characterized.The optimum pH and temperature of A1 and B2 were 7.0 and 40°C,respectively as determined with catechol as the substrate and,pH value less than 7.0 or more than 7.5 could cause serious activity loss of them.In contrast to the POD properties,the enzyme isoforms were sensitive to metal ions,when they exhibited PPO activities.The PPO activity of A1 increased dramatically in the presence of Cu²⁺ and Mn²⁺ at lmmol/L.The four purified PODs were digested by trypsin,and the peptide fragments were sequenced by matrix-assisted laser desorption/ionization time-of-flight（MALDI-TOF）MS/MS,respectively.Two of them digested from A1 and A2 shared the same sequences,which were GFDVVDSMK and TLANLNIPAPTDPLQTLQSK;the other two of the same fragments from B1 and B2,were MGNLSPLTGTDGEIR and SVFLSGGPSWSVPLGR,respectively.Mascot search results showed that A1 and A2 matched to the POD coded by the partial cDNA（GenBank accession number:DQ408430）in litchi and,Bland B2 matched to POD2（GenBank accession number:ACI03400）.These results suggested that A1 and A2,B1 and B2 are coded by two different cDNAs,and undergo different post-translational modifications,respectively.The full-length cDNA consisting of 1339 bp for A1/A2 was cloned by means of 3’-RACE and 5’-RACE technique.The cDNA contained an open reading frame of 1113 bp encoding a polypeptide of 370 amino acid residues.Litchi pericarp anthocyanins were extracted in 0.5 M HCl,and then purified by XAD-7 and Sephadex LH-20 column chromatography,respectively.The major anthocyanin in litchi pericarp was confirmed as cyanindin-3-rutinoside by using HPLC with a standard.It showed stability in 1%formic acid solution（pH2.07）but was degraded quickly by A1 and B2 in the presence of 1.0 mM H2O2.In the decoloration process,the optimum pH was 3.0 and,A1 and B2 showed maximum activity at 55℃ and 45℃,respectively.The Km values of A1 and B2 for cyanindin-3-rutinoside were 16.73 mM and 17.44 mM at pH2.07,13.34 mM and 12.4 mM at pH3.0,respectively.The isofroms and the changes of membrane/wall bound peroxidases located in pericarp during the growth and development of litchi fruit were investigated as well in this study.