Purification And Characterization Of Amranthus Tricolor Hydroperoxide Lyase

In higher plant, hydroperoxide lyase (HPL) catalyzed the cleavage of hydroperoxide, which converted from linoleic or linolenc acid by LOX, to give C6 volatile aldehyes together withω–oxoacids. These volatile aldehydes are important contributors to the distinctive scent of fresh fruits and vegetables, which are so-called''green notes''. C6 aldehydes is an important flavor additive in food industry and daily industry. This thesis began with the screen and stabilization of HPL, then the investigation were carried out by four steps: emzyme purification and characterization, critical amino acid in active site, enzymatic mechanism, the kinetics and stability of HPL in presence of high salts concentration.We began our work with a survey of 13-HPL activity in various commercial vegetables, and found amaranthus tricolor leaves were a particularly rich source for 13-HPL. When 13-hydroperoxy-linolenic-acid and 13-hydroperoxy-linoleic-acid were used as substrates, the activities of HPL based on fresh weight were 1.92±0.12 U g?1 and 0.92±0.08 U g?1, respectively. The stability of HPL extract was enhanced in the presence of glycerol and DTT, with approximately 80% activity retention after 20 days of storage at ?20°C. By the speraction of detergent and addition of 100 g L?1 sucrose and before lyophilisation, microsomal HPL activity remained almost unchanged. Moreover, lyophilised HPL with sucrose retained nearly 100% of residue activity after 40 days storage at ?20°C.The lyophilised HPL was purified to electrophoretic homogeneity by DEAE-Toyopearl ion-exchange chromatography, phenyl Sepharose HP column hydrophobic interactionn chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS–PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25°C, respectively.The HPL showed higher activity against 13-HPOT. Km value for 13-HPOT was 62.7μmol/L, and the corresponding Vmax was 178.5μmol·min-1·mg-1. The chemical modification results revealed that serine, lysine, cysteine was important for HPL activity. Moreover, the activity of HPL was significantly inhibited by 2(E)-hexenal but not by EDTA, hexanal, N-acetylimidazole, and 1-ethyl-3-(3-dimethylaminopropyl carbodiimide.On the basis of inhibition kinetics model, PCMB is an competitive inhibitor. From the results we concluded that thiol located in the active site of HPL and played an important part in the combined with substrate. Meanwhile, circular dichroism and fluorescence spectra reflected the conformational changes of HPL.The PCMB modified HPL resulted in the decrease ofα-helix structure and surface hydrophobicity. The ANS-binding spectra indicated that surface hydrophobicity decreased during HPL reaction. HPL was irreversibly inactivated by 13-HPOD, the inhibition consistented with pseudo-first model, the inactivation rate constant was 16.133M-1s-1. The antioxidant (DTT, cystein) and the free radical scavengers (BHT, BHA) had protection effect of HPL inactivation, which indicated that radical may produce in HPL reaction. The EPR experiment confirmed that alkyl and alkoxyl radical was generated in reaction mixture with intensive signals. The addition of DTT and BHT had radical scavenge effect.Inorganic ion had a significant activation and stabilization effect on HPL. Generally speaking, the effect was kosmotropic anion dependent. The influence of salt on the activity and thermal stability of HPL followed hofmeister series. The hydrophobic interaction, electric interaction and specific ion force was the major contribution to the effect. The mechnism involved was: (1) ion may affect the water molecules layer then interact with enzyme surface and internal structure to improve the enzymatic activity and stability, (2) in presence of 1.0 mol/L PBS, the enzyme formed a natural rigid state with a hydrophobic surface structure by hyrophobic interation, thus increased HPL stability.