| Lonicera japomcaThunb. is a kind of important Chinese medicine material, with the effects of antibacterial and anti-inflammatory. At present, severe browning phenomenon easily occurs during storage and processing of Lonicera japonica Thunb. Color is one important judgment standard for its commercial grade provided by the State Administration of traditional Chinese medicine and the health ministry. Therefore, it has the important significance to study the deterioration mechanism of Lonicera japonica Thunb. color and inhibition of degradation phenomena in the storage process. Polyphenol oxidase (PPO, EC.1.14.18.1) a copper oxide reductase, widely exists in microorganisms, plants and animals with complex structure. It plays an important role in the agricultural products browning process. Peroxidase (POD, EC.1.11.1.7) is widespread in a variety of animal, plants and microorganisms, its structure and mechanism of action is not exactly the same kind of heme-containing enzymes, usually containing iron or copper and other metal ions, the enzyme mainly catalyzed by H2O2and oxidized various organic and inorganic compounds.In this study, flower buds of shandong yate fine variety Lonicera japonica Thunb. are raw material, using ammonium sulfate precipitation, DEAE cellulose-52ion exchange column for separation and purification of PPO and POD. The characteristics of two enzymes were separately investigated, including the effects of temperature and pH value on the stability of enzyme activity, substrate specificity, metal ions and inhibitors on enzyme activity and molecular weight of the purified enzyme. This study could enhance the understanding of PPO and POD in Lonicera japonica Thunb. and provide theoretical foundation and scientific basis for the storage and processing. The main results are as follows:1, The purification multiple of LjPPO was30.76purified by ammonium sulfate precipitation and ion exchange chromatography, the recovery rate is33.8%and the molecular weight is about49kDa. The purification factor of LjPOD was28.95, the recovery rate is17.52and the molecular weight is about20.4kDa.2, The optimum temperature of LjPPO was30℃, with general thermal stability. The residual enzyme activity were80%and53%heating for60min at temperature of20℃and30℃, the residual enzyme activity was lower than40%at40℃and50℃heating for60min, the residual enzyme activity was10%heated at60℃for60min, the enzyme activity was almost completely lost at70℃. The optimum pH value of LjPPO is7.5, the enzyme is stable in pH value of7.5-9.5and the residual enzyme activity is greater than90%. The optimum temperature of LjPOD was30℃, with good thermal stability. The residual enzyme activity is more than90%dealing with60min at20℃and30℃and the residual enzyme activity is less than10%heated at60℃for60min. The optimum pH value of LjPOD is5.5, it is stable at pH8.3, LjPPO belongs to bisphenol oxidase, can oxidize the neighbor bisphenols (catechol, L-dopa, chlorogenic acid), but not monophenol (L-tyrosine) and hydroxyquinol (gallic acid, pyrogallol, methyl gallate). LjPOD, with wide substrate characteristics, oxidized phenols and aromatic amines (guaiacol, ferulic acid, catechol, coffee acid, chlorogenic acid, pyrogallol, o-dianisidine, benzidine) in the H2O2conditions.4, The metal ions Na+and K+had no effect on enzyme activity of LjPPO, Mn2+Mg2+, Ca2+and Zn2+activated, Cu2+and Co2+showed inhibitory effect. SDS activated PPO activity, sodium sulfite, citric acid, ascorbic acid, kojic acid and tropolone have different degrees of inhibition on PPO activity. Metal ion Na+had no effect on LjPOD enzyme activity, Ca2+and Zn2+activated, K+, Mn2+and Mg2+showed inhibitory effect, inhibitor (citric acid, ascorbic acid, kojic acid, tropolone, DTT, etc.) and surfactants (SDS, TritonX-100, Tween-80) showed inhibition to LjPOD. |
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