Studies On The Characters Of PAL And Its Correlation Between Flavones And DMY On Contents Diversification In Tengcha

PAL catalyze L-phenylanine to other hydroxybenzene substance,it offers front body for kinds of hydroxybenzene substance and the end product of Flavones.Under the optimized conditions for the PAL of Tengcha(Ampelopsis grossedentata W.T.Wang) extracting and activity analysis,we compared the correction of Juvenile leaves,mature leaves and Juvenile stem during different stages of PAL between Flavones and DMY on contents diversification.Furthermore,PAL in Tengcha leaves was purified and its characterization was studied.This will establish theoretical foundation for further research on the mechanism of flavones and the content of DMY in Tengcha in the future.1 Optimization of conditions for extracting and activity analysis of PAL in Tengeha leavesUsing L-phenylanine as substrate,the conditions for extracting and activity analysis of PAL in Tengcha leaves.The results indicated that the amount of polyvinypolypyrrolidone(PVP) had great impact on PAL in Tengcha when the enzyme was extracted.With the amount of PVP increasing,the enzyme activity had the trend to ascend.It attained the apex when the amount of PVP was double to that of fresh leaves. The results also showed that PAL activity was afected by different pH values.As pH value rose,the enzyme activity increased fist,and then decreased.The enzyme activity increased slowly when pH value was in pH8.0 to pH8.8.As pH value rose to 8.8,PAL activity increased to max.It was found that the enzymatic reaction was stable at 55℃in 120 min.In conclusion,the extracting condition of PAL were that the amount of PVP should be double to that of freh leaves using boracic acid(pH8.8) as extracting buffer. The enzymatic reaction should be performed at 55℃in 120min.2 Isolation and purification of PALBy the methods of ammonium sulfate precipetation and column chromatography on DEAE-fiber,PAL in the tengcha was purified.It was a single protein stripe by SDS-PAGE,It was futher purified 8.6 fold with aspecific activity of 11.8U^* 10^2*mg^-1 and a yield of 6.8%. 3 Research on the characterization of PALResearched on the characterization of the purified PAL proved that the enzyme showed optimal activity at pH 8.8 and 55℃.It was stable in pH range from 8.0 to 9.0 and temperature range from 30℃to 60℃.The survival rate of enzyme activity remained over 60%when stored 4℃and glycerin for two week.The enzyme consisted of four monomers,which moleculer weights might be 35KD.The optimum substrate concentration and Km value of PAL were 0.02mol/L,1.9×10^-2,0.966×10^-3.Heavy metal salts SrCl₂,AgNO₃(2mmol/L except)inhibited PAL activities in different degree.FeSO₄, CuSO₄,MnCl₂(2mmol/L),some metal ions promoted PAL activities significantly,but others as MgCl₂,CaCl₂,ZnSO₄ inhibited PAL activities in different degree.0.5mmol/L EDTA inhibited PAL activities,where as range from 1mmol/L to 4mmol/L EDTA enhanced PAL activities.SDS inhibited the PAL activities significantly.4 PAL activity and flavonoids,DMY changed content and relation correlation at different times and partsThe relationship between PAL activity and flavonoids,DMY changed content:as a whole,PAL activity and flavonoids,DMY changed content had a negative correlation in a year.But some had a positive correlation and some had a negative correlation at different times and different positions,the degree of correlation was a great distinction. Thus PAL activity was not only so simple to have positive correlation with specific secondary metabolites,but aslo it based different metabolic pathways in different stages.