| The characters and main influence factors of the phenylalanine ammonia-lyase (PAL, EC4.3.1.5) which is one of the key enzymes in flavonoidss synthesis according to the metabolism process of flavonoidss in Ginkgo biloba leaf were completely studied with the enzyme extract and off-body leaf. Research aim was to find out some effective ways to enhance flavonoids content via regulating PAL activity based on some PAL accelerants. These assayed materials came from Ginkgo Research Orchards in Hubei Agricultural College, and all results were summarized to the followings.1. Purifying of PAL and its general charactersThe recovery of protein was 0.35%, and that of PAL was 1.17%, and the purification multiple was 3.32 after series isolating and purifying process. Its optimum pH was 8.8 in boracic acid buffer, and 8.0,8.8 in Tris-HCl buffer. PAL activity remained 13.07%, 45.64% respectively after 5 min with pH10 and pH 4 Tris-HCl treatments, which showed it can't bear alkali and acid, especially former. PAL optimum temperature was 50℃ , and its activity remained about 50% under the following conditions: 30 min with 40℃, 50℃, 20min with 60 ℃, 70℃ , 80℃,10min with 90℃,5 min with 100℃. It indicated that PAL had some tolerance to high temperature. The optimum substrate concentration and Km value of PAL were 0.02mol/L, 8.568 × 10-3mol/L apiece.2. Effects of metal ions on PAL activitiesHeavy metal salts BaCl2, SrCl2, CoNO3 inhibited PAL activities in different degree, whereas 4mmol/L AgNO3 enhanced PAL activity and others inhibited slightly. MnSO4 , CuSO4, FeSO4 and ZnSO4 , some metal ions usual in enzyme activity center, promoted the PAL activities significantly, and the most effective concentration showed as FeSO4 2 mmol/ L, CuSO4 4 mmol/ L, ZnSO4 4mmol/ L, MnSO4 1mmol/ L respectively. The effects ranked as follows: FeSO4 ,CuSO4 , Zn (Ac) 2,>MnSO4, ZnSO4. However , the effects were not obvious for different concentrations Al2(SO4)3, CaCl2 and MgCl2. 4 mmol/L, 0.5 mmol/L EDTA inhibited PAL activities, and 2 mmol/L, 1 mmol/L EDTA enhanced PAL activities, whereas 4 mmol/L, 0.5 mmol/L KHB4 enhanced PAL activities, and 2 mmol/L,l mmol/L KHB4 inhibited PAL activities.3. Effects of the light and mechanical hurt on PAL activitiesOn one hand, in the room temperature, PAL activities decreased slowly at dark, while increased at the red light and white light. Under the light, PAL changed periodically about 24 hours. The effect of the white light was better than that of the red light on PAL activity increase, but there was no significant difference between them PAL activity began to increase, and then decrease after 15 hours under mechanical hurt. On the other hand, in low temperature, the influence of the light on PAL activity was similar to that in the room temperature. 4.Effects of plant growth regulators on PAL activitiesThe PAL activities in off-body leaves ate and flew obviously. The effects were ETH>2,4-D>IAA>ABA>GA3. ETH enhanced PAL activities with different degree. Amongthem 300mg/L ETH had the strongest effect. CCC also enhanced PAL activities, but the effects decreased along concentration increase, and 200mg/L CCC had the strongest effect. ABA but 100mg/L treatment enhanced PAL activities. The PAL activities with IAA treatments but 1 00mg/L were higher than CK. Of them, 70mg/L had the greatest effect, and resulted in higher PAL activities than CK all the time. 2,4-D enhanced PAL activities, and 40mg/L had the strongest effect. The PAL activities with GA3 treatments were lower than CK but 75mg/L. 5.Effects of substrate, terminal products on PAL activitiesPhenylalanine inhibited PAL activities when its concentrations were more than 2.5mmol/L, whereas enhanced from 2.5mmol/L, and 1.25mmol/L effect was the strongest. Trans- cinnamons of 4 concentrations all inhibited PAL activities, and 100mg/L effect was the strongest. 3,5-dihydro-cinnamons of 4 concentrations but 100mg/L inhibited PAL activities, and 200mg/L effect was the strongest. Asafetida acid of all concentrations but 100... |
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