藤茶PAL特性及其与黄酮和DMY含量变化相关性的研究
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摘要
苯丙氨酸解氨酶(PAL)主要催化由苯丙氨酸(Phe)到各种酚类物质,为多种酚类及类黄酮终产物提供前体,是酚类物质代谢的关键酶。本文在优化藤茶PAL粗酶提取和活性检测条件的基础上,对藤茶PAL的分离纯化和相关酶学性质进行了研究,并比较分析了江西、湖南、恩施来源的1芽1叶,3、4叶和茎在不同时期PAL的活性与黄酮和二氢杨梅素(DMY)含量变化的相关性,为今后深入研究藤茶黄酮和DMY形成机理提供理论依据。
1.藤茶PAL提取和活性分析条件的优化
以L-苯丙氨酸(L-Phe)为底物,对藤茶PAL粗酶提取和活性分析条件进行了研究。结果表明,聚乙烯吡咯烷酮(PVP)加入量对酶活性影响较大。随着PVP加入量的增大,酶活性呈现上升趋势,当加入量为叶量2倍时,酶活性达到最高。缓冲液pH值对酶活性也有较大影响。随着pH值的增大,酶活性先增后减,在pH8.0-pH8.8时,活性增加缓慢,pH8.8时酶活性达到最大值。酶促反应以55℃为宜,在此条件下反应120分钟内酶活性稳定性较好。据此,PAL提取条件为:加入2倍鲜叶量的PVP,以pH8.8的硼酸-硼砂作为提取缓冲液;PAL活性分析条件为:酶促反应温度为55℃,反应时间为120分钟内。
2.藤茶PAL的分离纯化
采用硫酸铵分级沉淀法和DEAE-纤维素柱层析法对藤茶叶PAL进行分离纯化,经SDS-PAGE电泳检测得到一条单一蛋白的弱带,该酶的纯化倍数提高了8.6倍,得率为6.8%,酶比活为11.8U*10~2mg~(-1)。
3.藤茶PAL酶学性质研究
PAL酶学性质研究表明,该酶最适pH为8.8,在pH8.0-pH9.0间比较稳定,酶活性在90%以上。最适温度为55℃,热稳定范围在30℃-60℃之间。在4℃和甘油条件下贮藏两周,酶活性仍在60%。最适底物浓度为0.02mol/L。PAL可能是由35KD的4个亚基组成;对底物的米氏常数:Km_1=1.9×10~(-2),Km_2=0.966×10~(-3)。重金属SrCl_2、AgNO_3都能不同程度地抑制酶活性;金属离子MgCl_2,CaCl_2、ZnSO_4对藤茶PAL有不同程度的抑制作用,而FeSO_4,CuSO_4,MnCl_2(2mmol/L)均促进酶活性;0.5mmol/LEDTA抑制酶活性,而在1mmol/L-4mmol/L范围内对酶活表现为一定的促进作用,SDS对酶活表现出很强抑制作用。
4.藤茶不同时期和部位PAL活性与黄酮和DMY含量变化的相关性分析
PAL活性与黄酮,DMY含量变化在年生长周期内整体上成负相关性,但是在不同时期和不同部位,有的成正相关,有的则成负相关,而且相关性程度也有很大区别。因此PAL活性并不简单地与特定次生代谢产物呈正相关,而是在不同的阶段以不同的代谢途径为主。
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 leaves
Using 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 PAL
By 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 PAL
Researched 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 SrCl_2,AgNO_3(2mmol/L except)inhibited PAL activities in different degree.FeSO_4, CuSO_4,MnCl_2(2mmol/L),some metal ions promoted PAL activities significantly,but others as MgCl_2,CaCl_2,ZnSO_4 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 parts
The 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.
1.藤茶PAL提取和活性分析条件的优化
以L-苯丙氨酸(L-Phe)为底物,对藤茶PAL粗酶提取和活性分析条件进行了研究。结果表明,聚乙烯吡咯烷酮(PVP)加入量对酶活性影响较大。随着PVP加入量的增大,酶活性呈现上升趋势,当加入量为叶量2倍时,酶活性达到最高。缓冲液pH值对酶活性也有较大影响。随着pH值的增大,酶活性先增后减,在pH8.0-pH8.8时,活性增加缓慢,pH8.8时酶活性达到最大值。酶促反应以55℃为宜,在此条件下反应120分钟内酶活性稳定性较好。据此,PAL提取条件为:加入2倍鲜叶量的PVP,以pH8.8的硼酸-硼砂作为提取缓冲液;PAL活性分析条件为:酶促反应温度为55℃,反应时间为120分钟内。
2.藤茶PAL的分离纯化
采用硫酸铵分级沉淀法和DEAE-纤维素柱层析法对藤茶叶PAL进行分离纯化,经SDS-PAGE电泳检测得到一条单一蛋白的弱带,该酶的纯化倍数提高了8.6倍,得率为6.8%,酶比活为11.8U*10~2mg~(-1)。
3.藤茶PAL酶学性质研究
PAL酶学性质研究表明,该酶最适pH为8.8,在pH8.0-pH9.0间比较稳定,酶活性在90%以上。最适温度为55℃,热稳定范围在30℃-60℃之间。在4℃和甘油条件下贮藏两周,酶活性仍在60%。最适底物浓度为0.02mol/L。PAL可能是由35KD的4个亚基组成;对底物的米氏常数:Km_1=1.9×10~(-2),Km_2=0.966×10~(-3)。重金属SrCl_2、AgNO_3都能不同程度地抑制酶活性;金属离子MgCl_2,CaCl_2、ZnSO_4对藤茶PAL有不同程度的抑制作用,而FeSO_4,CuSO_4,MnCl_2(2mmol/L)均促进酶活性;0.5mmol/LEDTA抑制酶活性,而在1mmol/L-4mmol/L范围内对酶活表现为一定的促进作用,SDS对酶活表现出很强抑制作用。
4.藤茶不同时期和部位PAL活性与黄酮和DMY含量变化的相关性分析
PAL活性与黄酮,DMY含量变化在年生长周期内整体上成负相关性,但是在不同时期和不同部位,有的成正相关,有的则成负相关,而且相关性程度也有很大区别。因此PAL活性并不简单地与特定次生代谢产物呈正相关,而是在不同的阶段以不同的代谢途径为主。
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 leaves
Using 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 PAL
By 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 PAL
Researched 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 SrCl_2,AgNO_3(2mmol/L except)inhibited PAL activities in different degree.FeSO_4, CuSO_4,MnCl_2(2mmol/L),some metal ions promoted PAL activities significantly,but others as MgCl_2,CaCl_2,ZnSO_4 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 parts
The 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.
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59. Jin Nakashima, et al. Immunocytochemical localization of phenylalanine ammonia21yase and cinnamylalcoho ldehydrogenase in differentiating tracheary elements derived from Zinnia mesophyll cells [J]. Plant Cell Physiology, 1997, 38 (2): 113-123
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61. Kataoka I, et al. Changes in L 2phenylalanine ammonia21yase activity and anthocyanin synthesis during berry ripening of three grape cultivars [J]. Journal of the Japanese Society for Horticultural Science, 1983, 52 (3): 273-279
62. Kouko 1 J. Conn E E. The metabolism of aromatic compounds in higher plants. Purification and properties of the 2phenylalanine deaminase of Herdeum vulagare [J]. Journal of Biology and Chemistry, 1961, 236: 2692-2698
63. Kozukue N. Change in the contents of phenolic substances,phenylalanine ammonia-lyase(PAL) and tyrosine ammonia-lyase(TAL) accompanying chilling-injury of egg plant fruit. Sci Hort .A, 1979,11(1):51-59
64. Lim H W, et al. Purification and properties of phenylalanine ammonia-lyase from leaf mustard [J]. Molecules and Cells, 1997,7 (6): 715-720
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66. Neish A C. Biosynthesis path way of aromatic compounds [J]. Annual Review of Plant Physiology, 1960,32(11): 15
67. Ritenour M A , et al. Phenylalanine ammonia-lyase (PAL ) activity and its relationship to anthocyanin and flavonoidlevels in New Zealand2grow n apple cultivars [J]. American Society for Horticultural Science, 1996,121 (3): 281-285
68. Rajagopal Subramaniam, et al. Structure, inheritance, and expression of hybrid poplar (Populustrichocarpa ×Populusdeltoides) phenylalanine ammonia21yase genes [J], Plant Physiology, 1993, (102): 71-83
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