【作者】 党尉；

【导师】 尉亚辉；

【作者基本信息】 西北大学 ， 植物学， 2003， 硕士

【摘要】 本文以新鲜葡萄（Vitis vinifera Linn．）的果皮为材料，对白藜芦醇合酶（RS）分离纯化方案及其主要生化信息进行了研究与探讨。实验显示：（1）应用正交实验方法筛选出RS的抽提方案：提取固液比（g/ml）为1：2，4℃下用0.1M磷酸盐缓冲液（PH=7.5，含5mM巯基乙醇）抽提12小时后，20000g，4℃离心15分钟得到粗酶液。（2）以蛋白质分离纯化的基本策略为基础设计RS的纯化程序是：先后用65％、90％饱和度进行硫酸铵分级沉淀；透析脱盐后依次进行DEAE-cellulose 52离子交换层析和羟基磷灰石吸附层析，平衡液均为缓冲液D（0.01M磷酸盐缓冲液，PH=7.5，含1mM巯基乙醇），用0.01—0.1M磷酸盐缓冲液（PH=7.5，分别含1mM、5mM巯基乙醇）进行梯度洗脱，两次层析的洗脱液流速分别为0.6ml/min、1.2ml/min。经过该程序后RS的电泳、层析图谱分别显示一条带、一个峰；RS的比活力由0.35U/mg上升至36.4U/mg，纯化倍数由最初设定的1.0增至104。（3）凝胶过滤法测得RS的相对分子质量为87000D；RS在中性稍偏碱性环境下活力较高，其最适PH值为7.5；两种底物香豆酰辅酶A、丙二酰辅酶A的K_m、V_max值分别为1.93μM，4.84μM/min和9.8μM，4.95μM/min，由此可知香豆酰辅酶A为RS的最适底物；两种底物的V-[S]曲线均接近于米氏酶的曲线，可初步判断RS为米氏酶。（4）利用生物学分析软件、数据库和网络资源对RS的生物信息分析结果：RS具有Chal_stil_synt和Chal_stil_syntC两个结构域；五种葡萄属源的RS氨基酸序列间具有95％以上的同源性，得到了葡萄属RS的保守氨基酸序列；并根据这些制作得到RS的三维结构图谱；RS在植物细胞中的细胞核、线粒体基质空间、微体（过氧化物酶体）、叶绿体类囊体膜中的分布概率分别为：88％、36％、31.3％、28％。更多还原

【Abstract】 In this paper, an improved procedure was described for separating and purifying resveratrol synthase(RS) from the peel of fresh grape(Vitis vinifera Linn.). At the same time the chief biochemical information of RS was studied. The experiment showed that: (1) The extraction program was refined by orthogonal design. The optimal conditions for RS extraction were: the type of buffer was potassium phosphate buffer at PH7.5 (0.1M/L, including 5mM mercaptoethanol); the solid-liquid ratio(g/ml) was 1 : 2. After extraction for twelve hours at 4℃, the supernate was the crude enzyme solution by centrifugation at 20000g, 4℃ for 15 minutes.(2) The purification procedure of RS was as follows: the crude enzyme was fractionally precipitated by 65%,90% degree of saturation; after dialysis and desalination ,then RS was isolated on DEAE-cellulose 52 column and hydroxylapatite column successively, the columns were equilibrated with buffer D (0.01 M/L potassium phosphate buffer at PH7.5, including 1mM mercaptoethanol)and done gradient elution with 0.01-0.1 M/L potassium phosphate buffer at PH7.5 (respectively including 1mM , 5mM mercaptoethanol), the elution speed were 0.6ml/min, 1.2ml/min.After the process, the electropherogram and chromatographic profile of RS showed one band and one peak; the specific activity reached 36.4 U/mg from 0.35 U/mg, the purification fold was from 1.0 to 104. (3) The relative molecular weight was 87 000D by applying gel filtration chromatography; RS had higher activity at neutral or slightly basic conditions, its optimal PH was 7.5; the Km,Vmax of the two substrates, 4-coumaroyl-CoA and malonyl-CoA were respectively 1.93μM,4.84μM/min and 9.8μM, 4.95μM/min, so 4-coumaroyl-CoA was the optimal substrate of RS. We could tentatively consider that RS was a Michaelis enzyme for the V-[S] curves of the two substrates were both similar to the one of Michaelis enzyme.(4) Applying biological analysis software, databases and internet resources, the bioinformaticanalysis of RS indicated: RS included two domains, Chal_stil_synt and Chal_stil_syntC. The amino acid sequences of five RS from grape had over 95% homology, we obtained the conservative sequences of RS and its three-dimensional model as well. The distribution probability of RS was respectively 88%, 36%, 31.3%, 28% in nucleus, mitochondrial matrix space, microbody(peroxisome) , chloroplast thylakoid membrane.更多还原