金丝桃素合成酶的基因克隆 表达及其酶学性质研究.docx

摘要
金丝桃素是贯叶连翘中重要的生物活性物质。具有抗抑郁、抗病毒、抗肿瘤、抗菌及提高免疫力等功能。目前金丝桃素主要是从贯叶连翘中提取而获得,提取物中金丝桃素的含量较低,且夹杂很多黄酮、酚类、丹宁等物质,这些物质往往是贯叶连翘提取物副作用的来源。。利用酶法生产金丝桃素具有反应条件温和、产物单一易纯化等优点,有良好的发展前景。
本论文主要结果如下
1、经RT-PCR获得了编码金丝桃素合成酶的基因(Hyp),将其克隆至原核表达载体pET30a 中。得到了重组质粒pET30a-Hyp。转化Ecoli B121得到工程菌株,工程菌经IPIG诱导, 高效表达了重组蛋白即金丝桃素合成酶(Hyp)。经检测判定Hyp具有催化大黄素合成金丝桃索的功能。
2、进行了工程菌发酵条件的优化,得到较为适宜的培养条件如下: TB培养基,接种量l%,37"(2 140r/(约3h)。然后加入IFrG ,诱导温度为30"C,转速为120r/min,诱导8b后收集菌体。
3、离心发酵液以收集菌体,用缓冲液悬浮菌体,经过超声破碎、硫酸铵分级沉淀和Ni-NTA 亲和层析,得到了电泳纯的Hyp。酶学性质研究结果表明,该酶适宜的反应温度为30"C, ,底物大黄素适宜处理方式为乙醇溶解,米氏常数K。=,
‰I=。
关键词:金丝桃素合成酶、金丝桃素、贯叶连翘、原核表达
Abstract
Hypcricin is the most ponent in L It has the function of anti-depression,anti—Vim8’anti-turnout and is produced by extracted from H perforatum I^which may cause the lack of wild plant msoure:e of//.perforatum L The extract contains small amount of hypericin,most of it am may account for some of the reported side synthetase(Hyp)is an enzyme which can catalyze emodin to
hypericin in plex ’s convenient to produce hypcricin by the enzyme.
The main result was as follows:
(1)Total RNA of//.perforatum L was extracted by gene was amplified by RT-PCR and cloned into the vectfff of pEI'30a,the binant piasmid pE'T30a-Hyp Was transferred into BL210)r巳3).The binant strain could express Hyp when it was induced by showed that Hyp had the ability of converting emodin to hypeficin.
(2)The fermentative condition of the binant strain was suitable condition of shocking battle of the strain was as pH of medium Was ,the amount of inoculation was l%and the strain Was cultured at 37"(2 for 3h,then H”Was induced to
expressby0。5mMIPTGat30"C、120r/rainfor8h.
(3)Hyp Was purified after cell collection,suspension,ammonium sulfate precipitation and Ni-NTA colum