【作者】 王力；

【导师】 孙君社；

【作者基本信息】 中国农业大学 ， 食品科学与工程， 2005， 硕士

【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。金丝桃素合成酶是金丝桃素合成过程中的关键酶,可以催化大黄素生成金丝桃素。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 BL21得到工程菌株,工程菌经IPTG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素的功能。 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:采用初始pH为7.0的TB培养基,接种量1%,37℃ 140r/min培养至OD₆₀₀为0.6左右（约3h）,然后加入IPTG至终浓度为0.5mM进行诱导,诱导温度为30℃,转速为120r/min,诱导8h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℃,适宜反应pH为6.0,底物大黄素适宜处理方式为乙醇溶解,米氏常数K_m=5.049μmol/L,V_max=0.0831μg/h·mg。更多还原

【Abstract】 Hypericin is the most active component in Hypericum.perforatum L. It has the function of anti-depression, anti-virus, anti-tumour and etc. Presently, hypericin is produced by extracted from H. perforatum L, which may cause the lack of wild plant resource of H. perforatum L. The extract contains small amount of hypericin, most of it are other compounds. These compounds may account for some of the reported side effects. Hypericin synthetase（Hyp） is an enzyme which can catalyze emodin to hypericin in a complex way. It’s convenient to produce hypericin by the enzyme.The main result was as follows:（1） Total RNA of H. perforatum L was extracted by Trizol. Hyp gene was amplified by RT-PCR and cloned into the vector of pET30a, the recombinant plasmid pET30a-Hyp was transferred into E.coli BL21（DE3）. The recombinant strain could express Hyp when it was induced by IPTG. Research showed that Hyp had the ability of converting emodin to hypericin.（2） The fermentative condition of the recombinant strain was studied. The suitable condition of shocking battle of the strain was as follows. The primary pH of medium was 7.0, the amount of inoculation was 1% and the strain was cultured at 37℃ for 3h, then Hyp was induced to express by 0.5mM IPTG at 30℃ > 120r/min for 8h.（3） Hyp was purified after cell collection, suspension, ammonium sulfate precipitation and Ni-NTA column chromatography. The enzyme exhibited higher activity when temperature was 30℃ and pH was 7.0. Mchihaelis constants of enzyme were K_m=5.049μmol/L, V_max= 0.0831 μg/h·mg。更多还原