【作者】 朱宏莉；

【导师】 宋纪蓉；

【作者基本信息】 西北大学 ， 化学工程， 2007， 博士

【摘要】 本论文旨在利用微生物转化法对底物结构进行修饰，从而生物合成天麻素及红景天苷。通过对转化菌株的诱变选育，同时兼顾转化条件的优化，为天麻素及红景天苷的合成提供一条行之有效的新途径。在此基础上，对相关糖基化酶进行分离纯化及性质研究，为以后其它天然产物的糖基化反应提供新的思路和方法。采用静息细胞法和分批培养法，结合TLC和HPLC从数十株霉菌、酵母菌、细菌中筛选到了能够将对羟基苯甲醛转化成天麻素的根霉LN-1和一株能将对羟基苯乙醇转化为红景天苷的假单胞菌Z-1。分别对LN-1及Z-1转化条件进行了优化，结果表明根霉LN-1转化合成天麻素的最适条件为：12g(湿重)静息细胞／120mL转化液，底物质量浓度2.3mg／mL，pH5.5，28℃转化24h，天麻素得率可达16％。Z-1转化合成红景天苷的最适条件为：以葡萄糖作为碳源，酵母膏或硫酸铵作为氮源，底物浓度为1.5mg／mL，pH7.0，33℃转化20h，红景天苷的得率可达10％。进一步开展放大试验，转化液经萃取、硅胶柱色谱分离等得到转化产物，经HPLC、NMR、EI-MS等的测定，证明所得转化产物为天麻素及红景天苷。同时，利用紫外、He-Ne激光复合诱变LN-1的孢子及原生质体，获得了转化率及天麻素得率均明显提高的突变株。采用硫酸铵沉淀、DEAE-Ceilulose52离子交换层析及Sepharose CL-6B凝胶柱层析等一系列纯化步骤获得了电泳纯的天麻素合成酶，并用SDS-PAGE的方法测定其分子量为63.2kDa。样品酶的比活力为174.2U／mg，纯化倍数为32.3，回收率为11.4％。通过分析该酶的性质，得出最适温度为50℃，在50℃以下基本保持稳定；最适pH6.0，在pH6.0-9.0的范围内基本稳定。微生物作为生物转化体系中一个重要组成部分，对研究天然活性成分的合成有着不可替代的作用。本研究利用微生物转化法合成了天麻素及红景天苷，摸索出了一条生产天麻素及红景天苷的新途径，具有广阔的应用前景。更多还原

【Abstract】 Gastrodin and salidroside, the major ingredient of the well-known Chinese medicine,were biosynthesized through microbial transformation, which is a new method different from chemical synthesis and traditional plant extraction. In this experiment, mutagenesis of the strain, optimazition of transforming condition, extraction and purification of the enzyme were discussed in order to find the more efficient and economical method for gastrodin and salidroside synthesis and offer a new channel for microbial transformation of other bioactive natural products.Using the method of batch culture and resting culture, Rhizopus LN-1 and pseudomonas Z-1, which have the ability to biosynthesize gastrodin and salidroside were sreened sequently from many fungi, yeast and bacteria according to their TLC and HPLC results. The optimal transforming condition was investigated: 12g resting cell （wet weight） incubated at 28℃for 24 hours, in a 500mL flask containing 120mL of medium, produced a good yield of gastrodin（16%） by adding 2.3mg/mL substrate（final concentration）.The optimal initial pH of the medium was 5.5. The maximum yield of salidroside（10%） was acquired under the condition: substrate concentration 1.5mg/mL, initial pH 7.0, incubated at 33℃for 20 hours on a rotary shaker using glucose as carbon source and yeast extract or （NH₄）₂SO₄ as nitrogen source.The preparative scale experiment was carried out under the ideal transforming condition, the products were obtained by extraction, chromatography, and identified on the basis of its ¹H, ¹³CNMR and EI—MS spectral data. On the basis of this, a mutant LN-B with high yield of gastrodin was selected after irradiation of UV and He-Ne laser on the spores and protoplast of the original strain. The isozymegram proved the existence of the mutation.After （NH₄）₂SO₄ precipitation, DEAE-cellulose column chromatography and sepharose CL-6B column chromatography, the enzyme for biosynthesis of gastrodin was purified to one band in SDS-polyacrylamide gel electrophoresis, and its molecular weight was about 63.2kDa. The optimum temperature of the enzyme was 50℃, and the pH was 6.0. The enzyme was stable at less than 50℃, and pH 6.0-9.0.Microorganism performs great roles in biotransformation of bioactive natural product. The fact that gastrodin and salidroside could be synthesized by microbial transformation implies a new approach to gastrodin and salidroside production. As a result, this research will be of a great economic and social value.更多还原