改造紫茎泽兰中关键倍半萜合酶以减少副产物的研究

摘要近年来从紫茎泽兰中分离得到具有抗肿瘤活性强、作用范围广、毒副作用小、不易产生耐药性等优点的倍半萜类化合物β-榄香烯。β-榄香烯由吉马烯A合成酶催化FPP生成吉马烯A，再经过一定外部条件如200℃下的分子内重排形成终产物β-榄香烯。吉马烯A合成酶是β-榄香烯生物合成途径中的关键限速酶。【目的】通过对紫茎泽兰中关键倍半萜合酶基因进行改造，从而改变酶活性，提高β-榄香烯产量，减少副产物含量。【方法】用PCR引入定点突变法对紫茎泽兰GAS做T400S定点突变，再将突变成功后的基因转化到BL21（DE3）感受态细胞中表达。最后利用GC-MS检测固相微萃取装置萃取头上吸附的样品量进行测定，研究pH值对β-榄香烯产量及副产物含量的影响。【结果】成功将紫茎泽兰中吉马烯A合酶T400S位点进行突变。经GC-MS检测固相微萃取装置萃取头上吸附的β-榄香烯的产量显示在反应条件为30℃，2h，pH值为8。0时，β-榄香烯的产量最高，副产物最少。【结论】在减少突变副产物的同时，突变引起了催化活性中心的构象变化，从而降低了催化活性。该实验对生物合成中吉马烯A合酶的改造提供了重要的参考价值。89582

Abstract In recent years。 We can get sesquiterpenoid compounds β-elemene from the Eupatorium adenophorum。 β-elemene has the advantages of strong anti-tumor activity, wide range of effect, and difficult to produce drug resistance。 Gemagene A is produced by Gemagene A synthase catalytics FPP, And then through a certain external conditions such as 200 ℃ within the intramolecular rearrangement, ,Finally generated β-elemene。 Gemma A synthetase is a key rate-limiting enzyme in the biosynthetic pathway of β-elemene。 [Objective] use PCR lead to Site mutation to do T400S site mutation。 And then the gene after the mutation was transformed into BL21 competent cells。 Finally, the amount of adsorbed samples on the extraction head of solid phase microextraction was measured by GC-MS。 The effect of PH value on the yield and byproduct content of β-elemene was studied。 [Result] The mutant of the Gemagene A synthase T400S site in Eupatorium adenophorum was successfully constructed , The yield of β-elemene adsorbed on the extractor by GC-MS showed that the optimum reaction conditions were 30 ℃, 2h, pH 8。0。[Conclusion] While mutagenizing byproducts is reduced, mutations cause a conformational change in the center of the catalytic activity, thereby reducing the catalytic activity。This experiment provides an important reference value for the transformation of gemma-A synthase in biosynthesis。

毕业论文关键词：紫茎泽兰；倍半萜合酶；定点突变；副产物源Q于W优E尔A论S文R网wwW.yOueRw.com 原文+QQ75201,8766

Keyword: Eupatorium adenophorum；sesquiterpene synthase； Site-directed mutation； by-product

1 引言 6

1。1 紫茎泽兰及榄香烯的简介 6

1。2 β-榄香烯的简介及药用价值 6

1。3 β-榄香烯的生物合成途径 7

1。4 倍半萜合酶的概述 8

1。5 固相微萃取的概述 9

1。6 本研究的技术路线 9

2 紫茎泽兰T400S的定点突变 9

2。1 实验材料 10

2。1。1 实验试剂 10

2。1。2 实验仪器改造紫茎泽兰中关键倍半萜合酶以减少副产物的研究:http://www.youerw.com/yixue/lunwen_191840.html