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异戊烯基化吲哚类生物碱广泛存在于麦角菌、青霉菌和曲霉菌中,具有一定的药理学活性,与未异戊烯基化的前体在生物活性方面具有明显的差异.曲霉菌中的某些异戊烯基化吲哚类生物碱具有抗癌活性,如烟曲霉毒素C(fumitremorgin C)、tryprostatin B,但其天然产量低且不易分离,利用化学酶合成法可很容易地将前体转化为异戊烯基化吲哚类生物碱.异戊烯基转移酶FtmPT1对二甲丙烯基二磷酸(dimethylallyl diphosphate,DMAPP)具有专一性,但可以接受不同的芳香族底物.早期研究发现,FtmPT1能接受含色氨酸的不同环二肽为底物,但以cyclo-L-Trp-L-Tyr和cyclo-L-Trp-L-Phe为底物时,酶的相对活性很低,其产物量少,无法用于合成产物.本实验通过优化酶反应条件来提高其产量.将已构建的含ftmPT1的质粒在大肠杆菌中诱导表达,经Ni-NTA亲和柱纯化后用于酶反应.实验结果表明,通过增加酶量(终浓度2.8μmol/L)、延长培养时间(37℃,24h),以cyclo-L-Trp-L-Tyr和cyclo-L-Trp-L-Phe为底物的酶反应产率分别达到49.3%和21.3%,产物经1H-NMR、1H-1H-COSY和ESI-MS鉴定,其结果与预期吻合.据检索,这2个化合物均为新化合物,分别命名为cyclo-C2-1′-DMA-L-Trp-L-Tyr和cyclo-C2-1′-DMA-L-Trp-L-Phe.
Prenyllated indole alkaloids are widely present in the cells of the genus Erysiphe, Penicillium and Aspergillus, and have certain pharmacological activities, showing significant differences in biological activity with non-prenylated precursors. Certain isopentenylated indole alkaloids in mold have anti-cancer activity such as fumitremorgin C and tryprostatin B, but their natural yield is low and they are not easily separated. It is easy to use chemical enzyme synthesis Converts prenyl to prenylenated indole alkaloids, and isopentenyl transferase FtmPT1 is specific for dimethylallyl diphosphate (DMAPP) but can accept different aromatic substrates Early studies showed that FtmPT1 can accept different tryptophan-containing cyclic dipeptides as substrates, but when using cyclo-L-Trp-L-Tyr and cyclo-L-Trp-L-Phe as substrates, Relative activity is very low, the amount of the product is small, can not be used for the synthesis of the product.In this experiment, by optimizing the enzyme reaction conditions to increase its production.It has been constructed containing the ftmPT1 plasmid induced expression in E. coli, Ni-NTA affinity column After purification for enzyme reaction, the experimental results show that by increasing the amount of enzyme (final concentration 2.8μmol L-Trp-L-Tyr and cyclo-L-Trp-L-Phe were 49.3% and 21.3%, respectively, and prolonged the culture time (37 ℃, 24h) The products were identified by 1H-NMR, 1H-1H-COZY and ESI-MS, and the results were in line with the expected results.According to the search, these two compounds were new compounds named as cyclo-C2-1’-DMA-L-Trp -L-Tyr and cyclo-C2-1’-DMA-L-Trp-L-Phe.