【摘要】：棘白菌素類抗真菌藥物米卡芬凈和阿尼芬凈的合成工藝中包括一個(gè)關(guān)鍵步驟:水解除去FR901379分子和棘白菌素B(echinocandin B,ECB)分子的脂肪酸側(cè)鏈,形成環(huán)狀的6元多肽核心。FR901379和ECB的水解可以分別被FR901379酰基酶和阿庫來菌素A酰基酶(aculeacin A acylase,AAC)催化,因此,FR901379�；负虯AC的發(fā)掘、表征和生產(chǎn)在米卡芬凈和阿尼芬凈的工業(yè)生產(chǎn)上具有重要的應(yīng)用價(jià)值。本研究首先篩選到了猶他游動(dòng)放線菌SW1311,發(fā)現(xiàn)該菌株發(fā)酵液具有�；富钚�,并摸索了不同發(fā)酵條件對(duì)酰基酶活性的影響。然后將猶他游動(dòng)放線菌SW1311中的AAC基因克隆到改造過的質(zhì)粒載體p IJ8660中,并將該質(zhì)粒轉(zhuǎn)化到天藍(lán)色鏈霉菌(Streptomyces coelicolor)A3(2)中對(duì)AAC基因進(jìn)行高表達(dá),得到重組菌株sSCO-AAC。最后將sSCO-AAC生產(chǎn)的AAC活性和所需培養(yǎng)時(shí)間與猶他游動(dòng)放線菌SW1311進(jìn)行比較,表征了sSCO-AAC的發(fā)酵液水解FR901379的反應(yīng)。結(jié)果表明,猶他游動(dòng)放線菌SW1311中的酰基酶具有水解FR901379和青霉素V的�；钚浴Ｓ弥亟M菌株sSCO-AAC生產(chǎn)的AAC活性比猶他游動(dòng)放線菌SW1311的高4.6倍,且該重組菌株所需的培養(yǎng)時(shí)間比猶他游動(dòng)放線菌SW1311縮短了30%。該結(jié)果不僅將ACC的應(yīng)用范圍從阿尼芬凈合成拓寬到了米卡芬凈合成,而且還揭示出天藍(lán)色鏈霉菌A3(2)可以作為一個(gè)良好的AAC表達(dá)菌株。本研究對(duì)阿尼芬凈和米卡芬凈的工業(yè)化生產(chǎn)具有潛在的應(yīng)用價(jià)值。
[Abstract]:One of the key steps in the synthesis of mikafenem and Amifen is the hydrolysis of fatty acid side chains of FR901379 molecules and B (echinocandin FR901379 molecules. The hydrolysis of FR901379 and ECB can be catalyzed by FR901379 acylase and acurylin A acylase (aculeacin A acylase,AAC, respectively. Therefore, the discovery of FR901379 acylase and AAC, Characterization and production have important application value in the industrial production of mikafennet and arnifen. In this study, first of all, we found that the fermentation broth of SW1311, had acylase activity, and explored the effect of different fermentation conditions on the activity of acylase. Then, the AAC gene from SW1311 was cloned into the modified plasmid vector p IJ8660, and the AAC gene was overexpressed in Streptomyces aquilinum (Streptomyces coelicolor) A3 (2). The recombinant strain sSCO-AAC. was obtained. Finally, the activity and culture time of AAC produced by sSCO-AAC were compared with that of SW1311, and the reaction of hydrolyzing FR901379 in sSCO-AAC fermentation broth was characterized. The results showed that the acylase in SW1311 had the activity of hydrolyzing FR901379 and penicillin V. The AAC activity of the recombinant strain sSCO-AAC was 4. 6 times higher than that of SW1311, and the culture time of the recombinant strain was 30 times shorter than that of SW1311. This result not only broadens the application range of ACC from ANFN synthesis to mikafennet synthesis, but also reveals that Streptomyces aquilinus A3 (2) can be used as a good AAC expression strain. This study has potential application value for the industrial production of arnifen and mikafennet.
【作者單位】： 華東醫(yī)藥集團(tuán)新藥研究院有限公司;浙江大學(xué)求是學(xué)院;浙江大學(xué)生命科學(xué)學(xué)院;
【基金】：Projects supported by Zhejiang Provincial Natural Science Foundation of China(No.LR16H300001) National Natural Science Foundation of China(No.31670008)
【分類號(hào)】：R914
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本文編號(hào)：2355125