本文選題：變鉛青鏈霉菌 + 棘白霉素脫酰酶��； 參考：《華東理工大學》2014年碩士論文

【摘要】：棘白霉素類藥物是一類重要的半合成抗真菌抗生素,批準臨床應用的有米卡芬凈、阿尼芬凈和卡泊芬凈。米卡芬凈和阿尼芬凈的合成過程均包含一步由猶他游動放線菌(Actinoplanes utahensis NRRL12052)執(zhí)行的脫�；磻�,該反應由棘白霉素脫酰酶(ECB脫酰酶)催化。當前,我國相關(guān)制藥企業(yè)在該步反應中仍然采用猶他游動放線菌野生菌發(fā)酵轉(zhuǎn)化的工藝,由于野生菌發(fā)酵生產(chǎn)的ECB脫酰酶表達量不高,催化反應的底物濃度僅能達到1-2g/L,無法滿足大規(guī)模工業(yè)化生產(chǎn)。因此,通過構(gòu)建基因工程菌生產(chǎn)ECB脫酰酶,提高該酶的表達量,具有重要的應用價值。 本課題旨在構(gòu)建可適應工業(yè)化需求的ECB脫酰酶高效表達基因工程菌。選擇與猶他游動放線菌近親且表達體系成熟的變鉛青鏈霉菌為表達宿主,從猶他游動放線菌中獲取ECB脫酰酶的基因序列,隨后與鏈霉菌表達載體pNW-S1連接,通過原生質(zhì)體轉(zhuǎn)化的方法獲得基因工程菌株。結(jié)果顯示,在搖瓶發(fā)酵水平上,ECB脫酰酶在基因工程菌中表達量為野生菌株的10倍,酶活為125U/L；且ECB脫酰酶在基因工程菌中為分泌表達,發(fā)酵液離心后的發(fā)酵上清可用于生產(chǎn),明顯優(yōu)于猶他游動放線菌的全細胞發(fā)酵轉(zhuǎn)化。通過發(fā)酵放大實驗,在300L發(fā)酵罐上,該基因工程菌的酶活可達到80U/L。以米卡芬凈的原料FR901379的脫酰反應為例,FR901379在基因工程菌發(fā)酵上清中的投料濃度可達到15g/L,摩爾轉(zhuǎn)化率為100%。此外,為了便于該酶的生產(chǎn)保存和應用,本課題還對該酶的粗純化及固定化等工作進行了研究。本課題對棘白霉素脫酰酶基因工程菌的研發(fā),勢必有助于升級改良當前棘白霉素類藥物的生產(chǎn)工藝。
[Abstract]:Echinomycin is an important class of semi-synthetic antifungal antibiotics. The biosynthesis of mikafennet and arnifen consisted of a deacylation reaction performed by Actinoplanes utahensis NRRL12052, which was catalyzed by the acanthomycin deacylase (Echinomycin deacylase), the actinoplanes utahensis NRRL12052, and the acylated actinoplanes of Actinoplanes utahensis NRRL12052. At present, the related pharmaceutical enterprises in our country still adopt the technology of fermenting and transforming from wild actinomycetes of Utah in this step, because of the low expression of ECB deacylase produced by fermentative production of wild actinomycetes. The substrate concentration of the catalytic reaction is only 1-2 g / L, which is not suitable for mass industrial production. Therefore, it has important application value to produce ECB deacylase by constructing genetically engineered bacteria to increase the expression of this enzyme. The purpose of this study was to construct ECB deacylase that could efficiently express gene engineering bacteria. The ECB deacylase gene sequence was obtained from Streptomyces mutans, which was a close relative and mature expression system of Streptomyces spp., and then was linked with Streptomyces expression vector pNW-S1. Genetic engineering strains were obtained by protoplast transformation. The results showed that the expression of ECB deacylase in genetically engineered strain was 10 times of that in wild strain, and the enzyme activity was 125 U / L at the level of shaking flask fermentation, and ECB deacylase was secreted in genetically engineered bacteria, and the fermentation supernatant of fermentation broth after centrifugation could be used for production. It is obviously superior to the whole cell fermentation transformation of the Utah motile actinomycetes. The enzyme activity of the genetically engineered strain reached 80 U / L on 300L fermenter. Taking the deacylation reaction of FR901379 as an example, the feed concentration of FR901379 in the supernatant of genetically engineered bacteria could reach 15g / L, and the molar conversion rate was 100g / L. In addition, in order to facilitate the production, preservation and application of the enzyme, this paper also studied the crude purification and immobilization of the enzyme. The research and development of Echinomycin deacylase genetic engineering bacteria will help to upgrade the current production process of Echinomycin.
【學位授予單位】：華東理工大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：Q78;Q93

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