棘白霉素脱酰酶基因工程菌的构建及应用研究

发布时间：2018-05-31 12:07

本文选题：变铅青链霉菌 + 棘白霉素脱酰酶　；参考：《华东理工大学》2014年硕士论文

【摘要】：棘白霉素类药物是一类重要的半合成抗真菌抗生素,批准临床应用的有米卡芬净、阿尼芬净和卡泊芬净。米卡芬净和阿尼芬净的合成过程均包含一步由犹他游动放线菌(Actinoplanes utahensis NRRL12052)执行的脱酰化反应,该反应由棘白霉素脱酰酶(ECB脱酰酶)催化。当前,我国相关制药企业在该步反应中仍然采用犹他游动放线菌野生菌发酵转化的工艺,由于野生菌发酵生产的ECB脱酰酶表达量不高,催化反应的底物浓度仅能达到1-2g/L,无法满足大规模工业化生产。因此,通过构建基因工程菌生产ECB脱酰酶,提高该酶的表达量,具有重要的应用价值。本课题旨在构建可适应工业化需求的ECB脱酰酶高效表达基因工程菌。选择与犹他游动放线菌近亲且表达体系成熟的变铅青链霉菌为表达宿主,从犹他游动放线菌中获取ECB脱酰酶的基因序列,随后与链霉菌表达载体pNW-S1连接,通过原生质体转化的方法获得基因工程菌株。结果显示,在摇瓶发酵水平上,ECB脱酰酶在基因工程菌中表达量为野生菌株的10倍,酶活为125U/L；且ECB脱酰酶在基因工程菌中为分泌表达,发酵液离心后的发酵上清可用于生产,明显优于犹他游动放线菌的全细胞发酵转化。通过发酵放大实验,在300L发酵罐上,该基因工程菌的酶活可达到80U/L。以米卡芬净的原料FR901379的脱酰反应为例,FR901379在基因工程菌发酵上清中的投料浓度可达到15g/L,摩尔转化率为100%。此外,为了便于该酶的生产保存和应用,本课题还对该酶的粗纯化及固定化等工作进行了研究。本课题对棘白霉素脱酰酶基因工程菌的研发,势必有助于升级改良当前棘白霉素类药物的生产工艺。
[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.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q78;Q93

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