【作者】 周晓芳；

【导师】 叶蕊芳；

【作者基本信息】 华东理工大学 ， 微生物学， 2010， 硕士

【摘要】 本文以花生粉和棉籽蛋白取代了原培养基中的黄豆饼粉,并采用响应面法对林肯霉素产生菌的发酵培养基进行优化。首先采用Plackett-Burman实验分析各因素的主效应,选出对响应值影响较大的3个因素,即花生粉、K2HP04和玉米浆。对这些因素做爬坡实验,确定三个重要因素的中心点浓度,并运用响应面分析法确定了这3个重要因素的最优水平,分别为熟花生粉18g／L、K2HP040.4g／L、玉米浆1.2g／L。采用优化后的培养基进行摇瓶发酵,林肯霉素摇瓶生物效价为4268μg／ml,比优化前提高了12-17%。最后分别比较优化前后培养基中金属离子和氨基酸含量的差别,可知金属离子和氨基酸对林肯霉素生物合成有较大贡献。使用紫外照射结合NTG处理的复合诱变处理,并选用具有糖苷结构的IPTG(异丙基-β-D-硫代半乳糖苷)和林肯霉素本身作为筛选因子,取得很好的结果,效价提高了12-30%。在发酵Oh添加0.02%的IPTG,使效价提高了10%左右。实验证明IPTG具有诱导作用,即诱导p-半乳糖苷酶将乳糖分解为半乳糖和葡萄糖,提供营养物质或者合成抗生素的前体。实验发现,乳糖和半乳糖对林肯霉素的生产具有积极意义,尤其是半乳糖,几乎在发酵任何时间段添加都有利于抗生素的合成。多次实验证实在发酵前期(0-48h)添加0.01%的半乳糖,效价可提高20%左右。通过对莽草酸途径的代谢流分析,找到途径中关键酶氨基苯甲酸合成酶的抑制剂三甲胺,通过三甲胺抑制氨基苯甲酸合成酶的活性,使代谢流更多地流向酪氨酸的合成方向。实验证明,在48h添加0.08%的三甲胺对林肯霉素的生物合成有积极的意义。使效价提高了20%左右。更多还原

【Abstract】 The soybean flour was replaced by cottonseed protein and peanut meal as main nitrogen sources for lincomycin production. The response surface methodology was used to optimize the biosynthesis medium of Streptomyces linconinensis. First, peanut meal, KH2PO4 and corn steep liquor were found to be the important factors by Plackett-Burman design. Then center concentration of the three important factors was determined by the steepest ascent search. And the optimal concentration of the three important factors was defined by response surface methodology. The result were 18 g/L peanut meal,0.4 g/L KH2PO4 and 1.2 g/L corn steep liquor. The average potency of lincomycin could reach 4268μg/ml by using the optimal medium, which was increased by 12%-17%compared with the original medium. Finally, We found metal ions and amino acid had effects on lincomycin production by comparison the differents between the original culture and the optimal one.Based on the structure of lincomycin, mutant strains, with resistance of feedback suppression of Isopropyl-β-D-thiogalactopyranoside (IPTG),which is an analog of methyl thiolincosamide, were thus selected and lincomycin production increased by 12-30%. IPTG was proved to be an inducer ofβ-D-galactosidase when it was added at the beginning of the culture. The production of lincomycin was increased by 10%with the addition of 2 mg/L IPTG at 0 h directly in the medium. IPTG can induce the degradation of the lactose to galactose, the addition of galactose was better than lactose, which can increase the yield of lincomycin by 20%,with the addition of 0.01%galactose at 0-48 h directly in the medium.The inhibitor of anthranilate synthetase—trimethylamine was found by shikimic acid pathway analysis, which change the metabolite flux, so that more metabolite flux turned to the direction of tyrosine synthesis. Experiments showed the potency was increased by 20%or so via adding 0.08%trimethylamine at 48h directly in the medium.更多还原