【作者】 吴品芳；

【导师】 陆茂林； 陶文沂；

【作者基本信息】 江南大学 ， 微生物与生化药学， 2006， 硕士

【摘要】 本论文对三孢布拉氏霉（Blakeslea trispora）发酵辅酶Q₁₀的生物合成途径及代谢调控作了初步研究。以三孢布拉氏霉JSF4为出发菌株,采用紫外线及⁶⁰Coγ射线诱变处理,以L-甲硫氨酸及D-酪氨酸或对氨基苯丙氨酸结构类似物抗性定向筛选,通过摇瓶初筛和复筛,获得了一株双标记抗性突变菌株JSF4-JB1,在未经优化的摇瓶发酵条件下,菌体中辅酶Q₁₀产量可达27.1 mg/L,较出发菌株JSF4提高了92%。对三孢布拉氏霉JSF4-JB1的发酵条件进行了优化研究,确定最佳发酵培养基为（g/L）:葡萄糖19,豆粉30,蛋白胨15,KH2PO4 1,MgSO4 0.1,MnSO4 0.4,VB1 0.01,豆油9,棉籽油18,初始pH 6.5。菌株于25℃在200 r/min的旋转式摇床上振荡培养72 h,接种量为8%,装液量为40 mL/500 mL三角瓶。结果表明辅酶Q₁₀产量达36.69 mg/L,比未经优化提高了35%。采用正交实验,对醇碱皂化法提取三孢布拉氏霉菌体中辅酶Q₁₀的各工艺条件进行了研究,确定最佳的工艺条件为: KOH加量为干菌体质量的1.9倍,醇碱浓度为10%,焦性没食子酸添加量为干菌体质量的1/2,在70℃下皂化60min,其平均提取收率达81.5%。采用薄层层析法分离纯化三孢布拉氏霉提取菌液中的辅酶Q₁₀,结合紫外分光光度法及高效液相色谱法对其进行定性定量分析。实验结果表明,薄层—紫外分光光度法操作简便,是一种快速准确的辅酶Q₁₀分离测定方法,其薄板回收率可达82.8%,RSD值为1.49%。更多还原

【Abstract】 Biosynthetic pathway of coenzyme Q₁₀ in Blakeslea trispora and its metabolized regulation were primarily studied in this study. The strain of Blakeslea trispora JSF4 was treated with ultraviolet and ⁶⁰Co irradiation. By re-screening, the mutant JSF4-JB1, with dual resistant markers, was obtained on the L-Methionine, D-tyrosine and p-aminophenylalanine resistant culture media. Its coenzyme Q₁₀ yield was improved 92% as the parent strain, reaching 27.1 mg/L.The fermentation conditions of Blakeslea trispora JSF4- JB1 was studied, and the optimum culture medium was obtained as follows（g/L）: glucose 19, pease 30, peptone 15, KH2PO4 1, MgSO4 0.1, MnSO4 0.4, vitamin B1 0.01, bean oil 9, cottonseed oil 18 and the originally pH 6.5. The mutant was cultured on the rotating shaking bed at the speed of 200 r/min under 25℃for 72 hours,with inoculum size 8% and medium volume 40 mL/500 mL. The results showed that the coenzyme Q₁₀ could be improved 35%, reaching 36.69 mg/L.The extracting coenzyme Q₁₀ from Blakeslea trispora using saponific ation was also studied by orthogonal test. And the optimum extracting conditions were obtained as follows: Wet culture was distilled at 70℃for 60min, added caustic potash up to 1.9 times of dry culture（w/w）, pyrogallol up to 1/2 dry culture（w/w）, and the concentration of alkali-methanol was 10%.The average recovery of CoQ₁₀ was 81.5%.Coenzyme Q₁₀ was separated by Thin-layer Chromatograph, with its average recovery of 82.8%, and was determined by Ultraviolet Spectrameter and High Performance Liquid Chromatograph. The results showed it a fast and accurate determination for CoQ₁₀.更多还原