【作者】 田玉庭；

【导师】 岳田利；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2010， 博士

【摘要】 辅酶Q₁₀是一种重要的生化药剂,临床上被广泛应用于心血管疾病的治疗。此外,辅酶Q₁₀因具备抗氧化性和消除自由基的功能,而今已扩展到食品和化妆品领域。当前,辅酶Q₁₀制备方法主要有化学法、动植物组织提取法和微生物发酵法。与前两种制备方法相比,微生物发酵合成辅酶Q₁₀具有成本低、无光学异构体及生物学活性高等特点,因而被认为是最具前途的生产方式。本研究以根癌土壤杆菌（Agrobacterium tumefaciens）为出发菌株,诱变育种结合定向筛选,成功获得一株高产辅酶Q₁₀突变株,后通过发酵过程优化控制,最大限度的发挥该突变株代谢合成辅酶Q₁₀的特性。主要研究内容和结果如下:（1）系统考察超声波破碎法、反复冻融法、研磨法、酸热法对根癌土壤杆菌细胞破碎和辅酶Q₁₀提取效果的影响。结果表明,酸热破碎法提取辅酶Q₁₀效果最好;其中,以盐酸破壁提取效果最佳,乳酸和硫酸次之。酸热破壁提取工艺的关键因子为:料液比、破壁温度和处理时间。中心组合设计和响应面分析结果显示,在3 mol/L盐酸处理条件下,酸热破壁提取最佳工艺参数为:料液比10.8 mL/g、破壁温度84.2°C、处理时间35.3 min。在最佳破壁工艺下,辅酶Q₁₀提取量能达到1.518 mg/g。（2）以根癌土壤杆菌1.2554为出发菌株,经高静水压诱变处理,筛选获得一株呼吸链抑制剂NaN3抗性突变株PN07。菌株PN07后经过紫外线和硫酸二乙酯复合诱变,获得一株酪氨酸缺陷型突变株MT18。菌株MT18后经高静水压、紫外线和硫酸二乙酯交替诱变,采用抗结构类似物（乙基硫氨酸、柔红霉素、维生素K₃）的抗性平板初筛,经摇瓶发酵复筛,最终获得一株辅酶Q₁₀高产菌株PK38,其胞内辅酶Q₁₀含量达2.30 mg/g干菌,较出发菌株提高51.51%。菌株PK38后经10次传代培养,辅酶Q₁₀含量无明显下降,表明PK38是一株遗传性状稳定的优良菌株。（3）采用单因素试验并结合Plackett-Burman试验设计,筛选出影响PK38细胞生长和辅酶Q₁₀产量的重要因子为:蔗糖、玉米浆干粉、硫酸铵、硫酸镁、生物素和茄尼醇。结合Box-Behnken设计和响应面分析,得到生产辅酶Q₁₀的最佳发酵培养基配方为:蔗糖37.2 g/L、玉米浆干粉37.1 g/L、硫酸铵7.0 g/L、硫酸镁0.4 g/L、生物素82.9μg/L、茄尼醇0.2 g/L、CaCl₂ 140 mg/L、FeSO₄·7H₂O 0.2 mg/L、ZnSO₄·7H₂O 8 mg/L、烟酸8 mg/L。在最佳发酵培养基下摇瓶发酵72 h,生物量和辅酶Q₁₀产量分别可达到9.25 g/L和28.44 mg/L。（4）采用摇瓶发酵试验,利用单因素试验考察了初始pH值、发酵温度、接种量和发酵时间对PK38细胞生长和发酵生产辅酶Q₁₀的影响。结果表明,辅酶Q₁₀发酵最佳初始pH值、发酵温度、接种量和发酵时间分别为:7.2、30°C、8%和72 h。后利用5 L发酵罐,研究转速和溶氧浓度对辅酶Q₁₀发酵的影响。结果表明,较高转速和溶氧浓度有利于细胞生长,但不利于PK38胞内辅酶Q₁₀的代谢合成。后经采用分段控制溶氧工艺（0-36 h为90-100%,36-72 h为30%）,最终辅酶Q₁₀产量为56.46 mg/L,比最优恒定转速下提高11.71%。（5）基于Logistic方程、Leudeking-Piret方程以及Luedeking-Piret-like方程得到了描述辅酶Q₁₀分批发酵过程的动力学模型和模型参数。经模型检验,结果表明,在蔗糖浓度为30 40 g/L范围内,该组模型适应性良好,能较好地拟合发酵过程,可反映菌株PK38分批发酵过程的动力学特征。（6）基于分批发酵动力学模型,以蔗糖为单一流加底物,分别考察了分批补料培养、恒速流加培养（高速、低速）和指数流加培养3种培养模式下,菌株PK38发酵生产辅酶Q₁₀的代谢特征。结果表明,指数流加培养策略为菌株PK38发酵生产辅酶Q₁₀最佳培养模式。该培养模式下,PK38最终生物量和辅酶Q₁₀产量分别达32.37 g/L和120.01 mg/L,与分批发酵相比,分别提高了126.11%和173.12%。更多还原

【Abstract】 Coenzyme Q₁₀ (CoQ₁₀, ubiquinone) is an important biochemical compound receiving increased attention as a nutraceutical dietary supplement for its known benefits in the prevention of aging and cardiovascular problems. Extensive attempts have been made to produce CoQ₁₀ to meet the growing demands. The production of CoQ₁₀ follows one of the three routes: extraction from biological tissues, chemical synthesis, and microbial fermentation. In the wake of environmental awareness, the first two options became least desirable due to the inherent uses of solvents and chemicals in the process. Microbial fermentation, on the contrary, offers an environmentally benign option based on the enzymatic catalysis at the cellular level for CoQ₁₀ assembly. Moreover, this approach is attractive to the industry because the process is easy to control at a relatively low production cost. In this study, A. tumefaciens 1.2554 was treated with HHP, UV, and DES, and strain PK38 was isolated using selection marker. The CoQ₁₀ production of PK38 was investigated using batch and fed-batch cultures. The main results are as following:（1） The yield of CoQ₁₀, an intracellular product extracted from Agrobacterium tumefaciens cells, is dependent on the effectiveness of cell lysis post fermentation. Various cell lysis approaches were investigated, including ultrasound, repetitive freezing/thawing, grinding, and acid-heat treatment. The acid-heat combination using hydrochloric acid was found the most effective in releasing CoQ₁₀, followed by lactic, sulfuric, phosphoric, and oxalic acids. The most significant processing parameters, namely the ratio of acid solution volume and bacteria weight （A/B ratio）, incubation temperature, and reaction time, were optimized by using the Central Composite Design （CDD） with a quadratic regression model built by Response Surface Methodology （RSM）. The highest CoQ₁₀ yield at 1.518 mg/g dry cell was attained using hydrochloric acid （3 mol/L） under optimal A/B ratio, temperature, and time at 10.8 mL/g, 84.2°C, and 35.3 min, respectively.（2） A. tumefaciens 1.2554 was subjected to a series of treatment including the high hydrostatic pressure （HHP） treatment, UV irradiation, and Diethyl sulfate （DES） treatment. Through these treatments, a mutant strain PK38 with high-yield of CoQ₁₀ was isolated, screened by selecting mutants resisting the respiration chain inhibitor （sodium azide） and the structure analogue （ethionine, daunomycin and Vk3）. The stability of the mutants was tested and the strain PK38 had specific CoQ₁₀ content between 2.318 and 2.321, which was 51.51% higher than the original strain.（3） Six nutritional factors, including sucrose, corn steep power （CSP）, （NH₄）₂SO₄, MgSO₄·7H₂O, biotin and solanesol were optimized for CoQ₁₀ production using response surface methodology （RSM）. The optimal medium for CoQ₁₀ production were （g/L）: sucrose 37.2 g/L, CSP 7.1 g/L, （NH₄）₂SO₄ 7.0 g/L, MgSO₄·7H₂O 0.4 g/L, biotin 82.9μg/L, solanesol 0.2 g/L, CaCl₂ 140 mg/L, FeSO₄·7H₂O 0.2 mg/L, ZnSO₄·7H₂O 8 mg/L and nicotinic acid 8 mg/L. Under the optimum condition, the CoQ₁₀ yield was 28.44 mg/L, while the biomass reached 9.25 g/L.（4） The optimal initial pH, culture temperature, inoculum dosage and culture time were 7.2, 30°C, 8%, and 72 h, respectively. Based on the kinetic analysis of the batch fermentation process, a two-stage agitation speed or dissolved oxygen （DO） control strategy was performed. When the agitation speed was gradationally controlled （300 rpm for 0-36 h and 100 rpm for 36-72 h） or the DO concentration was gradationally controlled （90¹00% for 0-36 h and 30% for 36-72 h）, both CoQ₁₀ production and specific CoQ₁₀ content were promoted. With DO controlled strategy, the maximal CoQ₁₀ yield and specific CoQ₁₀ content reached 56.46 mg/L and 3.94 mg/g-DCW, respectively, which were 11.71% and 6.78% higher than the best result controlled by constant agitation speeds.（5） A kinetic model of CoQ₁₀ production by batch fermentation with PK38 was studied. After observation of experimental data, a model based on the logistic equation of PK38 growth, CoQ₁₀ accumulation combined non-growth-associated and growth-associated contributions, and consumption of sugar for biomass formation and the maintenance of biomass, was developed. The optimal set of parameters was estimated by fitting the model to experimental data. The results predicted by the model were in good agreement with the experimental observations.（6） The metabolic characteristics under three operation ways of fed-batch fermentation, constant feeding rate fermentation, and exponential fed-batch fermentation were studied in a 5-L fermentator. And the influences of different CoQ₁₀ fermentation modes were investigated. The optimum fermentation mode among x them of CoQ₁₀ was exponential feeding fermentation. With this strategy, the final cell biomass, CoQ₁₀ production, and specific CoQ₁₀ production increased 126.11, 173.12, and 22.76% mg/g-DCW, respectively, compared to those of batch culture.更多还原