能产辅酶Q_（10）的土壤根瘤杆菌菌株选育及其发酵条件优化
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摘要
辅酶Q_(10)(Ubidecarenone，conezyme Q_(10))，又名泛醌，是呼吸链中一种重要的递氢体。辅酶Q_(10)具有抗氧化、防衰老、提高机体免疫力等功能，被广泛用于各类心脏病，高血压、肝炎等疾病的治疗。本文对产辅酶Q_(10)的菌种选育和发酵过程进行了初步研究。
研究了辅酶Q_(10)的定量分析方法：Craven试验显色法和高效液相色谱法(HPLC)。将Craven反应的溶剂改进为丙酮，使该反应的灵敏度提高了近50％。该方法特异性强，操作简单，可在短时问内测定大批样品；确立了HPLC法测定辅酶Q_(10)条件和相应标准曲线，测定方法准确可靠。
研究了可以批量提取样品中辅酶Q_(10)的化学破胞方法，工艺路线为EDTA预处理→NaOH作用破胞→丙酮抽提萃取。通过Plackett-Burman设计试验和最终优化实验得到较佳的提取工艺条件：0.1mol／L EDTA作用温度为30℃，1ml发酵液中2mol／L NaOH用量和生物量的关系为V=4.335m，R~2=0.971，其作用时间和温度分别为50min和52℃。与碱醇皂化、高压匀浆等方法提取结果比较，化学破胞方法因其条件温和、破胞彻底而提取效果更好。
以土壤根瘤杆菌AT-hh01为出发菌株，确定了紫外诱变的最佳条件：30W紫外灯，诱变距离为65cm，照射时间为20s；~(60)Co_γ射线诱变的较佳诱变剂量为300 Gy。经过多轮γ射线和紫外诱变，在亮氨酸和D—半乳糖的复合筛选平板中筛选得到了一株突变菌株AT-hh1201-4，辅酶Q_(10)产量为27.2mg／L，较出发菌株AT-hh01提高了51.96％，经过传代10次，证明其遗传性状稳定。
对土壤根瘤杆菌AT-hh1201-4的摇瓶培养及制备辅酶Q_(10)过程进行了优化。通过考察不同的碳源、氮源对辅酶Q_(10)产量的影响，确定了较好的碳源为葡萄糖和蔗糖，氮源为酵母膏和(NH_4)_2SO_4。以均匀试验设计对Plackett-Burman设计试验筛选所得关键因素一酵母膏、(NH_4)_2SO_4和种龄作了进一步优化，采用二次多项式逐步回归的方法对结果进行拟合，获得优化化条件为：酵母膏46.2g／L，(NH_4)_2SO_425.4g／L，种龄为24h，经实验验证，回归拟合结果较好。经优化后辅酶Q_(10)的平均产量为32.24mg／L，比初始的27.2mg／L提高了18.53％。
考察了发酵过程中溶氧对辅酶Q_(10)产量的影响。将发酵培养过程分成两个
阶段对溶氧条件进行考察，第一阶段获得以最大生长速率为目标的通气条件，第二阶段为在第一阶段培养结束后改变通气条件以进一步提高辅酶Q_(10)产量。以正交试验确定第一阶段培养条件为：装液量25ml／250ml，转速210rpm和摇瓶形状为C_3；以二次饱和D—最优设计试验得到第二阶段培养条件为：在第一阶段培养到104h时将转速调为180rpm。经过对溶氧条件的优化，发酵液中辅酶Q_(10)产量达到38.22mg／L，比优化前提高了18.5％。
Ubidecarenone (coenzyme Q_(10), Ubiquinone) is an important electron transfer conmponent in respiratory chain. Coenzyme Q_(10) exhibitating excellent medical and physiological functions such as antioxidant, prevention of senility and boost of immunity is widely used in the therapy on several heart diseases, hypertension, hepatitis and so on. In this paper, coenzyme Q_(10)-producing strain breeding and fermentation conditions optimization were studied.
Methods for quantitative determination of coenzyme Q_(10) were studed: Craven test and High Performance Liquid Chromatography (HPLC). Replacing the solvent of coenzyme Q_(10) with acetone in Craven test, the sensitivity of the test was improved by about 50%. The Craven test was of high specificity, easy to operate and able to deal with great number of samples in short time. Then the determination condition of coenzyme Q_(10) with HPLC and relevant standard curve were determined. The advantage of assay by HPLC was accurate and reliable.
Chemical disruption which could extract coenzyme Q_(10) in batch was studied. The process route was as follows: treatment of EDTA → treatment of NaOH → extraction with acetone. The optimum technology for extraction by chemical disruption was got by Plackett-Burman design experiment and final optimization experiment as follows: temperature of treatment with 0.1mol/L EDTA was 30°C; the relationship between 2mol/L NaOH and biomass in 1 ml fermentation broth
was V = 4.335m, R~2 = 0.971; the time and temperature of treatment of NaOH were
50min and 52°C, respectively. Compared with Saponification with alkali and methanol and high pressure homogenizer, the result of extraction by chemical disruption was better because of its mild and thorough treatments.
Selected AT-hh01 as the origin strain, the optimum technology for ultraviolet mutation was selected as follows: UV lamp 30W, distance and time of radiation 65cm and 20s; optimum dosage for ~(60)Coγ ray mutation 300Gy. With several circles of mutagenesis treatments and screening, a mutant strain AT-hh1201-4 was bred on the
composite plate of leucine and D- Glactose. Its yield of coenzyme Q_(10) was 27.2mg/L, which was increased by 51.96% compared with that of the origin strain. Its genetic character was proved to be stable after ten passages.
Optimization of fermentation conditions of coenzyme Q_(10) produced by AT-hhl201-4 was studied in shake flask cultures. By studying on difference of yield of coenzyme Q_(10) in different nitrogen and carbon sources, better nitrogen and carbon sources were glucose, sucrose and yeast extract, (NH_4)_2SO_4. Employed Uniform Design to optimize the significant parameters got by Plackett-Burman Design, fitted with Duality Quadratic Polynomial Stepwise Regression, the results of the optimization were yeast extract 46.2g/L, (NH_4)_2SO_4 25.4g/L, age of inoculum 24h. Verification experiment showed that the optimized condition leaded the average yield of coenzyme Q_(10) to be increased by 18.53% from 27.2mg/L to 32.24mg/L.
The influence of dissolve oxygen to the yield of coenzyme Q_(10) in flask culture was studied. The process of fermentation was divided into two stages: one achievement of aeration conditions for the biggest growth rate, the other changing aeration conditions following the first stage to further improve the yield of coenzyme Q_(10). The optimal culture conditions were determined by Orthogonal experiment as follows: liquid volume 25ml/250ml, rotate speed 210rpm and flask shape straight bottom with wider bottleneck. By Second Saturating D-Optimal Design the conditions for the second stage was set as adjusting rotate speed to 180rpm at 104h of fermentation. After optimization of dissolve oxygen in flask fermentation, the yield of coenzyme Q_(10) was 38.22mg/L increased by 18.5% compared with that not being optimized.
研究了辅酶Q_(10)的定量分析方法：Craven试验显色法和高效液相色谱法(HPLC)。将Craven反应的溶剂改进为丙酮，使该反应的灵敏度提高了近50％。该方法特异性强，操作简单，可在短时问内测定大批样品；确立了HPLC法测定辅酶Q_(10)条件和相应标准曲线，测定方法准确可靠。
研究了可以批量提取样品中辅酶Q_(10)的化学破胞方法，工艺路线为EDTA预处理→NaOH作用破胞→丙酮抽提萃取。通过Plackett-Burman设计试验和最终优化实验得到较佳的提取工艺条件：0.1mol／L EDTA作用温度为30℃，1ml发酵液中2mol／L NaOH用量和生物量的关系为V=4.335m，R~2=0.971，其作用时间和温度分别为50min和52℃。与碱醇皂化、高压匀浆等方法提取结果比较，化学破胞方法因其条件温和、破胞彻底而提取效果更好。
以土壤根瘤杆菌AT-hh01为出发菌株，确定了紫外诱变的最佳条件：30W紫外灯，诱变距离为65cm，照射时间为20s；~(60)Co_γ射线诱变的较佳诱变剂量为300 Gy。经过多轮γ射线和紫外诱变，在亮氨酸和D—半乳糖的复合筛选平板中筛选得到了一株突变菌株AT-hh1201-4，辅酶Q_(10)产量为27.2mg／L，较出发菌株AT-hh01提高了51.96％，经过传代10次，证明其遗传性状稳定。
对土壤根瘤杆菌AT-hh1201-4的摇瓶培养及制备辅酶Q_(10)过程进行了优化。通过考察不同的碳源、氮源对辅酶Q_(10)产量的影响，确定了较好的碳源为葡萄糖和蔗糖，氮源为酵母膏和(NH_4)_2SO_4。以均匀试验设计对Plackett-Burman设计试验筛选所得关键因素一酵母膏、(NH_4)_2SO_4和种龄作了进一步优化，采用二次多项式逐步回归的方法对结果进行拟合，获得优化化条件为：酵母膏46.2g／L，(NH_4)_2SO_425.4g／L，种龄为24h，经实验验证，回归拟合结果较好。经优化后辅酶Q_(10)的平均产量为32.24mg／L，比初始的27.2mg／L提高了18.53％。
考察了发酵过程中溶氧对辅酶Q_(10)产量的影响。将发酵培养过程分成两个
阶段对溶氧条件进行考察，第一阶段获得以最大生长速率为目标的通气条件，第二阶段为在第一阶段培养结束后改变通气条件以进一步提高辅酶Q_(10)产量。以正交试验确定第一阶段培养条件为：装液量25ml／250ml，转速210rpm和摇瓶形状为C_3；以二次饱和D—最优设计试验得到第二阶段培养条件为：在第一阶段培养到104h时将转速调为180rpm。经过对溶氧条件的优化，发酵液中辅酶Q_(10)产量达到38.22mg／L，比优化前提高了18.5％。
Ubidecarenone (coenzyme Q_(10), Ubiquinone) is an important electron transfer conmponent in respiratory chain. Coenzyme Q_(10) exhibitating excellent medical and physiological functions such as antioxidant, prevention of senility and boost of immunity is widely used in the therapy on several heart diseases, hypertension, hepatitis and so on. In this paper, coenzyme Q_(10)-producing strain breeding and fermentation conditions optimization were studied.
Methods for quantitative determination of coenzyme Q_(10) were studed: Craven test and High Performance Liquid Chromatography (HPLC). Replacing the solvent of coenzyme Q_(10) with acetone in Craven test, the sensitivity of the test was improved by about 50%. The Craven test was of high specificity, easy to operate and able to deal with great number of samples in short time. Then the determination condition of coenzyme Q_(10) with HPLC and relevant standard curve were determined. The advantage of assay by HPLC was accurate and reliable.
Chemical disruption which could extract coenzyme Q_(10) in batch was studied. The process route was as follows: treatment of EDTA → treatment of NaOH → extraction with acetone. The optimum technology for extraction by chemical disruption was got by Plackett-Burman design experiment and final optimization experiment as follows: temperature of treatment with 0.1mol/L EDTA was 30°C; the relationship between 2mol/L NaOH and biomass in 1 ml fermentation broth
was V = 4.335m, R~2 = 0.971; the time and temperature of treatment of NaOH were
50min and 52°C, respectively. Compared with Saponification with alkali and methanol and high pressure homogenizer, the result of extraction by chemical disruption was better because of its mild and thorough treatments.
Selected AT-hh01 as the origin strain, the optimum technology for ultraviolet mutation was selected as follows: UV lamp 30W, distance and time of radiation 65cm and 20s; optimum dosage for ~(60)Coγ ray mutation 300Gy. With several circles of mutagenesis treatments and screening, a mutant strain AT-hh1201-4 was bred on the
composite plate of leucine and D- Glactose. Its yield of coenzyme Q_(10) was 27.2mg/L, which was increased by 51.96% compared with that of the origin strain. Its genetic character was proved to be stable after ten passages.
Optimization of fermentation conditions of coenzyme Q_(10) produced by AT-hhl201-4 was studied in shake flask cultures. By studying on difference of yield of coenzyme Q_(10) in different nitrogen and carbon sources, better nitrogen and carbon sources were glucose, sucrose and yeast extract, (NH_4)_2SO_4. Employed Uniform Design to optimize the significant parameters got by Plackett-Burman Design, fitted with Duality Quadratic Polynomial Stepwise Regression, the results of the optimization were yeast extract 46.2g/L, (NH_4)_2SO_4 25.4g/L, age of inoculum 24h. Verification experiment showed that the optimized condition leaded the average yield of coenzyme Q_(10) to be increased by 18.53% from 27.2mg/L to 32.24mg/L.
The influence of dissolve oxygen to the yield of coenzyme Q_(10) in flask culture was studied. The process of fermentation was divided into two stages: one achievement of aeration conditions for the biggest growth rate, the other changing aeration conditions following the first stage to further improve the yield of coenzyme Q_(10). The optimal culture conditions were determined by Orthogonal experiment as follows: liquid volume 25ml/250ml, rotate speed 210rpm and flask shape straight bottom with wider bottleneck. By Second Saturating D-Optimal Design the conditions for the second stage was set as adjusting rotate speed to 180rpm at 104h of fermentation. After optimization of dissolve oxygen in flask fermentation, the yield of coenzyme Q_(10) was 38.22mg/L increased by 18.5% compared with that not being optimized.
引文
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