Breeding Of Coenzyme Q₁₀-Producing Agrobacterium Tumefaciens And Optimization Of Fermentation Conditions

Ubidecarenone (coenzyme Q₁₀, Ubiquinone) is an important electron transfer conmponent in respiratory chain. Coenzyme Q₁₀ 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 Q₁₀-producing strain breeding and fermentation conditions optimization were studied.Methods for quantitative determination of coenzyme Q₁₀ were studed: Craven test and High Performance Liquid Chromatography (HPLC). Replacing the solvent of coenzyme Q₁₀ 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 Q₁₀ with HPLC and relevant standard curve were determined. The advantage of assay by HPLC was accurate and reliable.Chemical disruption which could extract coenzyme Q₁₀ 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 brothwas V = 4.335m, R² = 0.971; the time and temperature of treatment of NaOH were50min 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 ⁶⁰Coγ ray mutation 300Gy. With several circles of mutagenesis treatments and screening, a mutant strain AT-hh1201-4 was bred on thecomposite plate of leucine and D- Glactose. Its yield of coenzyme Q₁₀ 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 Q₁₀ produced by AT-hhl201-4 was studied in shake flask cultures. By studying on difference of yield of coenzyme Q₁₀ in different nitrogen and carbon sources, better nitrogen and carbon sources were glucose, sucrose and yeast extract, (NH₄)₂SO₄. 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, (NH₄)₂SO₄ 25.4g/L, age of inoculum 24h. Verification experiment showed that the optimized condition leaded the average yield of coenzyme Q₁₀ to be increased by 18.53% from 27.2mg/L to 32.24mg/L.The influence of dissolve oxygen to the yield of coenzyme Q₁₀ 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 Q₁₀. 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 Q₁₀ was 38.22mg/L increased by 18.5% compared with that not being optimized.