Study On Screening Of High Yield Coenzyme Q₁₀ Strain

Coenzyme Q₁₀ also known as Ubiquinone 10 is a kind of fat-soluble quinone. Coenzyme Q₁₀ is an important biochemical compound and has been widely uesd in medical domain, food domain and cosmetics domain. In this paper, Rhizobium leguminosarum 1.1723 was used as an original strain and treated with UV-LiCl and Ultrasonic mutation in order to get a high yield strain. Then we studied the fermentation technology of coenzyme Q₁₀ of the strain. The main results were obtained as following:（1）The alkali saponification method was chosen to extract Coenzyme Q₁₀. The best conditions of alkali saponification were as fllows: The collected cells were suspended into 50mL/g（DWC） of 10% of KOH-CH3OH and 0.67g /g（DWC） of pyrogallic acid, then distilled at 90℃for 30 minutes. Finally, treated mixture was extracted by petroleum aether, 50 mL/g of biomass repeated 2 times. In the best conditon, the Coenzyme Q₁₀ content of Rhizobium leguminosarum 1.1723 was 0.389mg/g .（2） Using Rhizobium leguminosarum 1.1723 as starting strain, mutagenized with UV-LiCl and Ultrasonic. Results showed that 0.1% solution of LiCl could increase the positive mutation frequency and ultrasonic could change cell wall structure of the strain. A mutant C40-05 over producing coenzyme Q₁₀ was obtained through preliminary screening of Roxithromycin and re-screening of shaking flask fermentation, and its coenzyme Q₁₀ yield was 1.198mg/g, which was 208% higher than the original stain. A reduction of only 2.69% in the coenzyme Q₁₀ yield of the mutant was observed after five times of subculture. Therefore, the obtained mutant was a stable strain that was worthwhile to be studied further.（3）The fermentation medium of C40-05 was optimized for Coenzyme Q₁₀ production using signal factor experiment and Plackett-Burman design. Results showed that CaCl2, solanesol and carrot juice were the main influential factors. The optimum fermentation medium were: tryptone 10g/L, yeast extract 9.0 g/L, CaCl2 0.3g/L, carrot juice 13.53ml/L, solanesol 0.14g/L. The coenzyme Q₁₀ yield was 5.617mg/L under the optimum condition, which was 1.2 times than that of non-optimized medium.（4） The fermentation condition of C40-05 was optimized for Coenzyme Q₁₀ production using signal factor experiment and orthogonal experiment. The suitable fermentation medium of C40-05 was: the volume of medium was 12mL in 250 mL flask volume, initial pH 7.0, temperature 34℃, rotation speed 220r/min, inoculum size 3%. The coenzyme Q₁₀ yield was 28.166mg/L under the optimum condition, which was 4 times than that of non-optimized medium.