| The coenzyme Q10 is a prenylated benzoquinone lipid, and it is also named ubiquinone. The chemical name is 2,3-dimethoxy-5-methy-6-decaprenyl-1,4-benzoquinone. Members of the coenzyme Q10 are found in microorganisms, plants, and the heart, kidney, liver of animals, where they function are redox carriers in mitochondria or in association with respiratory processes of electron transport chains. It is an antioxidant produced naturally in the cells. Coenzyme Q10 exhibited excellent medical and physiological activities against various diseases such as heart diseases, cancer, hepatitis, Parkinson and so on. Microganisms are good source of coenzyme Q10 homologs. In this paper, we studied the extraction procedure and the fermentation condition of coenzyme Q10 from Agrobacterium tumefaciens to provide basic theory and make experimental instruction for further extending coenzyme Q10 production. The main research contents and results are as follows:(1) The quantitative assays for coenzyme Q10 by ultraviolet spectroscopy method, visible spectroscopy method and HPLC method were compared. The best chromatographic conditions are mixture of anhydrous ethanol and methanol (9/1, V/V) as mobile phase with flow rate being 1.0mL/min in column Hypersil ODS (5μm, 4.6mm×250mm, Germany) and UV detection at 275nm was used.The ultraviolet spectroscopy method, which was simple, economic and accurate was proposed to replace the HPLC method in the research of breeding of high producing strain and optimization of cultivation conditions.(2) In this paper, the effects of grinding method, freezing and melting repeatedly method, acid-heat method and ultrasonic method on cell-wall disruption and coenzyme Q10 extraction were studied. The optimum conditions of each disruption method were determined. The saponification technology and the extraction condition were also studied. The optimum extraction procedure for coenzyme Q10 was as follows: The collected cells were suspended into 10mL/g(DCW) of 3mol/L aqueous solution of hydrochloric acid, after 30 minutes of heat treatment at 90℃, cooling immediately, 2.5 times (V:V) of 10% aqueous solution of potassium hydroxide- methanol was added, followed by further 30 minutes of heat treatment at 60℃. Thus treated mixture was extracted by petroleum aether, 40 times (V/m) of biomass repeated 3 times. The organic solvent phase was separated and evaporated to dryness under reduced pressure in a rotary evaporator. The residue was dissolved in 50mL anhydrous ethanol.(3) Using Agrobacterium tumefaciens AT-01 as starting strain, mutagenized with UV and DES, screened by selecting mutants resisting structure analogue Vk3 and respiration chain inhibitor sodium azide, directly on gradient plate, a mutant AT- N10 overproducing coenzyme Q10 was obtained, which has much higher productive qualities than its parental strain. The coenzyme Q10 productivity of the mutant reached 5.854mg/L, which was 71.43% higher than the parent strain, and the coenzyme Q10 content in the dried cells reached 1.744mg/g, which was 47.05% higher than the parent strain. A reduction of only 2.61% in the coenzyme Q10 yield of the mutant was observed after five times of subculture. Therefore, the obtained mutant is stable strain that is worthwhile to be studied further.(4) The optimizing medium for coenzyme Q10 biosynthesis was formulated vas Plackett -Burman design and response surface analysis method (RSM). The optimum fermentation medium consisted of glucose 34g/L, yeast extract 10.40g/L, peptone 10g/L, FeSO4·7H2O 9.8mg/L, PHB 44.3mg/L(adding after 24 hours culture), K2HPO4 1.0g/L, KH2PO4 1.0g/L, MgSO4·7H2O 0.3g/L, MnSO4·H2O 50mg/L, ZnSO4·7H2O 10mg/L, biotin 100μg/L, nicotinic acid 50mg/L, inositol 20mg/L. Otherwise, the best fermentation condition was obtained based on the single factor test, which was initial pH 7.0, 6% amount of inoculation, 50mL fermention culture medium, fermentation time 96h, culture temperature 28℃. The coenzyme Q10 yield was 17.195mg/L under the optimum condition, which was 193.73% higher than that of non-optimized medium.(5) Based on the optimal fermentation media, shaking-flask fermentation dynamics model was constructed. A simple model was proposed using the Logistic equation for growth, the Luedeking-Piret equation for coenzyme Q10 biosynthesis and the Luedeking-Piret like equation for glucose consumption. The kinetic model provided a reasonable description for biomass, product and substrate consumption variation along with the fermentation process.
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