Coenzyme Q₁₀ Prodction By Sphingomonas Sp.ZUTE03 In A Two-phase Conversion System

Coenzyme Q10 (CoQ₁₀) is a prenylated benzoquinone lipid similar to vitamin. CoQ₁₀ is an important transferring-hydrogen carrier in biological respiratory chain and a naturall antioxidant. It has been widely used in the field of medicine,cosmetics and health products.In this paper, CoQ₁₀ production by Sphingomonas sp. ZUTE03 with Solanesol and PHB in two phase conversion system, CoQ₁₀ production by alginate gel-entrapped Sphingomonas sp. ZUTE03 in three-phase fluidized bed reactor, and CoQ₁₀ production by free Sphingomonas sp. ZUTE03 with BHT and LNA, was studied respectively.All these studies were the basis of application of CoQ₁₀ production by Sphingomonas sp. ZUTE03.Firstly, the optimization of CoQ₁₀ producing conditions by free Sphingomonas sp. ZUTE03 with Solanesol and PHB and precursor's concentration for gel-entrapped Sphingomonas sp. ZUTE03 was studied. In detail, we studied the effect of different amounts of soybean oil, different cell concentration, Ca²⁺ and Mg²⁺ on the yield of CoQ₁₀ produced by free cells. At the same time, we immobilized the cells with calcium alginate and studied the effect of PHB and Solanesol concentration on the bioconversion of CoQ₁₀ respectively. A double-factor orthogonal experiment using L9 (3)3 was designed to optimize precursors concentration. Results showed that when we used 2% soybean oil, the yield of CoQ₁₀ was the best; when the concentration of cells in two-phase conversion was 9.6 g/L and 16 g/L, the yield of CoQ₁₀ was 56.96,68.67 mg/L respectively; when we added 40 mmol/L Mg²⁺ and 20 mmol/L Ca²⁺ , the yield of CoQ₁₀ was 58.37,59.82 mg/L respectively. The better precursor's concentration by single factor experiment was 70 mg/L solanesol and 40 mg/L PHB respectively. The optimal precursors concentration by a double-factor orthogonal experiment was 70 mg/L solanesol and 35 mg/L PHB respectively.After 8 h conversion under optimized precursor's concentration, CoQ₁₀ yield reached 52.2 mg/L with conversion rate of 91% and 89.6% (on PHB and solanesol, respectively).We also studied CoQ₁₀ production by gel-entrapped Sphingomonas sp. ZUTE03 in a three-phase fluidized bed reactor. Single factor experiment showed the optimal conditions as follows: Ventilation volume was 1.2 L/min, optimal ratio of immobilized beads and conversion system was 75(mL):200(mL), and the optimal conversion temperature and time were 30℃and 8 h. Under the optimal conversion conditions, yield of CoQ₁₀ was 47.0 mg/L. Even after 15 repeats with 8 h one batch, the yield of CoQ₁₀ kept at a high level of about 45 mg/L. After reacting for 10 h, we reused the residual PHB in the water phase, changed the organic phase with new one which contained 70 mg/L Solanesol and continued the reaction with 8 h per batch until PHB was run out. The yield of CoQ₁₀ was about 43 mg/L each batch. After reacting for 10 h, we reused the residual PHB in the water phase, fed the solanesol that was used in each batch (8 h per batch) and continued the reaction until PHB was run out, the yield of CoQ₁₀ was up to 171.52 mg/L in the organic phase. After reacting for 10 h, we continued the reaction and fed solanesol and PHB to coversion system after each batch (8 h per batch). After 20 repeats, the yield of CoQ₁₀ was up to 441.65 mg/L in the organic phase.At last, we studied CoQ₁₀ production by Sphingomonas sp. ZUTE03 with BHT and LNA respectively and their combination with Solanesol, PHB as prucursors.We determined the most effective precursors, the optimal coversion time, conversion temperature, aqueous phase pH and the concentration of precursors by the yield of CoQ₁₀ and conversion rate. The results showed that most effective combination of precursors were BHT and solanesol,the optimal conversion time and temperature were 8 h and 37℃, the organic phase was hexane,water phase was acetate buffer(0.2 M, pH 4.6).Under the optimal conditions, the yield of CoQ₁₀ was 75.81 mg/L. Both single factors experiment and double-factor orthogonal experiment showed that 70 mg/L solanesol with 30 mg/L BHT was the optimal concentration for CoQ₁₀ conversion by Sphingomonas sp. ZUTE03 in the two-phase conversion system. After 8 h conversion under optimized precursor's concentration, CoQ₁₀ yield reached 76.1 mg/L with conversion rate of 92.6% and 92.4% (on PHB and solanesol, respectively).