Study On The Fermentation Production Of Coenzyme Q₁₀ By Photosynthetic Bacteria-Rhodopseudomonas Sphaeroides

The production of coenzyme Q₁₀ by microbe fermentation is the most potential method to be industrialized. Recently, coenzyme Q₁₀ has been used as not only medicine but also food supplements because of its physiological activities. Photosynthetic bacterial cells contain large amounts of coenzyme Q₁₀, so Rhodopseudomonas sphaeroides 1.1737T was used as an original strain in this paper. R. sphaeroides 1.1737T was mutagenized by using UV radiation and NTG mutation. A highly productive mutant R. sphaeroides PHBr-2 was obtained by rational screening with the roxithromycin and precursor resistant. The coenzyme Q₁₀ production by mutant PHBr-2 can reach 50.6mg/L. After five times of subculture, the coenzyme Q₁₀ production by mutant PHBr-2 is 49.5mg/L, which is 202.38% higher than the original strain. It has shown that this strain has great potential for the coenzyme Q₁₀ production.Coenzyme Q₁₀ was determined at 275nm under the UV method and the alkali saponification method was chosen. The extraction of coenzyme Q₁₀ was carried out as follows. The derived fermentation broth was subjected to centrifugation to collect the cells. After washing, 5mL of acetone was added and the cells were crashed under ultrasonic icy conditions. The cell debris was removed to the flask and added 10mL of acid water with pH3.0 and distilled at 90℃for one hour. Then 15mL of 10% NaOH solution was added and continued to distill for another one hour. Finally, 25mL of hexane was used to extract coenzyme Q₁₀. The organic phase was combined and concentrated to dryness. Then this extract was redissolved in 10mL of ethanol absolute.It was indicated that the trace elements Mg2+,Fe2+and Mn2+ play an important role in the coenzyme Q₁₀ production. Orthogonal experiments show that optimal culture medium was 1g/L of sodium acetate anhydrous, 2g/L of sodium citrate, 3g/L of peptone, 3g/L of yeast extract, 7g/L (NH₄)₂SO₄, 3g/L of KH2PO4, 2g/L of sodium chloride, 1g/L of NaHCO3 and 1mL/L of trace element solution and vitamin solution respectively. The fermentation medium was adjusted to pH 6.5 and cultivation was carried out under the temperature 30℃, inoculation at 4% and illuminate intensity at 3000Lx for 96h. Under these conditions, the coenzyme Q₁₀ production by R. sphaeroides PHBr-2 reach 74.4mg/L, which is 50.3% higher than the original conditions.The coenzyme Q₁₀ extract was separated and purified through silica gel column chromatography. In this method, the silica with 80-120 mesh was used as stationary phase, and the solution of hexane-ethanol absolute (8:2) was used as eluant. The partially purified coenzyme Q₁₀ was redissolved in small amount of ethanol absolute and recrystalized by gradually cooling down to 4℃. The yield of the coenzyme Q₁₀ was 77.08%.Under the different culture modes, it has shown that inoculated fermentation culture was cultivated at 30℃, with the rotation of 150rpm for 24h, and then cultivated at 30℃, 3000Lx illumination intensity under stationary conditions. Finally, the coenzyme Q₁₀ production can reach 78.36mg/L. By observing changes of dissolved oxygen and pH during cultivation, it has indicated that dissolved oxygen plays an important role in coenzyme Q₁₀ production. Therefore, coenzyme Q₁₀ production can be increased through changing culture modes and controlling the dissolved oxygen.