| Trisporic acid (TA) can stimulate P-carotene biosynthesis in Blakeslea trisopora. And it is a byproduct of this fermentation process can be extracted from the fermentation broth, returned to the broth to increase the production of P-carotene. Thus it is necessary to establish a simple and economical method to obtain large quantities of high purified TA for the purpose on the industrial production of P-carotene. Recently, the mechanism of TA is not clear, and more and more people pay attention to this study.In this dissertation, TA was analyzed by a Hitachi HPLC system, consisting L-7420 tunable absorbance detector, Hitachi model L-7100 pumps and a 5μm (250×4.6 mm) reversed-phase C18 column (DiamodsilTM). The mobile phase was methanol-water system containing 0.2% trifluoroacetic acid. The solvent system consisted of 50% methanol in water, and changed linearly over 35 min to 70% methanol to purge the column. Column oven temperature was set at 30℃. The flow rate was 1 ml/min, and 10μl sample was injected into the column. TA was detected by UV 325 nm. Results showed that was a prompt and concise method for quantification of TA concentration.In this dissertation, separation of TA by s liquid-liquid extraction was performed. And the purity of products was only 30%. After repeated the extraction steps between NaHCO3 and chloroforms, slightly higher purity of products can be obtained, but the purity was not more than 40%.In this dissertation, separation of TA by the Sephadex LH-20 chromatography was performed. The optimal conditions were as follow: the mobile phase was 40% methanol in water, the flow rate was 2 ml/min, and the purity of TA was 70%.In this dissertation, separation of TA by silica gel column chromatography was performed. The optimal conditions were as follow: the mobile phase condition was chloroform:ethyl acetate:petroleum ether:acetic acid (v:v:v:v)= 14:3:5:0.5, the loading amount should be less than 100 mg, and the purity of products was over 60%.In this dissertation, the performance of TA on macroporous resins was evaluated according to their adsorption and desorption properties. SP850 resin showed the best adsorption and desorption capacity for TA than other resins, and its adsorption data fitted well to the Freundlich and Langmuir isotherm. To optimize the separation process of TA from the fermentation broth, dynamic adsorption and desorption experiments were conducted with the column packed with SP850 resin. The optimal conditions were:the concentration of TA in the sample solution 15.30μg/ml, the sample solution pH 2, flow rate 6 BV/h, temperature 25℃, the processing volume 30BV. The gradient elution program was as follows:elution solvent ethanol-water (30:70, v/v) 3 BV then ethanol-water (60:40, v/v) 8 BV, flow rate 9 BV/h. After the SP850 resin treatment, the purity of TA was 70%.Then high purity of TA was obtained by preparative HPLC. The mobile phase was 55% methanol in water at the flow rate of 10 ml/min, and the loading amount was limited to less than 20 mg. The product was confirmed by MS and NMR.In this dissertation, TA,β-ionone and abscisic acid were added into the minus cultures of Blakeslea trispora to increase the production ofβ-carotene. When the concentration of TA, abscisic acid andβ-ionone were 9.10μM,0.909μM and 50μM, the productions of P-carotene were increased by 70.4%,43.4% and 47.2%, respectively.The effects of TA, abscisic acid andβ-ionone on two key genes carRA and carB transcript levels were investigated. The effects on carRA and carB genes transcript levels after the addition of TA were most significant, and the mRNA level of carRA and carB were up to the maxium at 6h and 3h,3.24-fold for carRA and 9.93-fold for carB. The mRNA level of carRA and carB after the addition of abscisic acid were both up to the maxium at 3h,1.97-fold for carRA and 7.58-fold for carB. The mRNA level of carRA and carB after the addition ofβ-ionone were up to the maxium at 6h and 3h,2.58-fold for carRA and 6.10-fold for carB. |
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