Multiple Resistance. Mycophenolic Acid Producing Strain Screening And Fermentation Process

Mycophenolic Acid is an antibiotic with hundred of years' history and a new indication has been founded in recent years as immunosuppressant. Mycophenolate Mofetil (the 2-morpholinoethyl ester of Mycophenolic Acid), the precursor and active metabolite of MMF in vivo, is now being widely used in clinical therapy of organ transplant, especially in prevention of acute allograft rejection in renal transplant recipients. In this paper, the strain-improving, extraction and purification as well as the biosynthesis and metabolism regulation of Mycophenolic Acid were discussed by using the producer-Ad-2 as a starting strain.First of all, Mycophenolic Acid can be biosynthesized by many Funguses. Investigation of different dosage UV mutagen was used to change the starting strain's gene. The screening models of tolerance to SDBS, Simvastadin, Clofibrate and Mycophenolic Acid were established. Confirmed by flask liquid fermentation, the result shows high-yield mutant strain can produce Mycophenolic Acid in the fermentation broth as 2.5 times as the starting strain.Secondly, Investigation of fermentation method leads to the following results: the optimized conditions of fermentation temperature(26℃), fermentation period(8d), seed age(96h), fermentation volume(50ml in 250ml shaking flask), pH4.0 before sterilization. The metabolic curve of fermentation was drawn. Based on the experiment of shake flask, submerged fermentation was investigated. In additional, the resting fermentation is proved to an affective to produce Mycophenolic Acid.In this paper, the simple and accurate methods for determination, extraction and purification of MPA in the fermentation broth were developed at the same time. The RP-HPLC method for the quantification of Mycophenolic Acid (MPA) in the fermentation broth was established. The determination was performed on a SinoChrom ODS-BP column(200 mm×4.6 mm, 5μm) using methanol-10 mmol·L^-1 ammonium acetate (67:33) as the mobile phase at a flow rate of 1.0 ml/min, with a UV detector setting at 220 nm. The calibration curves of MPA was linear over the range of 0.30-6.00 mg·mL^-1 (r= 0.999 7, n = 6) and the mean recoveries was 102.4% (RSD =1.7%). The method is accurate, simple with good reproducibility, which is suitable for the determination of Mycophenolic Acid in fermentation broth.Subsequently, the present study provides the systematic methods for biosynthesizing MPA, which could successfully amplify the yield of MPA both in laboratory and industrialized production.