Studies On The Co-production Of S-adenosylmethionine And Glutathione By Candida. Utilis

S-adenosylmethionine (SAM) and glutathione (GSH) are both important small molecular sulfur substances in vivo. SAM acts roles of methyl, sulfur and aminopropyl transfers while GSH significantly performed in the maintenance of the redox balance in cells. SAM and GSH were widely used in the fields of medicine, cosmetics and health products.In this thesis, Candida utilis was firstly used for the study of SAM and GSH co-production in domestic. The study was carried out in three major aspects: mutagenesis and screening for the positive mutant of SAM and GSH co-production; optimization of medium composition and culture conditions for the mutant by using statistical methods; exploration of the optimal conditions for the co-production on a 5 L fermentor level. The main conclusions obtained were as follows:1. The parent strain C. utilis SZU 07-01 was treated by two turns of UV and onceγ-ray irradiation for complex mutagenesis, following by cultivating and screening on the selective medium (YPD containing 1 g/L L-Met, 0.5 g/L ethionine and 2 g/L NaHSO₃) for a positive mutant C. utilisγ21, which was lately collected and numbered as CCTCC M 209298. The proliferation of C. utilis CCTCC M 209298 was then carried out in flasks, and the productions of S-adenosyl-L-methionine and glutathione were 324.0 mg/L and 220.5 mg/L, respectively, which were 131.4% and 26.0% higher than those of C. utilis SZU 07-01. In addition, it shows high genetic stability even after 10 generations of cultivation, which indicates that the mutant C. utilis CCTCC M 209298 obtained in this study has stable capacity for high co-production of SAM and GSH, and can be used as industrial strain in the near future. 2. Result of RSM tells that the optimal medium for SAM and GSH co-production was as follow (g/L): sucrose 35.4, (NH₄)₂SO₄ 10, KH₂PO₄ 12.3, MgSO₄·7H₂O 0.05, CaCl₂ 0.05, L-methionine 4.6. The validation results showed that the co-production of SAM and GSH reached 589.3 mg/L. Furthermore, orthogonal experimental results showed that the optimal conditions for the co-production of SAM and GSH were: 33℃, pH 6.5 with a volume rate of 40 mL/500 mL at a 200 rpm flask shaker. Finally, the co-production of SAM and GSH reached 630 mg/L, which was 15.7% increased compared with that before the optimization.3. A series of batch fermentation experiments were carried out in a 5 L Minifors fermentor, and the results showed that the optimal conditions were as follows: 30℃, pH 5.0, stirring speed 450 rpm, aeration 1 vvm. At the optimized conditions, co-production of SAM and GSH reached 598.7 mg/L. Then, the contradictions of SAM and GSH co-production countered in the batch fermentations were tried to be analyzed.