Fermentation, Purification And Study Of A Low Molecular Factor With Antimicrobial Activity By Fungi

The wildly use of antibiotics in clinic has saved plenty of lives. On the other hand, unreasonable application of antibiotics has caused the rapidly arise of resistant organism, which made some infective deseases to be a tough problem of clinic.Thus, the research and development of new drug which can control resistant pathogens became more essential. A kind of antibacterial peptide named AF2 was purified from the broth of fungi Penicillium sp.M03 which was screended out by state key lab of microbial technology at SDU, and some properties of AF2 was studied.Result of preliminary test showed that, Penicillium sp.M03 had the ability to produce some antibiotics, which can diffuse into culture medium. To increase the output of AF2, the fermentation condition of Penicillium sp.M03 was optimized. Then the best carbon source, nitrogen source, proportion of them, pH, temperature, inoculate quantity and fermental time was determened. Variety of culture medium could be used by Penicillium sp.M03 to produce AF2. Allowed for many factors, The optimum fermental medium of AF2 was as the following: Starch 30g, Na₂HPO₄ 1g, KC1 0.05g, (NH₄)₂SO₄18g, MgSO₄ · 7H₂O 0.5g, FeSO₄ 0.01g, distilled water 1L. The optimum cutrule medium was: Glucose 30g, Na₂HPO₄ lg, KC1 0.05g, (NH₄) 2SO₄ 18g, MgSO₄ . 7H₂O 0.5g, FeSO₄ 0.01g, distilled water 1L. And the optimum culture condition was : pH 6.5, inoculate quantity10%, 30℃ for 5 days.Then Penicillium sp.M03 was fermented by fermentor (BioFlo 410 , NBS). In this condition, the output of AF2 could be increased, and the fermentation time could be reduced. Because the growth of fungi is relatively slow, fermental process of fungi is easy to be infected by other microorganism. The last part of this chapter introduced how to analysis the causation of infection and resolve the problem.Then followed part of this thesis was the purification of AF2. The broth was dealt withfiltration, centrifuging and ultra-filtration. After freeze-dried, the crude AF2 product waspurifided by Sephadex LH-20 molecular sieve chromatography , Sepharose-DEAE FFand Sephadex G-15, a kind of pure component AF2 was got.Structure and molecular-weight of AF2 were studied through UV, ER, NMR and ESI-MS.The results showed: AF2 was a kind of peptides, Its molecular weight was about1155.The data of amino acid analysis showed that AF2 contained twelve kinds of aminoacids: Asp, Glu, Ser, Gly, Arg, Thr, Ala, Pro, Cys, Ile, Leu and Phe. AF2 was stable toβ -lactamase, pH and proteinase, its heat-resistance was also well. It could inhibit thegrowth of SA, MRSA and blue pus organism, thus, it was hopefu; AF2 be used as novelantibiotics to control resistant-bacteria.