Fermentation Media Screening Of Pleuromutilin-producing Strain And Product Research

Pleuromutilin and its derivatives are diterpenes antibiotics produced by Pleurotus mutilus and Pleurotus passeckerianus as well as Clitoilus passeckerianus. They cause bacterial death by binding to bacterial ribosome 50S subunit and then inhibiting protein synthesis. They are used for treating infection of Gram-positive bacteria, mycoplasma and spirochaetes.Pleuromutilin was first isolated and primary identified by Kavanagh, et al in 1951. In 1976, F.Knauseder, et al. investigated the fermentation conditions of pleuromutilin-producing strain, Chemical Structure of pleuromutilin, and biosynthesis pathway primarily. In the later 20 years, investigations were focused on structures and activities of its derivatives to screen drugs with high activity but low toxicity. There are derivatives veterinary antibiotics tiamulin and valnemulin, and human drug retapamulin has been approved by FDA. As a semisynthetic precursor, pleuromutilin shows a wide market requirement, so producing pleuromutilin with high production and purity has it realistic economic value.In this study, a fungi IMB P4-04 preserved by our lab was primarily identified by morphological observation and molecular identification. And the inhibition of its fermentation product had been carried out. HPLC and mass spectrum analyses were used to identify the product. The fermentation medium was screened to optimization, and the purifying condition was investigated.Morphological observation showed that mycelium of IMB P4-04 was septate. Sporophore couldn't be observed on the plate. Identification was also carried out by ITS conserved sequence. The sequenced PCR product was aligned in NCBI. Vector NTI suite9.0 and Clustal X were applied for phylogenetic tree analysis. The results reveal IMB P4-04 has the closest phylogenetic relationship with Basidiomycetes, Clitopilus sp. Clitopilus prunulus.Antimicrobial experiment showed the fermentation product of IMB P4-04 had the inhibiting activities to Staphylococcus aureus, Escherichia coli, Mycobacterium smegmatis, Micrococcus luteus, especially for S. aureus and M. luteus.The carbon and nitrogen single-factor tests results showed that the best carbon source and nitrogen source were flucose and bean powder. Since the cost of flucose is relatively high, so glucose was used in the following experiment. The optimal concentration of glucose and bean powder were 6% and 2%, respectively. Low concentration of FeSO₄ enhanced the production, while in high concentration, the enhancement was decreased. The optimal concentration of FeSO₄ is 0.04%. So screened media was composed with 6%glucose, 2%bean powder, 0.04%FeSO4, 0.04%MgSO₄, 0.4 % bean oil, CaCO₃ 0.1%. Production in this condition was increased by 34.6% than in basic media.Organic solvents with different polar were used to purify product and compare their recover rate and purity. Ethyl acetate had the best product purity and recover rate with purity 77.6% and recover rate 87.8%. It was found that the best pH value was pH7.0-7.5. The effect of extracting volume was remarkable in extracting parameters. The optimal extracting condition was extracting with the equivoluminal ethyl acetate. The extracted product was dissolved in methanol, followed with fractional crystallization. The crystal was harvested with a concentration 93.1%.HPLC analysis showed the product and pleuromutilin standard had the same retention time; the mass spectrum revealed the product had a very similar spectrum character. That the fermentation product is pleuromutilin was identified.