Studies On Enopeptin,A Depsipeptide Leading Compound Derived From Microorganisms

Bacterial resistance has become a global problem.The human face the threat of the "post-antibiotic age".Depsipeptides are nature polypeptides in which one or more amino acid is replaced by hydroxy acids,resulting in the formation of the ester bond.They possess various biological properties,antiviral,antibacterial,anti-cancer,antithrombotic and antimalarial.The acyldepsipeptide(?-cyldepsipetide,ADEP),a new antimicrobial peptide,has a unique resistance to resistant bacteria.It's target is highly conserved caseinolytic kinase(caseinolytic kinase,ClpP).By integrating with ClpP.By integrating with ClpP,ADEP makes the hydrolysis of bacterial proteins out of control and leads to the death of bacteria.Enopeptin A is a member of ADEP family with the resistance to gram positive bacteria and gram negative bacteria including MRSA.In recent years,scientists have modified the structure of Enopeptin through chemical synthesis and the antibacterial effect has greatly improved.In this paper,the techniques of fermentation,separation and purification of the Enopeptin A were studied to lay a foundation for further research of a new drug that has the double effect of anti-virus and anti drug resistant bacteria.This research has some original and practical value for the study of Enopeptin A.In this paper,the UV-Vis spectropHotometric and HPLC analysis method was established for the Enopeptin A.The Enopeptin A was detected by UV-Vis spectrophotometric in UV absorption at 375 nm.The HPLC anlysis method used column Hypersil ODS2(250×4.6 mm,5 ?m)with Acetonitrile-1‰ formic acid water(50:50)as a mobile phase with the flow rate of 1.0 m L/min.The detection UV wavelength was at 375 nm.The column temperature was 40?.The injection volume was 20 ?L.In this paper,the original strain SIIA-H7264(110 ?g / mL)was mutated by UV,NTG,EMS and atmospheric temperature plasma.A high yield and good genetic stability strain A-21 was obtained with a titer of 440 ?g/m L.The optimum fermentation medium was obtained by single factor screening and response surface methodology on the basis of optimization design of the seed technology and fermentation conditions.The fermentation medium was composed of: glucose 2%,dextrin 8.85%,soy flour 3%,KNO3 0.74%,NaCl 0.2%,KH2PO4 0.1%,CaCO3 0.2%,pH 7.5.The fermentation conditions were as follows: inoculation amount 3%,culture temperature 28 ?,250 m L shake flask culture medium loading 20 mL,shaking speed 220 r/min,fermentation cycle 138 h.Under the optimum medium and conditions,the yield of Enopeptin A was 1750 ?g/m L and 2.98 folds higher than that with the initial medium(440 ?g/m L).The yield of Enopeptin A reported in the literature was increased by 10.67 folds as much as 150 ?g/mL.The separation and purification of Enopeptin A was studied through the research of solvent and chromatography system,including filler and chromatographic conditions.The filtrate and mycelia were coarse step separated by macroporous absorption resin chromatography and solvent extraction.The crude Enopeptin A was purified by a combination of normal phase silica gel and reverse phase silica gel multi-step chromatography process.The cost of the extraction process is low and the use of toxic organic solvents is effectively reduced.The total yield of the overall process of separation and purification was about 44%.