Fermentation And Extraction Of Sisomicin By High-yield Micromonospora Inyoensis

Aminoglycoside antibiotics（AGs）are a kind of anti-infective drugs that have postantibiotic effect on a variety of pathogenic bacteria by inhibiting protein synthesis.As a kind of typical AGs,sisomicin is mainly produced by the fermentation of micromonospora inyoensis.Its chemical structure is only more double bonds than gentamicin,but its antibacterial activity in vivo is stronger than gentamicin.The third generation AGs synthesized with sisomicin as the mother nucleus,netilmicin and plazomicin,have lower ear and renal toxicity than sisomicin,which makes the clinical application of AGs more promising.The domestic industrial production capacity of sisomicin is only about 1000 U·m L^-1,which is far from meeting the needs of the world market.Therefore,in order to improve its titer,it is of great significance to study the medium composition and extraction process of sisomicin.In this study,M.inyoensis G2-24 obtained through the preliminary transformation of the laboratory was used as the starting strain to explore the effects of the optimization of culture medium and fermentation conditions on the fermentation process of sisomicin,and the extraction process of sisomicin was preliminarily explored.The main results are as follows:（1）The titer in fermentation broth was detected by standard curve method,the linear range was 1.80～10.74 U·m L^-1,R²=0.9993,and the RSD was less than 10%,which was not significantly different from that determined by two dose method;When determining the titer of high concentration aqueous solution with large sample volume,the polarimetry is selected,and the linear range is 100～10000 U·ml^-1,R²=0.9998,the average recovery was 100.11%（n=3）,RSD<1%,and the experimental results were reliable;Congo red photometric method is selected to determine the titer of aqueous solution with small sampling volume and low concentration,and the linear range is 1.80～10.74 U·m L^-1,R²=0.9907,the average recovery was99.76%（n=3）,RSD<2%,and the experimental results were accurate.（2）The modified bacteria were tested by single factor experiment.Firstly,the carbon source and nitrogen source of fermentation medium were optimized,and then the fermentation conditions were further optimized.The results of medium optimization showed that the dual carbon source fermentation of corn starch+maltose could increase the fermentation titer by20.09%to 1853.96±84.48 U·m L^-1;When using peptone as the main nitrogen source,the titer was further increased to 2046.85±71.91 U·m L^-1.The optimized medium was used to optimize the fermentation conditions,and the final optimization results were the initial p H 7.0,35℃and230 r·min^-1.The titer was increased by 33.72%(2120.78±51.04 U·m L^-1)compared with the starting strain after seven days of fermentation.（3）The maximum working exchange capacity of macroporous weak acid cationic resin DK-13（NH₄⁺）was 24.59×10⁴ U·m L^-1,and the desorption rate reached 99.47%.Wet column filling,height diameter ratio of 3:1 and fixed bed dynamic adsorption elution process were used to explore the best process of extracting sisomicin with DK-13 resin.The results showed that the optimum p H was 6.73 and the loading speed was 1 BV·h^-1;The optimum ammonia concentration of eluent was 0.65 mol·L^-1 and the elution rate was 3 BV·h^-1.The adsorption kinetics and thermodynamic behavior of sisomicin adsorbed by DK-13 resin showed that the adsorption of sisomicin by DK-13 resin accorded with Langmuir adsorption isotherm equation（R²>0.998）at 20～35℃;At 30℃,the adsorption kinetic data accorded with the pseudo second-order kinetic equation.The whole adsorption process was controlled by both particle diffusion and membrane diffusion;The adsorption process was a non spontaneous endothermic reaction and an entropy increasing process.The whole system tended to be chaotic.