Study Of Sisomicin High Producing Strain Breeding And Fermentation Optimization

Sisomicin is a new aminoglycoside broad-spectrum antibiotics, and has a strong antibacterial effect on both gram-positive and gram-negative bacteria. Its Semi-synthetic compound-Natimicin is an ideal product in a variety of aminoglycoside antibiotic with high efficiency and low toxicity characteristics. The sisomicin bulk drugs production in China accounts for about80%of the world production, and the demand of sisomicin and netilmicin continues to rise in domestic and foreign markets recently.In order to improve the yield of sisomicin and consolidate the monopoly position of China, study on the chemical mutation, the nitrogen ion beam implantation mutagenesis combined with resistance screening mutation breeding research of the marine Streptomyces sp. GB-2was carried out. Overproducing strain was obtained; the fermentation conditions and liquid submerged fermentation process were optimized. The main results are as following:1. The titer was determined by the paper strip method and the cylinder plate method, and three-dose method was used in the biological statistic test. The biological titer of Marine Streptomyces sp. GB-2was measured successfully as387.0289U/mL. The experimental results obtained by comparing the paper strip method and the cylinder plate method showed that two methods were both reliable and could make a good reproducibility. The paper strip method could be used for the primary screening to in the fermentation optimization process and mutation breeding of marine Streptomyces sp. GB-2. The selected results could use cylinder plate method for accurate determination. These two methods could be used combined addition to cut costsand increase the number of sample testing.2. PB design (Plackett-Burman Screening for Design) was used to screen the nine factors affecting the sisomicin production of marine Streptomyces sp. GB-2. The results showed that:the main factors affecting the fermentation production of sisomicin were soybean powder, glucose, temperature and speed. Based on this, the response surface method (Response Surface Methodology, RSM) was used to study the optimal level of the four significant factors. By solving the quadratic regression equation the correlation coefficient R2was informed as0.9833, under the condition that:soybean powder11.95g/L, glucose2.96g/L, temperature28℃, and speed183r/min, sisomicin titer increased from387.03U/mL to726.11U/mL, production increased by87.61%.3. Compound mutagenesis combined with resistance screening, N+implantation means were used in the marine Streptomyces sp. GB-2mutation breeding processing. After the single spore suspension pretreatment, at first the type and dose of mutagen were determined through the single-factor mutagenesis, and then mixed the LiCl solution and spore suspension at1:1, keeped in30℃water for30min. Then learned1mL handling liquid and mixed with NTG solution at1:1, keeped in30℃for another30min. Coat the mixture on the surface of the tablets mixing different concentrations and types of antibioticfor screening, rescreening and stability studies. The marine Streptomyces sp. GB-2strains and the high-yielding strains we had gotten were reprocessed by N+implantation mutagenesis, in the condition of doses25kev, injection time for0s,5s,10s,15s,20s,25s,30s. The survival rate showed a "saddle-shapped" curve with the injection time changes.61high-yielding strains were screened in mutation breeding, and the maximum mutation rate reached up to48.82%.4. Marine Streptomyces sp. FMB-34with high-yielding sisomicin production was screened by combined mutagenesis, resistance screening, and N+implantation, and the titer of the sisomicin production reached1354.07U/mL after media screening. To further enhance the yield, at first, single factor tests were taken to determine the types of carbon and nitrogen sources and the initial amount of glucose components, then Fractional Factorial Screening for the Design was used to quickly identify the three key factors (soy flour, wheat flour, and peptone) affecting the fermentation. The statistical analysis of the main effects and the interactions of various factors were obtained by the JMP software, and the optimum fermentation conditions were ascertained as:soy powder40.0g/L, wheat flour20.0g/L, peptone5.0g/L, the remaining components of glucose10.0g/L, CaCO36.0g/L, MgSO424.0g/L, FeSO4·7H2O1.5×10-4g/L, CoCl2·6H2O1.5×10-4g/L, in accordance with4%inoculum size,50/250mL liquid volume,33℃,180r/min of fermentation conditions for5days, the forecast value of the titer could up to3345±238.67U/mL.