Improvement Of Clavulanic Acid-producing Streptomyces Clavuligerus

In this thesis .during selection of clavulanic acid high-producing strain,using resistant to analog of end-production and using glycerol-tolertant and using auxotrophic mutants as selection pressure can obviate low-producing strain and increasing the successful rate of obtaining high-producing strain,it is better than traditional screening approach-Experiment showed that using Streptomyces clavuligerus CCRC11518(with the yield of clavulanic acid being 282.4ug/ml)as parent strain,after UV mutation ,the mutant CCRC11518- I 10 resistant to 14 mg/ml of subtam was obtained,the mutant produced 489.9ug/ml of clavulanic acid and increased by 73.5% compared with the parent strain CCRC11518.Subsequently, the mutant CCRC11518 - I 10 as parent strain,after DBS mutation ,the mutant CCRC11518- II29 tolerant to 15 percent of glycerol was obtained,the mutant produced 703.4ug/ml of clavulanic acid and increased by 43.5% compared with the mutant CCRC11518 - I i0.Lastly, the mutant CCRC11518- II 29 as parent strain,after NTG mutation and eutrophication with pentachlorophenol ,the auxotrophic mutant CCRC11518-Else was obtained, the mutant CCRC11518-III so produced 834.8ug/ml of clavulanic acid and increased by 18.6 % compared with the mutant CCRC11518 - II 29- ,the auxotrophic mutant CCRC11518 - IIIso increased by 196% compared with the parent strain Streptomyces clavuligerusCCRC11518.the auxotrophic mutant CCRC11518 - III 50 was stable in clavulanic acid production after continous subcultures for seven generations.After studying on fermentation conditions for high-yield the auxotrophic mutant CCRC11518-III50 .optimum fermentation conditions were established as following :250ml Erlenmeyer flask contained 20ml medium,containing glycerol 30g ,malt exact 7.5g ,soya bean protein 60g, FeSO4 -7H2O 1 g, K2HPO4 3H2O 1 g,MgSO4.7H2O 2 g,distilled water 1 liter ,pH6.5-6.7,inoculated with level of inoculum of 15%(v/v),incubated on a 220r . min-1 shaker at 25-26 for 72 hours .Under optimum fermentation conditions, the auxotrophic mutant CCRC11518 -III50 produced 1082.6 g/ml clavulanic acid ,increased by 29.6% compared with its initial fermentation conditions and increased by 283% than the original parent strain Streptomyces clavuligerus CCRC11518.Amplied experiment in 1000ml Erlenmeyer flask and 1600ml fermenter has been done,which can be used as reference in clavulanic acid industrial procuction.