Clavulanic Acid Producing Bacteria

In order to improve the production of clavulanic acid for the industrial need, the organism Streptomyces clavuligerus NR3585 was studied in this paper.Firstly, the bioassay method, TLC-Bio-autograph method and HPLC method of clavulanic acid were developed, so that clavulanic acid in the fermentation samples can be measured qualitatively and quantitatively. On the basis of studies on S. clavuligerus culture characteristic, colonies characteristic and sensitivity to various mutagens, physical and chemical mutagens including UV, X-ray, DES, HNO₂ and NTG were used in the mutagenesis of Streptomyces clavuligerus. Then, in order to accelerate the process of strain improvement, a glycerol-resistant screening model and an agar column solid fermentation screening method was established.A clavulanic acid high-yield strain Streptomyces clavuligerus U7D5XH-45 was obtained after several circles of mutagenesis, which produced 1139.4#/ml clavulanic acid in shaking flask fermentation, about 20 times higher than the productivity of initial strain. At the same time the content of clavulanic acid increased from about 70% to almost 100% measured by HPLC.The fermentation media for clavulanic acid production were also inspected and optimized in this paper. The optimal carbon source and nitrogen source was maltose and soybean powder respectively through the study. Feeding amino acids to fermentation medium from 5 mM to 15 mM at the beginning of fermentation showed no promoting effect on the biosynthesis of clavulanic acid. However Lys, Val, Tyr, Phe, Cyc and Trp showed strong inhibition effect on the biosynthesis of clavulanic acid. An unknown product but no clavulanic acid accumulated when Cys was added into the fermentation medium. The investigation of the effects ofinorganic salts and trace elements showed that KNO₃, Mn2+ and Mg2+ promoted the production of clavulanic acid. Experiment results also showed that feeding of 1.0#M PMS (a redox agent) to fermentation medium stimulated the biosynthesis of clavulanic acid. Fermentation medium of clavulanic acid was optimized through uniform design. The clavulanic acid titer increased obviously with the optimized fermentation medium.The optimized fermentation techniques of clavulanic acid production by Streptomyces clavuligerus were as follows: fermentation temperature, 26 癈 ; fermentation period, 80-84h; inoculation volume, 10% (v/v); fermentation volume, 40ml in 250ml shaking flask; pH, 6.5 before sterilization.Results showed that phosphate exerted obvious regulative effects on the biosynthesis of clavulanic acid. The optimal concentration of phosphate for the biosynthesis of clavulanic acid was 5-10mmol/L. Higher phosphate concentration depressed clavulanic acid production greatly. The catabolism pathway of saccharide also exerted regulative effects on clavulanic acid biosynthesis.When low concentration (0. lmg/100ml)iodoacetic acid was added to fermentation media in the beginning of fermentation to alter the catabolism pathway of saccharide, the biosynthesis of clavulanic acid was promoted obviously.