Mutation Breeding And Fermentation Optimization Of High ε-Polylysine-producing Streptomyces Albulus

ε-poly-L-lysine (ε-polylysine,ε-PL) was first discovered as a novel food antimicrobial preservative which was produced by microorganism in Japan in the 1980s. It is a homo-poly-amino acid characterized by the peptide bond between the carboxyl andε-amino residue of L-lysine. It can be digested to a single lysine for human nutrition. Moreover,ε-polylysine was recognized as a GRAS material by FDA in US. CAS Registry Number is 28211-04-3. Thus the poly-L-lysine has the non-toxic effects and safe characteristics as a food preservative.In the essay Streptomyces albulus SA was studied. Through the electrostatic reaction with charged dyes, Dragendorff reaction and amino acid analysis, it can be drawn from the analysis above thatε-polylysine was a polymer of lysine with positive charge and was a kind of alkaloids. It provided a simple and quick method for screening and confirming the strains which had the capacity forε-polylysine production.Streptomyces albulus SA was treated by UV and LiCl mutation firstly. The different doses of UV were studied and the death rate curve of UV was obtained. According to the genetic breeding experiences that a higher positive mutation was in low doses mostly, the optimum irradiation was selected as 25 s. After initial selection, the strains with significant inhibitory effects were quickly elected by drilling agar-inhibition in large quantities. The production was increased to 1.33 g/L that was higher than Streptomyces albulus SA by 27.9% after UV mutation.The mutant obtained by UV was treated by nitrous acid next. The optimum processing time was 20 min. After mutation, an AEC resistant mutant high-yield UN2-71 was obtained. The production ofε-poly-L-lysine was 1.64 g/L, with a production increased by 57.7%.In order to investigate the fermentation stability, the mutant UN2-71 was tested after continuous culture generation by generation. The result showed that the mutant had a good genetic stability. The cultivation characteristics and biophysiclogical and biochemical characteristics of Streptomyces albulus UN2-71 were observed.Different carbon and nitrogen sources had conspicuous differences in synthesis ofε-polylysine. The test indicated that soluble starch was the best carbon and the combination of (NH4)2SO4 with yeast extract were the best sources of nitrogen. On the basis of carbon and nitrogen sources screening, the elements of fermentation medium were optimized. In the Plackett-Burman Screening test, the eight factors which were ingredients of the fermentation medium such as soluble starch, ammonium sulfate, yeast extract, K2HPO4, KH2PO4, MgSO4·7H2O, ZnSO4·7H2O, FeSO4·7H2O were analysed. Soluble starch, ammonium sulfate and yeast extract were the significant factors. A three-factor and three-level Box-Behnken experiment was designed, and the optimization model was obtained by RSM analysis. The ingredients of the optimized medium were as follows: soluble starch 40 g, ammonium sulfate 8 g, yeast extract 6.4557 g, K2HPO4 0.8 g, KH2PO4 1.36 g, MgSO4·7H2O 0.75 g, ZnSO4·7H2O 0.06 g, FeSO4·7H2O 0.045 g. The predictive production ofε-polylysine was 2.54 g/L, the results for confirmation was 2.48 g/L.