The Selection And Breeding Of Strains Producing E-polylysine By Fermentation

ε-polylysine (ε-PL) consists of25-30L-lysine residues by linkage a-carboxyl group and ε-amino group. ε-PL is becoming a most popular preservative owing to its good solubility, wide range of inhibitory and edible quality. Today, ε-PL is used as a food preservative legally in many countries. The microbe fermentation of ε-polylysine has been industrialized in Japan, while in our country the fermentation technology is still in laboratory scale. So the study on screening of high-yielding strains and production technology of ε-PL possesses great values.In this paper we separate a new strain SAC23from the solid through an improved Nishikawa’s method, combing with specific precipitate reactions. We studied the characteristics of product that purified from the fermentation broth. Antibacterial test showed that the product has antibacterial activities on Gram-positive, Gram-negative bacteria and fungi. Thin layer chromatography (TLC) proved that the product is a lysine polymer. SDS-PAGE electrophoresis showed that the relative molecular weight of ε-PL is3.4KDa and the degree of ε-PL polymerization is approximately27. The strain SAC23was identified as Streptomyces ahygroscopicus by morphology, physiological and biochemical analysis and molecule identification of16S rRNA.Through the method of60Co mutagenesis, combining with ribosome engineering and methylene blue plates, we obtained two high yield mutants SAS12and SAS17with s-PL production of4.111g/L and4.384g/L. The mutants were genetically stable, which tested by passaging experiment. SAS12and SAS17were selected to carry out the protoplast fusion breeding. The strain SAP3was finally obtained with s-PL production of5.167g/L, improving18%compared with that of the strain SAS17.Through the orthogonal experiment, we got the optimization medium:5%glucose,1%yeast extract,1%(NH4)2SO4,0.136%KH2PO4,0.08%K2HPO4,0.04%ZnSO4-7H2O,0.003%FeSO4·7H2O, and superinduce with50uM scandium nitrate,5mM L-lysine, buffer pH4.0. The ε-PL yield of SAP3could reach to7.454g/L when inoculated in the best fermentation medium, improved44.3%compared with original medium.The ε-PL yield of mutant SAP3improved394percent compared with the original strain SAC23in optimization medium, which meant that60Co irradiation and protoplast fusion are effective methods to improve ε-PL yield of Streptomyces ahygroscopicus. The genetic stable SAP3strain has the potential to further expandation and industrialization.