Isolation And Breeding Of ε-poly-l-lysine Producing Strain And Enhancement On Production Process

s-poly-L-lysine (ε-PL) consists of 25-35 L-lysine residues by linkage between a-carboxyl group and s-amino group.ε-PL has been used as a safe and effective natural preservative because it has some incomparable advantages compared with chemical preservatives, such as strong water-soluble, heat stability, broad spectrum antibacterial, non-toxic edible, etc. In addition, it can be used as emulsifying agent, dietary agent, biodegradable fibers, highly water absorbable hydrogels, drug carriers, anticancer agent enhancer, biochip coatings and so on owing to the positive chargedα-NH2 on its branched chains. Therefore,ε-PL has broad prospects of development. The main contents of this paper are as follows:The screening method ofε-PL producers was improved. One kind of acidic dye (fancy red) was first reported to replace methylene blue and added into culture medium directly without effecting strain growth because it is non-toxic toward strain. If a strain can produceε-PL, a positively charged substance, this anionic dye will condensed around the colonies by electrostatic attraction. So the s-PL producers could be selected more quickly and conveniently. By this method,26 strains were found to haveε-PL production capacity, whose yield were 0.15～0.50 g/L. The hydrolytes of strains'broth were analyzed by thin layer chromatography (TLC) and proved that the product was a polymer polymerized by lysine.Study on protoplast fusion breeding of S. graminearus were performed. The main conditions of protoplast preparation and fusion were as follows:0.5%(W/V) of lysozyme as a broken enzyme; 30℃for 120min; protoplast were fused by 30%(W/V) of PEG6000 for 5 min. UV mutagenesis of S. graminearus were selected as fusion parents by resistance to sulfadoxine-guanidine. By protoplast fusion breeding, the highest yield of fusant we got was 0.91 g/L, which was 72% higher than that of parent strain.Effect of different amino acids on s-PL production by S. graminearus was studied. Results showed thatε-PL output was increased obviously by phenylalanine (Phe). Concentration and adding time of Phe were further optimized and 3 mM Phe could increase s-PL yield by 20-30% when added it at the beginning of fermentation. The metabolic flux analysis (MFA) of S. graminearus by glucose as carbon source was first established and described the mechanism of Phe for increased production ofε-PL. Indicated that metabolic flux distribution ofε-PL fermentation was changed by adding Phe. The flux of phosphoenolpyruvate(PEP) to shikimate pathway was inhibited, while the flux toε-PL was increased. Besides, When 1.37 g/L Lys was added at 48h of fermentation,ε-PL yield was increased by 64.8%. This result indicated that S. graminearus is possible to produceε-PL by adding exogenous Lys.