Regulation Of ε-Poly-L-Lysine Biosynthetlc Process By Streptomyces Sp. M-Z18

ε-poly-L-lysine (ε-PL) which consists of25~35L-lysine residues is an L-lysinehomopolymer biosynthesized extracellularly by minority actinomyces and has a uniquestructure linking between α-carboxyl and ε-amino groups. This characteristic polymer wasmainly produced as a Dragendorff-positive substance by actinomycete streptomyces isolatedfrom soil. Because of its cationic charge density of side-chain α-amino groups, ε-PL showsstrong antimicrobial activity against a wide spectrum of microorganisms including bacteria,fungi, and also some kinds of viruses. Since the safety of ε-PL was demonstrated inexperiments using rats and its character of water soluble, thermal stability, wide used range ofpH, it has been widely used as a natural food preservative in a number of countries includingJapan, the United States, and Korea. In this dissertation, a high ε-PL producing strain,Streptomyces sp. M-Z18was used as a model, the main results of this paper are as follows:1. A pH control strategy was proposed to solve the common problem, pH uncontrolled inflask for ε-PL production, resulting in degradation of cells and low ε-PL production by ε-PL-producing strains. The effect of sodium citrate-citric acid, sodium dihydrogen phosphate-citricacid, acetic acid-sodium acetate and glycine-hydrochloric acid on pH stability and ε-PLfermentation were investigated. The results showed that pH was maintained at about3.8wellin flask through addition of0.1mol/L sodium citrate-citric acid（pH3.5）at18h of cultivationusing Streptomyces sp. M-Z18. Under pH control strategy in flask by citrate,5.00±0.05g/Lε-PL and6.05±0.17g/L DCW were achieved, which were167%and75%higher than thoseunder natural pH fermentation, respectively. Furthermore, ε-PL fermentation performed in5Ljar-fementer by pH control using NH4OH showed that sodium citrate-citric acid play animportant role in pH control during Streptomyces sp. M-Z18cultivation in flask for ε-PLfermentation. It was proved that this pH control strategy has wide applicative scope by testingwith two original ε-PL producing strains.2. The synthesis of ε-PL was performed by adding different auxiliary energy materials.The effect of NADH, methanoic acid and citric acid on ε-PL fermentation were investigatedusing Streptomyces sp. M-Z18. The results showed that the contribution of citric acid to thefermentation of ε-PL was obvious. The optimum dose, addition time of citric acid and batchfermentations at various pH values were studied. Under the optimized condition, pH3.8and20g/L citric acid in5L jar-fementer, the production of ε-PL reached9.50g/L and theproductivity of ε-PL was4.40g/（L·d）, which was enhanced by46.4%and48.1%，respectively, compared with original batch fermentation strategy. Moreover, the fed-batchproduction of ε-PL reached26.89g/L,26.7%higher than that of control.3. Microparticle-enhanced cultivation was applied as a novel method to changemorphology and diameter of Streptomyces sp. M-Z18to appropriate size for improvingbiomass and the production of ε-PL. We have investigated the effect of talc powder with D50of5μm on ε-PL production in flask and found that by addition of10g/L talc powder theproduction of ε-PL reached2.01±0.06g/L and DCW was7.86±0.72g/L at the end offermentation, which was59.5%and119.6%higher than control, respectively. Simultaneously, we found the morphology of pellet was changed and its diameter got half ofusual. Under proper condition, pH3.8and10g/L talc powder, batch fermentation wasperformed in5L jar-fementer. For the activity of mycelium was enhanced, glycerol wasexhausted in39h. The production of ε-PL reached6.47g/L which was59.8%higher thancontrol of the same time. Throughout the process, the productivity of ε-PL reached3.98g/（L·d）which was32.3%higher than control, and became more and more higher over time.The productivity of DCW was also highly promoted, and at the end of fermentation theproduction was14.26g/L which was45.9%higher than that of control. Finally, fed-batchfermentation achieved maximum ε-PL concentration,30.57g/L ε-PL was accumulated at theend of the fermentation, which was44.1%higher than that of control.