| Laboratory screening of high yield strain Streptomyces with griseolus poly lysine as the starting strain fermentation production of epsilon, study its fermentation medium components for the best, optimization of fermentation process, to improve the polylysine production purposes, to expand the set of pure fermentation, reasonable process, verify the validation of the synthetic product. Search known producing poly- L- lysine strain No. AB385841 landing pls fragment obtained from Gene Bank(gene for epsilon-poly-L-lysine synthase) were amplified by genomic sequence design primer lab, by means of a preliminary study on sequencing. The main conclusions are as follows:Shake flask fermentation, the optimum culture medium on the basis of experiment supplemented with D152 resin in situ separation fermentation experiment, found that when the glycerol and xylose mixture was D- in accordance with 3:7 as carbon source,(NH4) 2SO4 and yeast powder as nitrogen source was mixed according to 1:1.25, the highest yield was 1.76g/L, the fermentation yield, compared to before optimization. The amount of fermentation and the fermentation efficiency ratio were increased by 30.1%, 18.8% and 17.1%.In situ separation experiments also prove that the feedback inhibition effect is not very obvious poly-L-lysine was added, but the use of D152 resin, compared with blank control poly-L-lysine production increased by 24.8%. Prove that the D152 resin has eliminated some feedback strong produced in the process of fermentation inhibiting substances, comparison of reducing sugar consumption, it was found that the efficiency is improved by 13.38%.Poly-L-lysine with a positive charge, purification and recovery of the cation exchange resin D152 as an adsorbent for product recycling process, the adsorption of 30 min resin for: shock under neutral conditions of 25 DEG C; eluent concentration of HCl was 0.1mol/L, the elution conditions of 150r/min, temperature 30, time 60 min using acetic ether(; 2:1) precipitated, filtered through Sephadex G-25 gel column dissolved, the flow rate was 4ml/L, with dd H2 O after elution, collecting the eluent of vacuum freeze concentration, that is pure. The process, adsorption capacity of the resin is 101.6g/L, desorption rate is 91.6%, the final calculation of the recovery rate was 77.5%, the purity of the product can reach 94%.The purified sample by thin layer chromatography, confirmed that the sample hydrolysates with lysine, poly-L-lysine standard hydrolysis product with the same RF value, confirm that the product lysine polymers, followed by nuclear magnetic resonance spectroscopy verified, product of peak and peak area ratios are consistent with the literature, confirm that the product is epsilon poly lysine.Fermentation in shake flask fermentation stage of experiment, the two phase control method of p H fermentation process control, focusing on the effects of p H value and stirring rate on fermentation system, optimization of fermentation conditions for 350r/min, 30 degrees p H5.75, phosphate regulating initial fermentation, fermentation process of natural p H dropped to 3.75 by 10% ammonia solution maintain the p H until the end of fermentation, the initial dissolved oxygen was 100%, to be reduced to 30% after the end of dissolved oxygen, adjusting ventilation ratio until fermentation. The biomass yield of experimental 12.6g/L, 2.75g/L e-PL, optimization of fermentation production of the biomass was 7.48g/L, the yield of 2.17g/L was-PL, and the laboratory of shake flask fermentation of biomass and-PL yield was 4.32 g/L and 1.78 g/L, the optimized production increased by 54.5%.Homology at the level of gene verification experiment screening strains and standard strains, simple experimental design was amplified and sequenced the gene sequence, compared to standard bacteria strains using Yoshimitsu Hamano compared with the Gene Bank accession number is AB385841, the primers were designed to amplify DNA sequencing found that laboratory strains were screened for the homologous 42.6%. |
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