Studies On Cleaned Fermentation Of L-Tryptophan

L-tryptophan,one of the essential amino acids in human and animal life activities,is widely used in the pharmaceutical,food and feedstuff industries.With the development of fermentation technology,the market demand of tryptophan continues to increase and tend to exceed supply.Therefore,it is of practical significance to research on fermentative production of L-tryptophan.In this article,L-tryptophan producing strain Escherichia coli TRTH was used as original strain under the guidance of metabolic engineering theory,the problems of culture medium,fermentation regulation,separation and extraction during tryptophan fermentation were studied.The active components such as amino acid,nucleotides and vitamins in yeast powder,the common organic nitrogen sources for L-tryptophan fermentation,was determined by HPLC to construct a new clear medium for L-tryptophan fermentation,and the medium was-optimized by two-level orthogonal test and response surface analysis.The results showed that:lysine,guanosine and inosine had a great influence on L-tryptophan cleaned fermentation medium.The optimal addition of lysine 1.02 g/L,guanosine 0.16 g/L and inosine 0.19 g/L were obtained by response surface analysis.The fermentation conditions of L-tryptophan clear medium were studied at shake flask level,the results showed that the optimum culture conditions were as follows:the optimum culture volume was 25 mL in 500 mL triangular flask,the pH was 6.8-7.2,the culture temperature was 36-37 ? and the speed of the rocking bed was set to 220 r/min.The effects of L-tryptophan clear fermentation medium on L-tryptophan fermentation were studied by fed batch fermentation in 30 L fermenter.The fermentation biomass and L-tryptophan yield of clear medium were 45.8g/L and 40.8g/L,which increased by 8.6%and 24.39%respectively compared to the ordinary fermentation medium.Besides,the final acetic acid accumulation of clear fermentation medium was 5.05g/L,which was 29.1 g/L lower than that of ordinary fermentation medium(7.12g/L).The metabolic flow equilibrium model of L-tryptophan biosynthesis was established by metabolic flow analysis,and the metabolic network was quantitatively analyzed using the detection data of fermentation process.The results of metabolic flux analysis of L-tryptophan fermented with clear fermentation medium showed that the metabolic flux(r2)of EMP pathway decreased from 81.3 to 76.8,the metabolic flux r11 of HMP pathway increased from 18.7 to 23.2,the metabolic flux of acetic acid branch decreased by 44.63%,and the metabolic flux of the tryptophan synthesis pathway increased by 18.71%.A new method of L-tryptophan extraction was constructed based on the research of clear fermentation medium broth,and the decolorization as well as adsorption properties of various decolorizing resins used in L-tryptophan extraction were investigated.The final results showed that the content of mycelium protein in the clear fermentation medium broth was 24.6%which was 11.83%lower compared with the ordinary fermentation broth(27.9%),the light transmittance of the clear fermentation medium broth was obviously higher than that of the ordinary fermentation broth.After ultrafiltration,the clear fermentation medium broth was clarified and its transmittance reached 94.2%after the decolorization of the resin,which allowed it to be purified directly,while the ordinary fermentation broth was still turbid after ultrafiltration,and its transmittance after decolorization of the resin was only 78.6%.The L-tryptophan extraction process,which was re-established on the basis of the advantages of fermentation in clear medium,contained only few steps such as centrifugation,ultrafiltration and decolorization.The resin for decolorization was optimized,SQD817 showed great advantages in decolorization rate(92.5%)and L-tryptophan adsorption rate(7.9%).The optimum working conditions were as follows:the temperature was 25 ?(room temperature),the pH was 6.0 and the flow rate was 1.5 ml/min.The extraction experiment of L-tryptophan was carried out under the optimum process,the yield of the product was 72.8%and the purity reached 98.9%,which was 11.66%and 1.33%higher than the traditional process,respectively.