Separation And Purification Of 5-Aminolevulinic Acid From Recombinant E.coli Fermentation Broth

5-Aminolevulinic acid(ALA) is generally known as an essential intermediate in the biosynthesis of tetrapyrroles such as porphyrin,chlorophyll,and vitamin B₁₂.It has important application in agriculture,medicine and biotechnology.Recently,the technology of producing ALA by fermentation using recombinant E.coli has been researched widely,however,there are very few researches about separating ALA from fermentation broth.The aim of this work is to establish a novel procedure for separating and purifying ALA from recombinant E.coli fermentation broth,obtain the product with high purity and lay the technological foundation for industrial production of ALA.In this work,di(2-ethylhexyl)phosphoric acid(D2EHPA) and n-hexanol were chosen as the optimum extractant and diluter respectively,the effect of D2EHPA concentration,initial aqueous pH,initial ALA concentration,temperature,volume ratio of organic phase to aqueous phase(R) and series on extraction equilibrium had been discussed respectively,HCl solution was selected as the stripping reagent,the effect of HCl concentration,temperature,volume ratio of aqueous phase to organic phase(r) and series on back extraction equilibrium were also discussed respectively. D2EHPA concentration of 50%(v/v) in n-hexanol,an initial aqueous pH value of 6.5, a phase ratio R value of 0.5,and 20℃were found to be the optimum extraction conditions.After two-stage extraction,the organic phases were mixed together for one-stage back extraction.Back extraction of organic phase was carried out with 1 mol/L HCI solution at 30℃,with a phase ratio r value of 0.5.The fermentation broth with recombinant E.coli was filtrated by the hollow fiber ultrafiltration membrane module to remove the bacterium with an ALA recovery efficiency of 86.5%and a decoloring efficiency of 54.4%.Then the filtrate was concentrated by reverse osmosis,an ALA recovery efficiency of 93.2%was achieved here.The concentrate after reverse osmosis was then extracted in the optimum extraction conditions,with a recovery efficiency of 79.8%and HPLC purity of 36.2%. The main factors which impacted ALA separation were investigated.The content of precursors and pigments could be decreased by prolonging fermentation time and decoloration,respectively.In the decoloration process the ALA recovery efficiency was 99.3%,the decoloring efficiency was 89.8%.The total recovery efficiency during the rough separation of ALA reached 63.9%.The purification of 5-aminolevulinic acid hydrochloride(ALA·HCl) was investigated.The aqueous phase after back extraction was concentrated by vacuum evaporation,with a recovery efficiency of 99.2%.The concentrate was then crystallized by cooling down.After two stage crystallization,the ALA recovery efficiency was 80.1%.The total recovery efficiency was 79.5%during the purification process.After the recombinant E.coli fermentation broth had been treated by membrane filtration,reactive extraction,concentrate and crystallization,the overall yield and the product purity of ALA·HCl reached 50.8%and 99.5%,respectively.