| 5-Aminolevulinic acid(ALA) is a precursor of pyrrole compounds(the heme of hemoglobin, chlorophyll, and vitamin B12, etc.) of the important intermediates, microbial cells in animal and plant cells and synthesis.As a green agricultural chemicals,it was widely used in the herbicides, pesticides and plant growth regulator, etc. And it has already been widely used in agricultural field. In recent years, ALA plays an important role in photodynamic therapy on the treatment of cancer.More and more people pay attention to the field of cancer therapy, ALA has its unique advantages: good treatment effective and less side effects, Such as skin cancer, bladder cancer, rectal cancer and other kinds of cancer are widely used in clinical treatment. ALA unique is in line with the green chemistry, green environmental protection, will in the near future of great help to human's life.The ALA synthetic methods mainly include both chemical synthesis and microbial fermentation, among which, the chemical synthesis process,due to its steps and trival, serious pollution, lower yield shortcoming increasingly was replaced by microorganism fermentation. Microorganism fermentation.it is through mutated strain or restructuring engineering strains, through the optimization of fermentation conditions to make it a substantial accumulation of ALA. The main biosynthesis of ALA metabolic pathways are C4 and C5 way two pathways.In this paper by R. palustris(Rhodop seudanonas palustris) genomic DNA as a template, after optimize the codons of hem O gene, and then design with specific primers Nde I and Hind? enzyme restrictive sites of upstream and downstream primers hem O-FOR and hem O-REV,Use Nde I and Hind III enzyme restrictive sites plasmid p ET21 a respectively and purpose gene after rigation after winning p ET21a-hem O into E. coli BL21 bacteria to express in the internal, screening of positive single colony, double enzyme used for identification and sequencing identification. We will build a genetic engineering bacteria fermentation culture at the same time, using IPTG inducing expression of recombinant proteins,ALAS,by optimizing the various fermentation conditions, to determine the optimal parameters, maximum enzyme activity and synthesis efficiency, in order to achieve the purpose of the experiment.The optimazed parameters of fermentation conditions are:0.2 m M IPTG, 0.06 M glycine,0.08 M succinic acid,19.4 m M levulinic acid and 1.95 g/L glucose, induction at the temperature of 28°C, obtaining the greatest ALA accumulation of ALA(2.35 g/L).Innovative experiment adopted by physical method(repeated freezing and thawing, ultrasonic processing) and the chemical method(normal hexane, carbon tetrachloride and toluene) on the change of E.coli cell membrane permeability, so that the precursor substances entering to E.coli cells easily, which can achieve the purpose of increasing yield of ALA, finally get in 25 KHZ, deal with ultrasonic processing under 200 W, 80 s, the increase of the ALA is about 14.0%(from 2.31 g/L to 2.63 g/L), the effect is best. Adding 0.4% amount of nomal hexane organic reagent experiment effect is best, can promote the ALA yield about 18.6%(from 2.21 g/L to 2.62 g/L).Because of the the time is limited, the experiment have no time to test the fermentation condition optimization experiment more factors may affect the yield of ALA(such as the choice of culture medium, the effect of dissolved oxygen, etc.), the income of the ALA yield is affected by these factors failed to achieve the best level, expect to complement in the subsequent students experiment. By the change of the final fermentation liquid color, we suggesting that ALA along the metabolic pathway generated protoporphyrin and other substances, expect to the following study in the future. |
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