| 3-hydroxypropionic acid (3-HP) is an important platform compound, which is the rawmaterial of many chemical products and daily supplies and the monomer of many largemolecular compounds and polymer. It is one of the 12 most development potential of thethree carbon chemical products. Microorganism transformation method for production of3-hydroxypropionic acid has many advantages such as, short production cycle, abundant rawmaterials, strong adaptability, low environmental protection, low production cost. So researchscholars at home and abroad pay close attention to it. While current research on the microbialproduction of 3-hydroxypropionic acid is still at the initial stage, and the relevant reports areseldom.We collected 27 samples (17 soil samples from Hainan and 10 human feces samples) and124 strains were screened. High performance liquid chromatography was used to assay3-hydroxypropionic acid in the fermentation broth of the 124 strains. The result showed that23 strains were able to produce 3-hydroxypropionic acid and the highest yield was 4.78 g/Lfrom the strain which was called Y-11. 3-hydroxypropionic acid was determined by highperformance liquid chromatography-mass spectrometry. Based on Characteristics andIdentification of Yeasts,physiological and biochemical identification and sequence analysis of18S rDNA were performed, and Y-11 was identified as Candida rugosa.UV-NTG-60Coγwas used to generate mutations in Y-11 and a genetic stability mutant5-13B was obtained. The 3-HP production of mutant 5-13B is 11.43 g/L, which is 2.39 timesof the primitive strain(Y-11). The fermentation characters of primitive strain and mutant strainwere compared. The results showed that both the accumulation of 3-hydroxypropionic acidand tolerance of propionic acid of mutant strain were better than primitive strain.At the same time, the fermentation medium and culture conditions with single factorexperiment and response surface analysis experiment were performed. The single factorexperimental results showed that the suitable fermentation medium components (g/L) wereglucose 30-40, yeast extract 10, (NH4)2SO4 15-25, KH2PO4 5, K2HPO4 9, MgSO4·7H2O 1,FeSO4·7H2O 0.015, CaCl2 0.12, propionate 15-25, TES 5 mL, pH 5.5-7.0. The bestfermentation condition: 8% inoculation quantity, 220 r/min, liquid volume 50 mL/250 mL,culture temperature 28°C, fermentation time 4 d. According to the results, four factors andthree levels experiment (A: glucose concentration, B: the concentration of ammonium sulfate,C:sodium propionate concentration, D: the original pH) , with 3 -hydroxypropionic acid yieldas response value, was designed. When the glucose was 35.6 g/L, ammonium sulfate was 16.3g/L, propionate was 15.2 g/L, fermentation initial pH was 6.4, the maximum predicted valueof 3-hydroxypropionic acid is 13.3 g/L, with the experimental values was 12.5 g/L.3-hydroxypropionic acid production was 9.2% higher than that of before.The metabolic process of 3-hydroxypropionic acid by Candida rugosa was explored. Itshowed that propionic acid was the substrate and it could be catalysised topropionyl-coenzyme A by propionyl-coenzyme A synthase. Propionyl-coenzyme Asynthetase is more affinity on substrate of propionic acid than acrylic acid. The optimal temperature of the enzymatic reaction and the most suitable temperature of 3-hydroxypropionic acid coincide. HPLC-MS of enzymatic reaction liquid analysis showed that m/z 822was characteristic absorption peak of propionyl-coenzyme A. Consequently, we presumedthat the metabolic pathway of 3-hydroxypropionic acid by by Candida rugosa might exist areaction, that is, propionic acid—propionyl-coenzyme A—3-hydroxy propionic acid. |
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