表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

Purification and Production of the Extracellular 5-aminolevulinate from Recombiniant Escherichia coli Expressing Yeast ALAS

摘要:

5-氨基乙酰丙酸（5-aminolevulinate，ALA）由5-氨基乙酰丙酸合酶（5-aminolevulinate synthase，ALAS）催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达，在摇瓶培养条件下，分析了胞外ALA的产量，重组菌的生长状况和细胞中ALAS的活性，利用两种国产树脂纯化ALA，毛细管电泳分析确定ALA纯度在LB培养基中，初始pH 6.5，含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸，37℃下诱导培养12h，胞外ALA的产量为162mg /L培养基。纯化的ALA纯度达到90%。

Abstract:

Aminolevulinic acid (ALA) is biosynthesized by the enzyme ALA synthase (ALAS). The ALA production has been studied using the overproducing ALAS from several bacteria in Escherchia coil, respectively. However, ALAS from eucaryote expressed in E.coli for producing ALA in the culture is not known. The extracellular ALA production and cell growth were investageted respectively using the recombinant E. coli overproducing Saccharomyces cerevisiae ALAS in shake-flask fermentations. The ALAS activity from the cell extract was assayed. The extracellular ALA was purified by the national-made large-dimension resins and confirmed by the capillary electrophoresis measurements. At 12h after induction at 37℃, the extracellular ALA production was up to 162mg per liter LB culture at initial pH 6.5 with exogenous levulinate, succinate and and glycine at the concentration of 20mmol/L respectively. The purity of ALA after purification is up to 90%.