Expression,Characterization And Application Of5-aminolevulinic Acid Synthases From Different Organisms

5-Aminolevulinic acid (ALA) is a common precursor of tetrapyrroles such as porphyrins, chlorophyll, heme and vitamin B12, widely existing in microorganisms, plants and animals. ALA has been used in a number of applications, i.e. as a new kind of green pesticide in agriculture, and as a photodynamic medicine for photodynamic diagnosis and therapy of cancer.In this study, to obtain a5-aminolevulinic acid synthase with excellent enzymatic properties, we investigated the differences of the hemA genes from different organisms resulting in further enhancement of ALA production.To begin with, four kinds of5-aminolevulinic acid synthase (ALAS) genes (hemA) from R. capsulatus, B. japonicum, R. acidophilus and P. denitrificans, were cloned and expressed in E.coli Rosetta (DE3). Among them, the R.capsulatus hemA gene was high-level expressed with the highest activity and ALA productivity, indicating its good potential for ALA production.Then, the recombinant ALAS (RC-ALAS) expressed by E. coli Rosetta (DE3)/pET28a-RC.hemA was purified by affinity purification on Ni-NTA agarose and gel filtration chromatography on Sephadex G-25Medium resin. And the enzymatic properties of the purified RC-ALAS were studied. Compared with ALASs encoded by the hemA genes from Agrobacterium radiobacter (AR-ALAS) and Rhodobacter sphaeroides (RS-ALAS), the specific activity of RC-ALAS reached up to198.2U/mg, which was about31.2%and69.5%higher than AR-ALAS (151.1U/mg) and RS-ALAS (116.9U/mg), respectively. The optimum pH values and temperatures of the three abovementioned enzymes were all pH7.5and37℃. respectively. Moreover, RC-ALAS was more sensitive to pH, while the others were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) on the three ALASs were also investigated, and the results indicated that they had the same effects on the activity of the three ALASs.The specificity constant (kcat/Km)S-CoA of RC-ALAS was1.4989, which was higher than both AR-ALAS (0.7456) and RS-ALAS (1.1699), showing its high catalytic efficiency and good industrial application prospects.Finally, up to8.8g/1ALA was produced by the recombinant strain E.coli Rosetta (DE3)/pET28a-RC.hemA in a15L fermenter, showing its high ability for ALA accumulation.