Screening,expression,characterization And Application Of Thermostable 5-aminolevulinic Acid Synthase

5-aminolevulinic acid(ALA)is the precursor for the biosynthesis of tetrapyrroles and has broad applications in agriculture and medicine with high additional value.5-aminolevulinic acid synthase(ALAS)is the key enzyme for ALA synthesis,therefore,the research of ALAS has been received extensive attention.The objective of this study was to synthesize ALA through the method of cell free multi-enzyme catalysis.We screened thermostable ALASs and they were expressed in E.coli BL21(DE3).Then ALASs were purified and their enzymatic properties were assayed.Finally,thermostable ALAS was used for cell free synthesis of ALA.The main results were as follows:Three ALASs from thermophiles,GT-ALAS(ALAS from Geobacillus thermoglucosidasius),LS-ALAS(ALAS from Laceyella sacchari),PA-ALAS(ALAS from Pseudomonas alcaliphila)and two previously studied ALASs,RP-ALAS(ALAS from Rhodopseudomonas palustris)and RS-ALAS(ALAS from Rhodobacter sphaeroides)were expressed in large quantities in E.coli.Then these five enzymes were purified and characterized.The optimum temperature for all five enzymes was the same at 37°C and optimum pH was also the same at 7.5.Three enzymes from thermopiles(GT-ALAS,LS-ALAS and PA-ALAS)were thermostable and had almost no activity loss after 60 h.The residual activity of LS-ALAS was the highest among these three thermostable ALASs after 3h at 55°C(43%).Moreover,the specific activity of LS-ALAS was the highest among these five ALASs(7.8U/mg)and it was the most insensitive to temperature and pH.LS-ALAS was used for cell free synthesis of ALA from succinate and glycine because of its high stablity and specific activity.The multi-enzyme system contained three enzymes: LS-ALAS,succinyl-CoA synthase(Suc)and polyphosphate kinase(Ppk)that was used for regeneration of coenzyme A(CoA)and ATP from polyphosphate.Pyridoxal 5'-phosphate(PLP)was also added as an enzyme cofactor.Succinate and polyphosphate were added to the reaction system in a fed-batch mode to avoid enzyme inhibition.An ALA concentration of 498mg/l was obtained.Due to the consumed polyphosphate has the ability to synthesize ATP,only succinate was added to the reaction system in a fed-batch mode and the final concentration of ALA was 707mg/l.This was the first reported work on developing cell free processes for enzymatic production of ALA.