Cloning,Expression,Purification And Partial Enzymology Characterization Of Acetyl-CoA Synthetase (ACS) From Dunaliella Tertiolecta

Acetyl-CoA synthetase(ACS) catalyzes the synthesis of acetyl-CoA, it is one of the important hubs of fat metabolism and acetate metabolism. According to reports, over expression of ACS will lead a large amount of acetate converted to acetyl CoA, thus to support subsequent glycolytic way, fatty acid metabolism, amino acid metabolism and gluconeogenesis of action, and accelerate the cell growth and material accumulation.In this paper, we take Dunaliella tertiolecta as the main object of study. Firstly, the 1175 bp DtACS EST fragment was amplified with the reverse transcription polymerase chain reaction(RT-PCR), the fragment was inferred as DtACS gene fragment by sequence alignment. Two fragments corresponding to the 5′ ends(758 bp) and 3′ends(784 bp) were isolated by rapid amplification of(RACE) technique, a 2464 bp sequence was obtained by piecing those three fragments above together, the predicted length of open reading frame(ORF) is 2184 bp, and 727 amino acids are encoded by its ORF. Sequence alignment showed that DtACS shares higher identities with ACS from chlorophyta(Chlamydomonas reinhardtii, 68% identity and Volvox carteri f. nagariensis, 70% identity).The results of bioinformatics analysis showed that the molecular weight of DtACS is 79.72 kDa, the isoelectric point is 6.7, and the instability index is 36.57, which indicates that DtACS is a stable protein. Its main structural domains are AMP-binding_C, ACS, PRK00174, Ac_CoA_lig_AcsA and AMP-binding. The phylogenetic tree showed that DtACS shares a higher intimate relationship with ACS in green algae and streptophytina.pET-32a(+) with the thioredoxin tag(Trx-tag) was selected as the prokaryotic expression vector, and DtACS was cloned into pET32a(+) to construct pET-32a(+)-DtACS. Then transformed it into E. coli BL21(DE3), meanwhile, transformed the blank vector pET-32a(+) into E. coli BL21(DE3) as well, thus, the recombinant bacteria pET-32a(+)-DtACS-BL21(DE3) and the control strains pET-32a(+)-BL21(DE3) was successfully constructed, respectively.DtACS with Trx tag and His tag fusion protein was expressed by the recombinant bacteria under the condition of 18℃, 0.6 mmol/L IPTG(final concentration) for 12 h, its molecular weight(about 100KDa) was detected by SDS-PAGE, and the result is consistent wich the predicted result(79.72 kDa+20 kDa, tag protein is about 20 kDa).In addition, the recombinant protein was purified via Ni2+ column, and the specific enzyme activity of purified protein is 52.8725 U/mg. When potassium acetate was used as substrate, the Km value of DtACS was 3.597 mM, and Vmax was 61.73 U/mg. When Sodium acetate was used as substrate, the Km value of DtACS was 4.673 mM, and Vmax was 59.88 U/mg. The optimum pH of DtACS was 8, and the optimum temperature was 37℃.