Gene cloning，expression and characterization of malate-CoA ligase in the polymerization pathway of polymalic acid from Aureobasidium pullulans

Abstract:

Abstract:［Objective］To clone and characterize the malate-CoA ligase in the polymalic acid biosynthetic pathway from Aureobasidium pullulans CCTCC M2012223.［Methods］ The malate-CoA ligase gene was cloned into the expression vector pET-Mcl by IPCR technique，and expressed in Escherichia coli BL21 (DE3)．After purified with Ni-NTA column chromatography，the protein was characterized.［Result］The full-length of malate-CoA ligase gene was 1498 bp，and composed with 440 amino acids containing 4 exons and 3 introns. The optimal temperature and pH was 25℃ and 8.0，respectively，but the high substrate concentration of ATP could obviously inhibited the enzyme activity．The monomer selectivity showed that the enzyme catalyzed the substrates of oxalic acid，oxaloacetic acid，butyric acid，and malonic acid.［Conclusion］ The malate-CoA ligase gene in the polymerization pathway of polymalic acid from Aureobasidium pullulans CCTCC M2012223 was successfully cloned，which will be helpful in deeply understanding the polymerization pathway and producing new polymers．