Molecular cloning and characterization of α-amylase/subtilisin inhibitor from rhizome of Ligusticum chuanxiong.

OBJECTIVES: To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine. RESULTS: The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment. CONCLUSIONS: This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.

Molecular cloning and characterization of caffeic acid 3-O-methyltransferase from the rhizome of Ligusticum chuanxiong.

OBJECTIVES: To clone and characterize caffeic acid 3-O-methyltransferase (LcCOMT) from the rhizome of Ligusticum chuanxiong, a traditional medicinal herb having a high content of ferulic acid. RESULTS: LcCOMT encoded an ORF of 362 amino acids with a calculated MW of 39,935 Da and pI of 5.94. Polygenetic tree indicated that LcCOMT was attributed to a new member of COMTs in plants. The recombinant LcCOMT was expressed in E. coli. HPLC and (1)H NMR analyses of purified LcCOMT protein confirmed that it could catalyze caffeic acid to produce ferulic acid in vitro. The further site-mutagenesis proved that His268 was one key catalytic residue. In addition, the substantial changing expression level of LcCOMT under chilling treatment suggested that LcCOMT might play important role in the accumulation of ferulic acid under chilling treatment. CONCLUSIONS: This is the first report of the isolation and characterization of a COMT clone from traditional medicine containing high contents of pharmaceutical ferulic acid.