Molecular cloning and functional analysis of bergaptol-O-methyltransferase from Angelica dahurica (Bai Zhi) and using it to efficiently produce bergapten in E. coli

Shu-Chin Lo; Pei-En Chung; Co-Shine Wang
April 2012
Botanical Studies;2012, Vol. 53 Issue 2, p197
Academic Journal
Bai Zhi (Angelica dahurica), a Chinese herb, has long been used as a face cream for skinwhitening purposes. One of the known skin-whitening components, 8-hydroxybergapten is a hydroxylated product of bergapten that is converted from bergaptol by bergaptol 5-O-methyltransferase (BMT) in Bai Zhi. The complementary DNA of BMT was cloned from Bai Zhi root using a pair of degenerate primers designed from the highly conserved regions of other plant O-methyltransferases (OMTs). RT-PCR analysis indicated that a single band of DNA fragment corresponding to AdBMT sequence was obtained. The tandem 5′- and 3′-rapid amplification of cDNA ends via polymerase chain reaction was used to obtain the full-length cDNA sequences. The AdBMT cDNA contains an open reading frame of 1,080 bp encoding a BMT polypeptide of 359 amino acids with a calculated molecular mass of 39 kDa and a calculated pI of 5.9. Sequence alignment revealed the considerable sequence similarity of AdBMT to those of other plant OMTs. The AdBMT sequence contains conserved region I-V, similar to other plant OMTs. His-tagged AdBMT was expressed in E. coli and partially purified by ammonium sulfate precipitation. The recombinant AdBMT is most active in potassium phosphate buffer at pH 7.5 and 35°C. The enzyme does not require a divalent cation for activity and the addition of Cu2+, Ni2+, and Co2+ at concentrations even as low as 0.1 mM severely inhibits enzyme activity. A simple and efficient production of bergapten in the E. coli culture overexpressing AdBMT was performed. The bergapten yield is approximately 13-fold higher than that produced by enzymes in the ammonium sulfate-purified fraction. With the supply of bergaptol in the medium, E. coli cells can be used as a potential bioreactor to produce bergapten.


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