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Molecular Cloning and Functional Analysis of Bergaptol-O-Methyltransferase from Angelica Dahurica (Bai Zhi) and Using It to Efficiently Produce Bergapten in E. coli

白芷佛手柑內醇甲基轉移酶之分子選殖和功能分析以及利用大腸桿菌有效率的量產佛手柑內酯

摘要


白芷中草藥古早以來具有面霜潤膚之美白功效。其中已知的一個美白成分,氫氧基化佛手柑內酯(8-hydroxybergapte)是佛手柑內酯(bergapten)氫氧基化的產物,而佛手柑內酯是由佛手柑內醇甲基轉移酶(bergaptol 5-O-methyltransferase,BMT)轉化佛手柑內醇(bergaptol)而成。從來自於其他植物BMT高保留區的序列中設計一對退化引子(degenerate primers),再從白芷根中選殖出單一的AdBMT DNA片段。利用5’-與3’-RACE-PCR對白芷DNA片段進行聚合酶連鎖增幅反應以取得全長的cDNA序列。此AdBMT可編譯區為1,080個核苷酸,可轉譯出359個胺基酸之蛋白質,預測分子量為39 kDa。經序列比對分析顯示AdBMT和其它植物O-methyltransferases具有相當程度的相同度(identity),序列中含有的I-V區域全部保留。將AdBMT轉形至大腸桿菌可表達出含His-tag的融合蛋白質,進一步硫酸胺鹽沈澱以局部純化蛋白質。AdBMT於pH 7.5之磷酸鉀緩衝液和35°C的反應溫度下的活性最佳,且其活性不須要二價離子的存在。AdBMT的活性受到Cu(上標 2+)、Ni(上標 2+)及Co(上標 2+)等離子的嚴重抑制,即使濃度低至0.1mM。在培養液中含有AdBMT基因之大腸桿菌可簡便有效率的轉化佛手柑內醇(bergaptol)成大量的佛手柑內酯,其產量比經過硫酸胺鹽純化之酵素所能催化的量還多出13倍。大腸桿菌可成為有潛力的生物反應器,以生產佛手柑內酯。

並列摘要


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 Cu(superscript 2+), Ni(superscript 2+), and Co(superscript 2+) 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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