白芷Cinnamate 4-hydroxylase基因之選殖、功能性分析及8-氫化佛手柑內酯美白物質量產之研究

Cloning and Functional Expression of Cinnamate 4-hydroxylase Gene from ＂Angelica dahurica＂ (Bai Zhi) and Producing 8-hydroxybergapten in Yesat

羅淑卿(Shu-Chin Lo) ； 鍾佩恩(Pei-En Chung)

《台灣農業研究》 63卷3期 (2014/09) Pp. 205-215

https://doi.org/10.6156/JTAR/2014.06303.03

摘要

Cinnamate 4-hydroxylase（C4H）為苯基丙烷類代謝路徑（phenylpropanoids）之酵素，合成許多植物重要二級代謝產物，包括白芷藥性成分呋喃香豆素（furanocoumarins）。為探討白芷C4H基因及酵素特性，由其它物種C4H高保守性區域序列中，設計退化性引子，從白芷根中選殖出單一的＂AdC4H＂ cDNA部分片段。利用5'-與3'-RACE-PCR對此白芷＂AdC4H＂片段進行聚合酶連鎖增幅反應以取得全長cDNA序列（accession no. KJ453107）。此＂AdC4H＂ cDNA之可編譯區含1,518個核苷酸，可轉譯為505個胺基酸，分子量預估為57.9kDa。AdC4H經胺基酸序列分析比對顯示和其它植物C4H有74%以上的相同度，具cytochrome P450特有的A–F保守性區域及6個受質辨識區。將＂AdC4H＂可編譯區cDNA建構在pYES2/NT載體，另外選殖阿拉伯芥cytochrome P450 reductase ＂ATR1＂ cDNA全長並建構於pYES3/CT載體，以缺乏色胺酸及尿嘧啶之培養基篩選，取得併存二載體（pYES-C4H及pYES3-ATR1）之轉形酵母菌（＂Saccharomyces cerevisiae＂），大量表現融合蛋白質後，進一步純化融合蛋白以探討酵素特性。酵素生化特性研究顯示AdC4H於25℃、pH 6.0之磷酸鉀緩衝液具最佳活性。AdC4H在ATR1存在下，催化cinnamate羥基化效率明顯增加，產物ρ-coumaric acid含量由偵測不到提升至62.9 μM。具備二載體之轉形酵母菌亦可催化bergapten為8-hydroxybergapten，培養液具抗酪胺酸酶活性，IC_(50)為1.35 mg mL^(-1)。本研究證實AdC4H屬cytochrome P450第二群家族成員，可能為膜蛋白，酵素活性在不同物種之還原酶存在下依然可以顯現。

關鍵字

白芷 ； 細胞酵素P450

並列摘要

Cinnamate 4-hydroxylase (C4H) is an enzyme of phenylpropanoids pathway, which can synthesize plant secondary metabolites, including furanocoumarin, a type of pharmaceutical components in Bai Zhi. To characterize ＂C4H＂ gene, the complementary DNA of ＂AdC4H＂ was cloned from Bai Zhi root using degenerate primers designed from the highly conserved regions of other plant ＂C4H＂. The tandem 5'-and 3'-rapid amplification of cDNA ends via polymerase chain reaction was used to obtain the full-length ＂AdC4H＂ cDNA sequences (accession no. KJ453107). The ＂AdC4H＂ cDNA contains an open reading frame of 1,518 bp encoding a polypeptide of 505 amino acids with a molecular mass of 57.9 kDa. Amino acid sequence alignment demonstrated over 74% identities on protein level between AdC4H and its homolgous proteins among plant species. They possess all cytochrome P450- featured conserved region A–F and six substrate recognition sites. The full length of ＂AdC4H＂ coding region was introduced into pYES2/NT vector. Besides, the full length of cytochrome P450 reductase ＂ATR1＂ cloned from Arabidopsis thaliana was introduced into pYES3/CT vector. The transformed yeast cell (＂Saccharomyces cerevisiae＂) harboring two different plasmids (pYES-C4H and pYES3-ATR1) were obtained by growing in the medium without containing tryptophan and uracil. The recombinant AdC4H protein was overexpressed in yeast and partially purified. The recombinant AdC4H revealed optimum activity in pH 6.0 potassium phosphate buffer at 25℃. It suggested that ＂AdC4H＂ together with ATR1 overexpression showed highly efficient catalysis of 4-hydroxylation of cinnamate, the ρ-coumaric acid was increased from undetectable to 62.9 μM. The yeast harboring two plasmids also catalyzed bergapten to 8-hydroxybergapten. The IC_(50) value of tyrosinase inhibition in the medium was 1.35 mg mL^(-1). The ＂AdC4H＂ enzyme that belongs to a member of cytochrome P450 ClassII family may be a transmembrane protein and its enzyme activity can be detected with reductase from different plant species.

並列關鍵字

＂Angelica dahurica＂ ； Cinnamate 4-hydroxylase ； Cytochrome P450