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

Abstract

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.