甘草甜素及阿拉伯芥中細胞色素P450 氫氧化酶之分子選殖、蛋白質表現及其功能性分析

Abstract

Triterpene saponins constitute a family of compounds with a triterpene aglycone and sugars structures. They were widely distributed in various species such as food, echinoderm, fungi and plants. Recent researches have demonstrated that triterpene saponins exhibited different biological activities from antiinflammatory, antifungal, antimicrobial, antiparasitic, and antitumor activity. Biosynthetic studies suggested that the structure diversity of triterpene saponin is generated by (1) the cyclization of oxidosqualene to various triterpene skeletons, (2) the subsequent hydroxylation or oxidation at multiple positions of triterpene, and (3) glycosylation to add sugar moieties through an ether or ester glycoside linkage. Based on our previous investigations on the S. cerevisiae oxidosqualene-lanosterol cyclase (ERG7), we have identified several particular amino acid residues associated with the cyclization and acquired various novel intermediated structures via proceeding with mutation on OSC. According to the biosynthesis of triterpene saponins, the first modification, followed by triterpene cyclization, is hydroxylation. In order to further diversify the scaffold of triterpene saponins for the potential pharmaceutical applications, in this thesis, we cloned and functionally expressed three hydroxylases, CYP88D6 (Glycyrrhiza uralensis, C-11 hydroxylase), CYP90B1 (Arabidopsis thaliana, C-22 hydroxylase), and CYP90D1 (Arabidopsis thaliana, C-23 hydroxylase) for activity study. Various expression systems including E. coli or yeast, coupled with a series of expression vectors, have been used to clone these artificial genes successfully. Even most of them failed to produce the recombinant proteins as predicted, the CYP88D6 from pRSETa-CYP88D6 plasmid and CYP90B1 from pET30a-CYP90B1 plasmid were over-expressed in E.coli BL21(DE3)pLysS strain and were confirmed the protein identity by MALDI-TOF/MS spectrum. Moreover, we have examined the potential hydroxylation activity of CYP88D6 and CYP90B1 by using the reconstituted assay with human NADPH-cytochrome P450 reductase and NADPH. The microsome fraction from E. coli expressing G. uralensis CYP88D6 or A. thaliana CYP90B1 exhibited their catalysis ability in transforming a hydroxyl group into the scaffold of the substrates, β-amyrin or campesterol, to form the respective hydroxyl products with predicted molecular mass from the ion extraction of GC–MS analysis, implying a functional activity of expressed cytochrome P450 hydroxylase, respectively. The partial purification of G. uralensis CYP88D6 or A. thaliana CYP90B1 were carried out via Ni-NTA column purification or Q-sepharose purification, and characterized by SDS-PAGE electrophoresis as well as UV-vis spectrum analysis at its Soret band absorption at 416 nm. Various modifications on the production and purification of functional cytochrome P450 hydroxylase are on the process in hope of acquiring the large scale of purified protein for studying their reaction mechanism and being subjected for sterol compounds’ tailoring reactions to produce various sterol derivatives, that carrying various biological activities to be applied in pharmacology. Moreover, mutagenic effect on product specificity as well as on the reaction efficiency will also be explored for tuning the expressed cytochrome P450 hydroxylase in future.