- 學位論文
牛樟芝抗氧化酵素—細胞色素c過氧化酶與銅鋅型超氧歧化酶之分子選殖、蛋白質表現及功能性分析
Molecular cloning, characterization, expression and functional analysis of anti-oxidative cytochrome c peroxidase and copper/zinc superoxide dismutase from Antrodia cinnamomea
摘要
牛樟芝(Antrodia cinnamomea) 為生長於台灣特有種之牛樟樹(Cinnamonum kanehirae Hayata) 樹心空胴壁處之珍貴且特別的醫藥菇蕈。牛樟芝具有多種有益生理活性，其中包括強抗氧化成份而使其具備良好抗發炎效果。抗氧化與抗發炎可有效抑制活性氧物種(reactive oxygen species, ROS)，而牛樟芝抗氧化成份中含不少抗氧化酶，其細胞色素c過氧化酶(cytochrome c peroxidase, CcP)、銅鋅型超氧歧化酶(superoxide dismutase, Cu/Zn SOD) 均具清除自由基之抗氧化效果。超氧歧化酶具催化超氧自由基(超氧陰離子) 成過氧化氫和氧分子的能力，目前超氧歧化酶因其活性位所結合的金屬不同可分為四種類型：銅鋅型、錳型、鐵型、鎳型。細胞色素c過氧化酶則具催化過氧化氫成水分子的能力。由分析先前已建構完成之牛樟芝(Antrodia cinnamomea) cDNA library，推測可能為細胞色素c過氧化酶、超氧歧化酶者，將兩者 cDNAs 上傳於 NCBI GenBank 與 Swiss-Prot database 上比對鑑定確實符合該酵素特性，且歸屬於可知該超氧歧化酶類型應為銅鋅型；同時收集分析相近物種之酵素胺基酸序列，以找出保守性胺基酸位置。接著，再由多方面生物資訊分析該基因與胺基酸特性，以預測該蛋白質結構，以及是否具 signal peptide、transmembrane domain、醣基化等資訊，作為異源表現兩種酵素的依據；同時，對牛樟芝之細胞色素c過氧化酶與銅鋅型超氧歧化酶進行類緣分析，結合所得之生物資訊學的資料，從中發現有關這兩個酵素在演化上可能代表的意義，並進而解釋牛樟芝之序列獨特性。為更近一步探究牛樟芝細胞色素c過氧化酶和銅鋅型超氧歧化酶，我們將牛樟芝 gDNA 與 cDNA 全長已解序之兩種抗氧化酶開放讀碼區(open reading frame, ORF) 建構於適合表現的載體上，並將其轉入大腸桿菌(Escherichia coli) 系統中，誘導重組蛋白之表現，再以 Ni-NTA 親和性管柱純化出該重組蛋白。牛樟芝細胞色素過氧化酶在大腸桿菌系統中有較好重組蛋白之表現，對其進行酵素活性測試測得該酵素在酸鹼值 pH 7與 pH 5，溫度42 ℃有最佳的活性；在最佳條件下進行酵素動力學之分析，得到 Vmax=1.06×10-5 M/min 及 Km=0.161 M。牛樟芝細胞色素c過氧化酶具有清除過氧化氫的能力，這樣的現象也同樣可印證在正常倉鼠卵巢細胞(chinese hamster ovary cell, CHO cell) 模式上，清除細胞外添加之過氧化氫有助於避免細胞內產生過量之活性氧物種，達到保護細胞免受胞內過量活性氧物種之傷害。牛樟芝細胞色素c過氧化酶被設計以褐藻膠包覆進行微膠囊化，以期待該酵素能有儲藏之穩定性。然而，微膠囊化在製備的過程卻會導致該酵素活性幾乎喪失，且儲藏之穩定性僅能維持兩周，整體來說，該酵素若要進一步進行微膠囊化之實際應用，改善過程中大量活性喪失的情況是最主要的關鍵所在，故酵素方面的微膠囊化方面仍有待研究探討。
並列摘要
Antrodia cinnamomea is an unique precious medicinal mushroom, inhabiting on the inner cavity wall of Cinnamonum kanehirae Hayata, an endemic species of Taiwan. Many beneficial biological activity of Antrodia cinnamomea have been reported. One of them is anti-oxidation and anti-inflammation against the reactive oxygen species (ROS). Cytochrome c peroxidase (CcP) and Cu/Zn superoxide dismutase (Cu/Zn SOD) both are anti-oxidative enzymes. Superoxide dismutase is capable of catalyzing the dismutation of the superoxide ion (O2•−) to hydrogen peroxide and molecular oxygen. Based on the affinity of metal elements which bind to the active sites, superoxide dismutase can be classified into four types：Cu/Zn, Mn, Fe, and Ni. Cytochrome c peroxidase is a mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water. The putative SOD and CcP genes were accessed in our previously constructed Antrodia cinnamomea cDNA library, and were demonstrated homologous to Cu/Zn SOD and CcP while blasting NCBI GenBank and Swiss-Prot database. The amino acid sequences were used to acquire the bioinformatics from bioinformatics resource portal, in order to figure out the characterizations of the two anti-oxidative enzymes and find the conditions of heterologous protein expression. Meanwhile, the phylogeny analysis of cytochrome c peroxidase and Cu/Zn superoxide dismutase show the significance of species evolution and can be used to explain the unique characteristics of Antrodia cinnamomea by the bioinformatics of amino acid sequences. The putative AC-Cu/Zn SOD and AC-CcP were cloned, characterized, and their gDNA and cDNA full-length was worked out. The open reading frame of AC-Cu/Zn SOD and AC-CcP were constructed into E. coli expression vector. The recombinant protein was purified to homogeneity by Ni-NTA affinity chromatography. The recombinant protein of AC-CcP was well expressed in E. coli expression system. In enzyme acivity test, AC-CcP had the best activity at pH 7, pH 5 and best reaction temperature at 42 ℃. In the best reaction condition, the result of the enzymatic kinetics got AC-CcP Vmax=1.06×10-5 M/min and Km=0.161 M. AC-CcP had ability to eliminate hydrogen peroxide. It also can find that AC-CcP prevent ROS in CHO cell (chinese hamster ovary cell) from overproducting. Therefore, AC-CcP should had the ability to protect cell from damege that induced by hydrogen peroxide. The microencapsulation of AC-CcP were coated with alginate and hope to provide the enzyme with stability of storage. However, the process led to loss the activity almost. The storage stability of the microcapsulated AC-CcP was just 2 weeks. In the case of microencapsulating enzyme, how to enhance activity of enzyme was an importance issue to be investigated.