芽孢桿菌屬ER1酪胺酸酶基因之蛋白質表達與活性分析

摘要學號 : M10418021 論文題目：芽孢桿菌屬ER1酪胺酸酶基因之蛋白質表達與活性分析總頁數：183 學校名稱：國立屏東科技大學所別：生物科技系畢業年月：研究生：廖科捷指導老師：徐志宏論文摘要內容：生物暴露在嚴重紫外光傷害之下，使皮膚表皮中之黑色素生成，黑色素不僅影響皮膚美觀使皮膚變得黝黑，過量黑色素會造成皮膚病變，嚴重導致黑色素腫瘤產生。酪胺酸酶 (tyrosinase) 為形成黑色素重要的關鍵，藉由陽光、發炎和自由基而產生活化，將體內中的酪胺酸 (tyrosine)轉換成多巴 (dihydroxyphenylanine，L-Dopa)，在轉換形成多巴色素(dopachrome)，接著透過化學反應機制與酵素催化的作用下，就形成了黑色素；因此抑制酪胺酸酶的活性，就能降低黑色素的形成，透過基因上的調控，控制酪胺酸酶的產生也是個值得研究的方法。本研究是採用本實驗室從篩選得到芽孢桿菌 ER1菌株中選殖出酪胺酸酶基因Tyr以及兩種過氧化物酶調控轉錄基因OhrR與PerR，其開放閱讀框架 Tyr為894 bp可編碼出297個胺基酸蛋白質，OhrR為450 bp可編碼出149個胺基酸蛋白質，PerR為435 bp可編碼出144個胺基酸蛋白質。接著分別構築於原核表達載體 pET28a與pET29a上，接著轉型至大腸桿菌表達菌株中進行重組蛋白質異源性表達，利用Ni2+親和性層析管柱來進行蛋白質純化，最後得到分子量Tyr為36.3 kDa、OhrR為19.87 kDa和 PerR為18.38 kDa可溶性重組白質。將Tyr純化後蛋白質進行活性分析，發現Tyr蛋白質於pH4弱酸、溫度40℃環境下為最適活性條件，有助於日後生產高活性之蛋白質，在未來酪胺酸酶 (Tyr) 可作為美白化妝用品之應用，例如運用在測試美白化妝品之標準樣品，此外透過四種抑制劑arbutin、 kojic acid、 tropolone與 glutahione進行抑制實驗，測試結果都能成功抑制酪胺酸酶活性，最高達到約98% 抑制率。後續研究 OhrR與 PerR兩種調控轉錄基因是否跟 tyrosinase之調控有關，如果這兩種基因影響 tyrosinase之生成，或許可控制 tyrosinase的產生，對於未來相關生物技術之運用上極具潛力。關鍵字：芽孢桿菌、酪胺酸酶、過氧化酶、黑色素、調控

關鍵字

芽孢桿菌；酪胺酸酶；過氧化酶；黑色素；調控

並列摘要

Abstract Student ID: M10418021 Title of Thesis: Protein expression and activity analysis of a Tyrosinase gene from Bacillus sp. ER1 Total page :182 Name of institute: National Pingtung University of Science and Technology Graduate Date : December , 2018 Degree Conferred: Master Name of student: Ke-Jie Liao Adviser: Douglas J. H. Shyu, Ph.D. The Contents of Abstract in This Thesis : Tyrosinase (E.C. 1.14.18.1) is an important key enzyme for the formation of melanin in melanocyte in mammals. It converts tyrosine into L-DOPA and then DOPAquinone, followed by several enzymes in the melanogenesis pathway. Therefore, inhibition of tyrosinase activity has been developed as a standard test for the evaluation of whitening cosmetics to prevent the melanin production.In this study, a tyrosine gene Tyr and two peroxidase regulatory genes OhrR and PerR, these was cloned from the environmental bacteria, Bacillus ER1 strain. The Tyr gene have total length of 894 bp open reading frame encoding for a 297 amino acids protein,OhrR have 450 bp open reading frame encoding for a 149 amino acids, OhrR have 435 bp open reading frame encoding for a 144 amino acids.The tyr, OhrR and PerR gene was cloned into a prokaryotic expression vector pET28a and pET29a for the production of recombinant His-tagged Tyr protein in E. coli BL21(DE3).The recombinant fusion protein was purified by using Ni2+ affinity chromatography . eluted as a soluble TyR protein with a molecular weight of 36.3 kDa,OhrR and PerR respectively 19.87 kDa and 18.38 kDa. Enzyme activity under 40℃temperature and 4 pH Weak acid environment conditions It is the most suitable active condition to help produce highly active protein, In the future, tyrosinase (Tyr) can be used as a whitening cosmetic, for example, as a standard sample for testing whitening cosmetics. In addition, inhibition experiments were carried out through four inhibitors, arbutin, kojic acid, tropolone and glutahione. The test results can successfully suppress the activity of tyrosinase, up to about 98% inhibition. Follow-up study Whether OhrR and PerR regulate transcription genes are related to the regulation of Tyrosinase, if these two genes affect the production of Tyrosinase, it may control the production of Tyrosinase. There is great potential for the use of related biotechnology. Key word: Bacillus, tyrosinase, peroxidase, melanin, regulatory