| 摘要: | 近五年來，全球的酵素市場以每年10-15 %的年增率成長，2002 年時市場總值已達到26 億美金。中華民國的工業酵素市場則以12 %的年增率成長，1999 年時市場總值達到5 億新台幣。除工業用外，高附加價值的酵素主要用途則在研究與醫藥的應用。目前世界上以酵素作為綠色科技催化劑有上升的趨勢。化學反應在加速工業製程上常常需要在高溫條件下表現；然而，並非很多的酵素對熱具穩定。能夠在加熱的過程中突破現今的限制並讓蛋白質維持活性和穩定性為工業應用上之研究趨勢。此外，從中溫菌、嗜熱菌、超嗜熱菌的同功蛋白質之間，結構與序列的差異是基礎研究中極重要的課題。微生物可生長於範圍寬廣的環境溫度中，蛋白質若是來自於生長於高溫的生物，其活性與穩定性更佳。微生物根據他們的最適生長溫度可以區分為：超嗜熱性(>80 °C)、嗜熱性(45-80 °C)、嗜中溫性(20-45 °C)與嗜冷性(<20 °C)四類。本研究擬挑選3 株微生物，是根據2006 年8 月31 日前基因體序列已解碼的28 株古細菌(太古生物)與279 株細菌，去除90％的致病性細菌以及中低溫菌，依其最適的生長溫度(>45 °C)、新穎的生理與生態特性及應用在生物科技上的潛力，進行熱穩定蛋白質的高通量篩選。所有原核生物都將培養在他們的最適溫度下，並在對數生長期取出，胞內蛋白體和胞外蛋白體經在 55-125 °C 間，以每10 °C 為間隔加熱處理，經超高速離心方法，製備不同熱處理而不會沉澱之可溶性蛋白質，經一維或二維電泳分離，以介質輔助雷射脫附離子化四極棒飛行時間式質譜儀，高通量分析經過胰蛋白?水解後的勝?序列，可快速鑑定蛋白質。已完備的基因體序列提供我們在大量篩選目標蛋白之高準確性鑑定，這些被確認的蛋白將進一步透過文獻和已知基因功能或生物資訊的預測（如多序列比對法、同功蛋白質家族演算法、酵素資料庫、代謝途徑資料庫等）以判定其功能。因為所挑選之菌株除具有在高溫生長之特性外，亦能在極端pH 值或高鹽度下生存，在可能找到之酵素中，除熱穩定性外，亦可能具有在不同極端環境中之活性，可增加工業生物製程的工業化酵素產品，並提升其應用上之廣度。利用已知基因體序列的微生物，篩選具有潛力的生物技術蛋白質其另一個優點為，在未來可直接從這些中選蛋白質，進行基因選殖或進行蛋白質工程的研究。從300 種嗜熱菌及超嗜熱菌當中，我們初步選擇３種菌作為第一年研究標的。 Geobacillus kaustophilus HTA426 經過30 分鐘、85oC 的熱處理後，還有18 個可溶性酵素存在，在這18 個酵素中，superoxide dismutase, peroxiredoxin 及thioredoxin 將做為選殖目標以確認它們的熱穩定性。Thermus thermohpilus HB8 在經過30 分鐘、100oC 熱處理後，有26 個熱穩定蛋白質被鑑定出來，pullulanase, transaldolase 及superoxide dismutase 將做選殖來定性。此外，一系列熱處理蛋白質體的蛋白質將利用二維膠體電泳分離與質譜之鑑定。這些獲得之蛋白質經高通量生物資訊分析，以進一步篩選熱穩定蛋白，之後可進行基因選殖、表達和定性研究。 The global enzyme market is 2.6 billions US$ in 2002 with a 10-15 % average annual growth for the past five years. The market for industrial enzymes in R.O.C. is 0.5 billions NTD in 1999 with a 12 % average annual growth. The high added value enzymes are major used in research and medical applications. The world trend in using enzymes as catalysts in green technology is increased. Chemical reactions often need to be performed at high temperatures to accelerate industrial processes. However, not many enzymes are stable to heat. Research is required to make proteins remain active and stable while being heated to overcome current limits on their industrial applications. Additionally, investigations of differences between the structure and sequence of homologous proteins from mesophilic and thermophilic/hyperthermophilic organisms are crucial topic in basic research. Microorganisms have been found to grow in the environment with a very wide range of temperatures. Proteins from an organism grown in high temperature are more active and stable in such temperatures. Microorganisms have been grouped into growth temperature classes in relation to their temperature optima: hyperthermophiles (>80 °C), thermophiles (45-80 °C), mesophiles (20-45 °C), and psychrophiles (<20 °C). According to Comprehensive Microbial Resource (CMR, www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl) by August 31, 2006, there are total 28 species in Archaea and 279 species in Bacteria, which genomes were completely sequenced. Among them, 90% of the bacteria are pathogens, mesophiles and psychrophiles which are not the focus in this proposal. Three species from thermophile or hyperthermophile prokaroytes are selected based on their optimal growth temperatures, physiological and ecological novelty and potential application in biotechnological processes. All the bacteria will be grown at their optimal temperatures and harvested at log phase. The proteomes prepared from secretomes and cell lysate were prepared and treated at temperatures from 55-125 °C (with 10 degree interval) followed by ultracentrifugation to obtain the soluble proteins. After 1D or 2D-gel electrophoresis to separate the soluble thermal stable proteins, matrix-assisted laser desorption ionization (MALDI)-Q-Tof mass spectrometer is used for high-throughput protein identification by peptide sequences from the trypsin-digested peptide mixtures. The available microbial genomes allow us to accurately identify the target proteins in a high-throughput formate. The identified proteins are further analyzed for their function either through literature, known gene annotation or bioinformatics prediction (such as multiple sequence alignment, protein family identification algorism, enzyme batabase and pathway database etc.). Enzyme products with higher operation temperatures and/or extreme pH or salt concentration are anticipated to be selected to be applied in many industries. One more advantage of using organisms with known genome sequences is that the identified thermal proteins with potential biotechnological applications can be cloned directly and engineered in the future. Among the 300 thermophiles and hyperthermophiles, we preliminarily selected three species for the first year study. Eighteen enzymes from Geobacillus kaustophilus HTA426 were shown to be remained in soluble form at temperature≧85 oC for 30 minutes. Among them, superoxide dismutase, peroxiredoxin, and thioredoxin are the candidates for our future cloning work to confirm their thermal stability. For Thermus thermohpilus HB8, there are total of 26 soluble proteins were identified to be stable from 100oC/30 min-treated sub-proteomes. Pullulanase, transaldolase and superoxide dismutase will be cloned for characterization. Further proteomic identification of proteins from the heat-treated proteomes will be conducted by 2D gel electrophoresis. High-throughput bioinformatics analysis of identified proteins will be anticipated to screen the protein thermal stability before individual cloning is conducted. 研究期間：9601 ~ 9612 |
