| 摘要: | 全球對酵素的需求未來將以每年7%的成長至2009年的50億美元左右，高附加價值的酵素主要用途在工業、醫藥與研究的應用。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性。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。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，其在高溫之活性與穩定性更佳。微生物根據他們的最適生長溫度可以區分為：超嗜熱性（>80℃）、嗜熱性（45-80℃）、嗜中溫性（20-45℃）與嗜冷性（<20℃）四類。本研究建立以一嗜熱性細菌進行高通量篩選熱穩定蛋白質之新穎方法。選取Geobacillus kaustophilus ATCC8005將其培養在最適溫度（55℃），並於對數期取出菌體，去除細胞膜部份，將胞內蛋白體經由熱處理，再經超高速離心，取得經熱處理但不會沉澱之可溶性次蛋白體，經三種蛋白體學方法（一維或二維電泳分離蛋白質之膠體內胰蛋白脢消化或直接將次蛋白體消化），以兩種不同質譜儀分析不同來源蛋白質之胜肽序列，可高通量且快速鑑定蛋白質。已完成基因定序的G.kaustophilus HTA426提供我們在大量篩選熱穩定蛋白之高準確性鑑定。G. kaustophilus ATCC8005 胞內蛋白體經過不同溫度（55、65、75、85、95及100℃），30分鐘的熱處理後，分別篩選出330、207、114、75、44和32個熱穩定蛋白質，酵素各佔了74、70、58、45、34和31%。依照不同溫度鑑定到的蛋白質以COG進行功能分類：三類功能之蛋白質（轉譯和蛋白質後修飾、代謝及伴隨蛋白和無機離子代謝），較具熱穩定性。另外，在G. kaustophilus ATCC8005熱穩定蛋白體中小分子量（>30 kDa）、pI值在3-4.9和9-11.2的蛋白質較具熱穩定性。在驗證我們的方法上，分三個方向進行：（一）以17個蛋白質在不同溫度之量的變化所預測的Tm值，與其熱處理溫度比較；（二）以目前已發表純化自Geobacillus屬的13個蛋白質，與本研究鑑定到相同的熱穩定蛋白質比較，其中11個的Tm值或最適反應溫度與本研究熱處理篩選的溫度相近；（三）以生物資訊方法，預測所得熱穩定蛋白質之Tm或熱穩定的機率，進行相關性探討。 這些被確認的蛋白質進一步透過文獻與專利搜尋，推薦高附加價值的熱穩定酵素進行基因選值與智慧財產保護。 The world demand for enzymes is expected to increase 7 percent per year to nearly US$5 billion in 2009. The high value-added enzymes are most needed in industries, medical and research applications. 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. Accordingly, investigations of the key features on structure and sequence from thermophilic and mesophilic proteins are crucial topic in basic research. Microorganisms have been found to grow in environments at a very wide range of temperatures. Proteins from an organism grown at high temperature are more active and stable in such temperatures. Microorganisms are grouped into different classes according to their temperature optima: hyperthermophiles (>80℃), thermophiles (45-80℃), mesophiles (20-45℃), and psychrophiles (<20℃). This research plans to establish a high-throughput screening of thermostable proteins by using proteomic approach. Geobacillus kaustophilus ATCC8005, a thermophilic bacterium was grown at its optimal temperature (55℃) and harvested at log phase. The crude cell extracts were obtained followed by a 55-100℃ treatment for 30 min. The soluble sub-proteomes were obtained after ultracentrifugation to remove aggregated proteins. After separating by 1D and 2D-gel electrophoresis, the proteins were digested by trypsin in-gel. Another approach is the proteomes of which the proteins were digested without separation. The MALDI-Q-Tof MS and ESI-Q-Tof MS were used to identify proteins from 2D PAGE and 1D PAGE/in-solution digestion, respectively. The sequenced genome from G. kaustophilus HTA426 allows us to accurately identify the target proteins in a high-throughput format. There were a total of 330, 207, 114, 75, 44 and 32 thermostable proteins identified at 55, 65, 75, 85, 95 and 100℃, respectively. There are three methods to confirm the accuracy of our research: (1) the Tm of 17 thermostable proteins were predicted by semi-quantification of spots volume change from different heated temperatures; (2) comparison of 13 thermostable proteins from current studies to purified proteins (Geobacillus sp.): 11 thermostable proteins were shown to possess very close properties with the literature report; (3) the Tm of 17 thermostable proteins were predicted to be reasonable with the heat treatment results. The identified proteins are further analyzed through literature and patent for their potential in applications. Among them, peroxiredoxin, thioredoxin, superoxide dismutase, acetyl-CoA acetyltransferase, branched-chain amino acid aminotransferase and hypothetical protein GK2713/GK2820 (contained cellulase conserved domain) are the candidates for our future cloning work to confirm their thermal stability. |
