| 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2007年6月30日前基因體序列已解碼的41株古細菌(太古生物)與486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43種嗜熱菌及超嗜熱菌當中，我們初步選擇5株菌作為研究標的。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。截至目前，95年1月至96年9月之執行成果，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%。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，其中27個較具工業應用價值的熱穩定酵素（GK2713和GK2820具纖維素分解酵素保守區域之假定蛋白質）將進行後續基因選殖和表現。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，其中pullulanase, transaldolase及superoxide dismutase將進行選殖以定性之。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，其中oligo-1,6-glucosidase, alpha-galactosidase, Xaa-Proaminopeptidase, pullulanase, 3-hydroxybutyryl-CoA dehydratase, transaldolase及superoxide dismutase將進行選殖以定性之。Methylococcus capsulatus Bath在經過30分鐘、75oC熱處理後，有29個熱穩定蛋白質被鑑定出來。第三年將持續從其中三株菌，共選定10個基因優先進行選殖、表達並進行專利申請。此外，我們將以所建立的平台應用在嗜超高溫菌Sulfolobus acidocaldarius DSM 639 (Topt.:80°C)和Aeropyrum pernix K1 (Topt.:90°C)的抗熱酵素篩選。在高溫環境中獲得之混合菌及分泌混合蛋白體方面，則將以metaproteomics方法進行分析。故97年將以之前建立的平台為基礎，增加環境微生物混合菌之蛋白體之製備與分析平台。 ; 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 June, 2006, there are total 41 species in Archaea and 486 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 43 thermophiles and hyperthermophiles, we preliminarily selected five species for the study. 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℃ from G. kaustophilus ATCC8005, respectively. The identified proteins are further analyzed through literature and patent for their potential in applications. Among 50 identified thermostable enzymes with high added value (GK2713 and GK2820 contained cellulose conserved domain) 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. Oligo-1,6-glucosidase, alpha-galactosidase, Xaa-Proaminopeptidase, pullulanase, 3-hydroxybutyryl-CoA dehydratase, transaldolase and superoxide dismutase will be cloned for characterization. For Methylococcus capsulatus Bath, there are total of 29 soluble proteins were identified to be stable from 75oC/30 min-treated sub-proteomes. Methanol dehydrogenase will be cloned for characterization. After an extensive patent search and survey, 2D heat stable protein from threethermophiles will be cloned and expressed for patent application. In 2008, the following works are anticipated: 1. Cloning, expression and characterization of 10 identified enzymes from 3 thermophiles. 2. Identification of thermal stable proteins from two hyperthermophile (A. pernix K1 and S. acidocaldarius DSM639) by using the established plate form. 3. Metaproteomics analysis of mixed culture from thermal environmental samples. 4. Establish a database containing prokaryotic thermal stable proteins (PTSPdb). ; 研究期間 9701 ~ 9712 |
