張文章2006-07-252018-07-062006-07-252018-07-062004-07-31http://ntur.lib.ntu.edu.tw//handle/246246/102531. 台灣嗜熱菌種源庫的建立與應用：已有六十五座溫泉當中的微生物被調查與研究，其 中已有超過一千餘株的嗜熱細菌被分離、純化與培養，並建立起本土台灣嗜熱菌種源 庫。所收集菌株包含革蘭氏陽性的Bacillus sp.， Geobacillus sp.， Alicyclobacillus sp.， Brevibacillus sp.， Deinococcus sp.， Rubrobacter sp. 及 Paracraurococcu sp. 等屬。革 蘭氏陰性的分離株則包括Thermus sp.， Meiothermus sp.， Rhodothermus sp.， Chloroflexus sp.， Roseiflexus sp.， Thermonema sp. 及 Pseudoxanthomonas sp. 等屬 (Chen. 2002)。在這些眾多的嗜熱菌分株中，許多分離株是屬尚未在文獻中發表的新 種，如具絲狀細胞的 Meiothermus taiwanensis WR-30T、具輻射線抗性的Rubrobacter taiwanensis LS-293T、具高溫脫氮活性的Pseudoxanthomonas taiwanensis CB-226T 等 (Chen et al., 2002a；Chen et al., 2002b; Chen et al., 2004)。 2. 具高酵素活性嗜熱菌株之篩選與熱穩定性蛋白酶基因之選殖：對本土嗜熱菌所作進行 酵素活性的分析結果中，發現有20% 菌株具澱粉水解酶活性，25%菌株具蛋白酶活性 及38%菌株具脂質水解酶活性。其中分離株 Brevibacillus thermoruber WR-249 的 Lon protease 基因已被選殖出並研究其特性。B. thermoruber Lon gene (Bt-lon)大小為 88-kDa ， 結構上包含N-terminal domain 、ATPase domain 、SSD (sensor- and substrate-discrimination) domain 及 C-terminal protease domain 等四個domain. Bt-lon 是 一個產物具六個單元體的熱誘導性基因，具chaperone-like 的活性，在70 oC 相當穩定 且最適催化溫度為 50 oC， 在細菌的生理調控上扮演相當重要的角色。 3. 嗜熱菌熱穩定性轉胺酶菌株之篩選與基因之選殖：我們選取四種轉胺酶作為研究之對 象， 這四種轉胺轉胺酶包括aspartate aminotransferase (AspAT) 、phenylalanine aminotransferase (PheAT)、branched-chain amino acid aminotransferase (BCAT)及D-amino acids aminotransferase (DAT)等四種。在初步的研究結果中，抗紫外線與輻射分離株 Deinococcus taiwanensis 407T 及 D. taiwanensis 539 具高 AspAT 及 BCAT之活性. 高 溫脫氮菌 Pseudoxanthomonas taiwanensis CB-225 具高AspAT 及PheAT 之活性。 4. 熱穩定性酵素基因的選殖載體構築與表現系統的建立：目前已利用快速篩選質體的方 式，篩選出53 株具質體的高溫菌株，並利用當中NTU-103 菌株質體建構出一個shuttle vector-p103，未來將以其為基礎，嘗試表現由本土嗜熱菌所選殖出熱穩定酵素的基因， 以達到大量表現酵素的目的。 5. 高抗性菌株之研究與利用：在研究中本研究群發現許多具高紫外線及輻射線抗性的菌 株，如具多樣酵素活性與高紫外線抗性及高γ-radiation 抗性的新種菌株 Rubrobacter taiwanensis LS-293T 及 Deinococcus taiwanensis WR-407T，極具作為表現載體的潛力。 Deinococcus taiwanensis NTU 407T 及其它分離株除了是從台灣地熱溫泉區中所分離的 本土新種菌株外，其最大的特色是耐高溫、抗輻射線及抗紫外線。分離菌株 NTU 407 T 紫外線抗性達 1500 Jm-2，約為 E coli 的 50 倍，而迦瑪輻射線的抗性則達到 15 kGy，約為 E. coli 的 200 倍。多樣的生理生化特性，顯示此本土菌株在研究與應用上 的高度潛力。1. Isolation of thermophilic bacteria and construction of strain library: We have surveyed 65 hot springs in Taiwan to collect and analyze the bacteria contained therein. The bacterial strains were isolated, purified, cultured and identified. Up to date we have obtained over 1000 bacterial strains. These are deposited in Academia Sinica, National Taiwan University and Tzu-Chi University. These bacteria were identified by Gram stain, electron microscopy and 16S rDNA sequencing and enzymatic analysis. They were found to belong to Bacillus sp.， Geobacillus sp.， Alicyclobacillus sp.， Brevibacillus sp.， Deinococcus sp.， Rubrobacter sp., Paracraurococcu sp., Thermus sp.， Meiothermus sp.， Rhodothermus sp.， Chloroflexus sp.， Roseiflexus sp., Thermonema sp. and Pseudoxanthomonas sp. 2. Enzymatic assay: Analyses of the enzymatic properties revealed that 20 % of the thermophilic strains contain amylase activity, 25 % contain protease activity and 38 % contain lipase activity. These strains are being analyzed in order to find bacteria with higher enzymatic activity. 3. Thermostable aminotransferases: Four aminotransferases were selected for study, including aminotransferase (AspAT), phenylalanine aminotransferase (PheAT), branched-chain amino acid aminotransferase (BCAT) and D-amino acids aminotransferase (DAT). In preliminary studies, Deinococcus taiwanensis 407T and D. taiwanensis 539 with highly enzymatic activities of AspAT and BCAT. Pseudoxanthomonas taiwanensis CB-225has highly enzymatic activities of AspAT and PheAT. 4. We have isolated several new strains with remarkable resistance to UV and gamma-radiation, such as Rubrobacter taiwanensis LS-293T and Deinococcus taiwanensis WR-407T. By using high dose of UV and gamma-radiation to treat the thermophilic bacteria we also found 87 strains of thermophilic and radiation-resistant bacteria, which can grow at temperatures from 50~70 °C. By 16S rDNA sequencing we found some of these bacteria to belong to Deinococcus sp., Rubrobacter sp., Bacillus sp., Geobacillus sp., Thermus sp. 5. Gene cloning of thermophilic enzymes and construction of a shuttle vector: We have screened 53 bacterial strains which contain plasmids and constructed a shuttle vector by using the plasmid existing in NTU-103. This shuttle vector will be useful for the construction of expression vector for the large-scale expression of thermophilic enzymes.application/pdf158586 bytesapplication/pdfzh-TW國立臺灣大學生化科學研究所reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/10253/1/922317B002027.pdf