杜 鎮

杜 鎮
Tu, Jenn
退休研究員

德國慕尼黑大學博士 (1982)

TEL: 

+886-2-2787-1185 (Office)
+886-2-2787-1076 (Lab)

研究主題:應用與演化微生物學;酵素學;蛋白質工程學

多醣類分解細菌的分離與酵素工程
 

(1) Paenibacillus木聚醣酶選殖及其酵素工程

與清除污染和生質再利用的生物資源技術是我門有興趣的課題 之一。為此目的,我們由製漿、洗漿流程的黑液中篩選出具有多種 多醣類水解能力的菌株Paenibacillus campinasensis BL11 (基因編 碼: DQ232773)並選殖了它的木聚醣酶(endo-1, 4-β-xylanase,基 因名稱:xylX,基因編碼:DQ241676)。這個酵素的基因由1,131個 核苷酸組成,產生一個大小約41 kDa的酵素。它的N-端有一個由39 個胺基酸組成的訊息胜肽。酵素圖譜分析顯示BL11的木聚醣酶是一 個分泌型蛋白,粗酵素的最佳反應活性出現在60°C、pH 7。在理想 狀況下該酵素的活性為2392 IU/mg,即使在pH 11中仍保有517 IU/ mg。為了因應工業需求,我們建造了一個木聚醣酶的突變基因庫並 從中篩選抗熱型的突變株。從這個基因突變庫中分離到兩個抗熱突 變株,一個是胺基酸取代突變株(酥胺酸44丙胺酸);另一個是下游片 段缺失突變株。下游片段缺失突變株遺失了該酵素大部分的糖水化 合物附著區(圖一)。在67.5oC 和 70 oC之間,胺基酸取代突變株的活 性為原來兩倍;而下游片段缺失突變株的活性為原來三倍。此外, 我們也分離到一個活性下降的胺基酸取代株(酥胺酸44甲硫胺酸)。這 個結果顯示酥胺酸-44 對木聚醣酶的抗熱性是有決定性的。

(2) Bacillus 聚醣酶選殖與酵素工程

在另一項研究中,我們從台北縣烏來鄉南勢溪溫泉裡分離到 耐熱菌Bacillus subtilis WL-A12。它具有分解多種胞外受質的能 力。WL-A12的葡聚醣酶(1,3-1,4-β-glucanase,又名地衣醣酶)被選 殖轉入大腸桿菌中以獲得具耐熱性質的酵素。該酵素為一分子量約 27 kDa的多胜鏈;訊息胜肽位於該酵素的N-端,長度為28個胺基 酸。酵素在pH 6及 50oC的條件下擁有最佳的反應活性;其活性約 17.72 IU/mg。這個葡聚醣酶目前正在進行耐熱改善和最終產物(葡萄 糖)低抑制突變的實驗。


圖一、 XylX 缺失型突變株與野生XylR 的結構比較。

All publication list

 

Selected publication list

 

  • Ko,C.H., Tsai,C.H., Tu,J., Tang,S.H., and Liu,C.C. (2011). Expression and thermostability of Paenibacillus campinasensis BL11 pectate lyase and its applications in bast fibre processing. Ann. Appl. Biol. (in press).
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  • Ko,C.H., Tsai,C.H., Tu,J., Lee,H.Y., Ku,L.T., Kuo,P.A., and Lai,Y.K. (2010). Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11. Process Biochemistry. 45:1638-1644.
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  • Ko, C.H., Tsai, C.H., Lin, P.H., Chang, K.C., Tu, J., Wang, Y.N., and Yang, C.Y. Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli. BIORESOURCE TECHNOLOGY. 101(20), 7882-7888,2010-06.
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  • Ko, C.H., Tsai C.H., Tu, J., Lee, H.Y., Ku, L.T., Kuo, P. A., and Lai, Y.K.. Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11. PROCESS BIOCHEMISTRY. 45, 1638-1644, 2010-08.
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  • Ko, C.H., Lin, Z.P., Tu, J., Tsai, C.H., Liu, C.C., Chen, H.T., and Wang, T.P. Xylanase production by Paenibacillus campinasensis BL11 and its pretreatment of hardwood kraft pulp bleaching. INTERNATIONAL BIODETERIORATION & BIODEGRADATION. 64(1), 13-19, 2010-01.
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  • Huang, C.Y., Hsieh, S.P., Kuo, P.A., Jane, W.N., Tu, J., Wang, Y.N., Ko, C.H. Impact of disinfectant and nutrient concentration on growth and biofilm formation for a Pseudomonas strain and the mixed cultures from a fine papermachine system.INTERNATIONAL BIODETERIORATION & BIODEGRADATION. 63(8), 998-1007, 2009-12.
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  • Y. C. Charng, K. T. Li, H. K.Tai, N.S. Li, and Tu, J., An inducible transposon system to terminate the function of a selectable marker in transgenic plants. MOLECULAR BREEDING. 21(3), 359-368, 2008.
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  • C. H. Ko, W. L. Chen, C. H.Tsai, W. N. Jane, C. C. Liu, and Tu, J., Paenibacillus campinasensis BL11: a wood material-utilizing bacterial strain isolated from black liquor. BIORESOURCE TECHNOLOGY. 98(14), 2727-2733, 2007.
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  • Charng YC, Wu G, Hsieh CS, Chuan HN, Huang JY, Yeh LC, Shieh YH, Tu, J., The inducible transposon system for rice functional genomics. . Botanical Studies. 48(1),1-11, 2007-01.
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  • Charng, Y. C., Li, H., Ping., Li, K. T., Hseu, T. H., and Tu, J. ,Fusion of the transposase with a classical nuclear localization signal to increase the transposition efficiency of Ac transposon. Bot. Bull. Academia Sinica 45:267-274,2004.
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  • Tsai, P.-J., Tu, J., and Chen, T.-H. Cloning of a Ca2+/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides. FEMS Microbiol. Lett. 212: 7-13,2002.
     
  • Yang, Y. C., Yang, M. K., Kuo, T. T., and Tu, J.,Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri. Mol. Gen. Genet. 265: 316-326,2001.
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  • Charng, Y. C., Pfitzner, A. J. P., Pfitzner, U. M., Charng-Chang, K. F., Chen, C. M., Tu, J., and Kuo, T. T. Construction of an inducible transposon, INAc, to develop a gene tagging system in higher plants. Mol. Breed. 6: 353-367,2000.
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  • Liu, C. C., Huhne, R., Tu, J., Lorbach, E., and Droge, P. The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases. Genes Cells 3: 221-233,1998.
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  • Charng, Y. C., Ma, C., Tu, J., and Kuo, T. T. A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase gene drives higher transposition of a Ds element than the native PR-1a promoter fusion drives. Plant Sci. 130: 73-86,1997.
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  • Cheng, C. M., Tu, J., Yang, P. C., and Kuo, T. T. Rifampicin: an inhibitor of Xp12-specific protein phosphorylation in Xanthomonas campestris pv. oryzae. FEMS Microbiol. Lett. 143: 141-149,1996.
     
 
Research Assistant 博士研究助理

Chung-Hong Tsai 蔡忠宏
Ph.D. Student 博士班學生
Pei-An Kuo 郭倍安
Cheng-Fang Li 李承芳

MS student 碩士生

Chun-Lin Sung 宋君陵
Guan-Yu Ke 柯冠宇

temporary Research Assistant 臨時研究助理

Sun-Shih Xin 孫世昕