嗜熱菌酵素中金屬離子的調控及其生物催化作用

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Title:	嗜熱菌酵素中金屬離子的調控及其生物催化作用
Other Titles:	Thermophilic enzymes regulated by metal ions and their biocatalysis
Authors:	吳昱嫺;Wu, Yu-hsien
Contributors:	淡江大學生命科學研究所碩士班鄭建中;Cheng, Chien-chung
Keywords:	嗜熱菌酵素;金屬酵素;生物催化反應;Thermophilic enzymes;Metalloenzymes;biocatalysis;Meiothermuc taiwanesis NTU30;phosphoryl catalyzed hydrolysis reaction
Date:	2005
Issue Date:	2010-01-11 02:31:32 (UTC+8)
Abstract:	生物催化是未來在資源使用及環境保護上的重要課題之一。一般化學反應常需高溫條件，酵素的使用雖然可以改變其反應性及產率，但也常因溫度影響而失去活性。本論文是利用高溫菌的特性，使其產生不同性質的金屬酵素，以改善在高溫中對化學反應的催化活性。在台灣特有種嗜熱菌Meiothermus taiwanensis NTU30的培養基中，改變金屬離子成份，Mn(II)、Zn(II)、B(III)、Cu(II)、Mo(VI)及Co(II)，發現僅一種或缺少單一種金屬離子時，對其生長速率影響不大，但所誘導出之蛋白質，經由SDS-聚丙烯醯胺膠體電泳（SDS-PAGE）的分析結果，卻明顯地具有不同消長的差異性。經破菌、硫酸銨沈澱及透析後所得之整體蛋白質，先行測試磷酸根水解的催化反應（dephosphorylation）。根據紫外光-可見光分子吸收光譜及高效率液相層析資料顯示，M. taiwanensis NTU30整體蛋白質於60℃下仍具有催化磷酸根水解反應的活性。然而在相同條件下，Escherichia coli的整體蛋白質則因高溫變性而無法進行水解作用。更有趣的是，當缺乏Cu(II)、Mo(VI)及Co(II)這些金屬離子時，則此高溫催化水解反應即無法進行。本實驗結果顯示，由嗜熱菌所分離出之酵素在高溫下仍能維持催化活性，而金屬離子則扮演了嗜熱菌蛋白質催化反應成敗的重要角色。本論文的研究結果，不但提供了新的耐高溫金屬酵素的製備觀念，對未來發展耐高溫的生物催化劑也具有相當重要的指標意義。 Biocatalysis is an important topic on resource regeneration and environmental protection. The use of enzyme is capable of improving the reaction rate and product efficiency. However, the enzyme easily losses its activity at high temperature. In our laboratory the thermophilic micro-organism, Meiothermuc taiwanesis NTU30, produces a variety of metalloenzymes to catalyze the chemical reactions at high temperature. The growth rate of M. taiwanesis NTU30 did not alter significantly in the absence or presence of either one of metal ions, Mn(II), Zn(II), B(III), Cu(II), Mo(VI) and Co(II). However, the protein patterns differed obviously from each other revealed by the analysis of SDS-PAGE. After sequential procedures of cell disruption, ammonium sulfate precipitation, and dialysis, the total proteins were obtained and ready to analyze the dephosphorylation activities. Nitrophenyl phosphate was successfully hydrolyzed by the total proteins from M. taiwanesis NTU30 at 60°C based on the UV-visible electron spectrum and HPLC data. In contrast, the total protein Escherichia coli did not show such reactivity in 3 hours. The catalytic activity was suppressed in the absence of Cu(II), Mo(VI) or Co(II). Therefore, thermophilic metalloenzymes have been demonstrated to retain their activities at high temperature. As well, the specific metal ions play an important role in the hydrolysis of phosphate monoester. These results not only provide a novel method in the process of metalloenzymes in thermophilic micro-organisms, but also develop a new vision of the high temperature-resistant biocatalysts.
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