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中國畜牧學會會誌46(增刊) |
,2017 |
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J. Chin. Soc. Anim. Sci. 46 (Suppl.) |
174
96.活性污泥多源酯酶基因次選殖與表現分析
廖仁寶(1) 陳若菁(1) 李佳音(2) 吳明哲(1)
(1)行政院農業委員會畜產試驗所 (2)國立臺灣大學農業化學系
由活化污泥多源基因體中所得10個推定的酯酶基因（est1, est2, est3, est4b, est5, est6, est10, est11, est12A, est13）已成功藉由PCR選殖至pET-52b(+) 3C/LIC表現載體上。重組質體分別命名為pET52b-Est1, -Est2, -Est3, -Est4B, -Est5, -Est6, -Est10, -Est11, -Est12A, -Est13。10個推定的酯酶基因可在Escherichia coli BL21 Star (DE3)中大量表現，但除rEst6外，其他表現的酯酶以不溶性形式存在。當使用Escherichia coli ArcticExpress (DE3)作為表現宿主並在低溫下培養時，大多數酯酶除了Est4B之外被大量表現為可溶形式。然而，只有在E. coli BL21 Star (DE3)中表達的rEst6可以用Strep•Tactin純化試劑組純化而得。雖然推定的酯酶可以在E. coli ArcticExpress (DE3)中表達為可溶形式，但問題為大多數酯酶無法使用Strep•Tactin純化試劑組和His•Bind純化試劑組純化。當使用Strep•Tactin純化試劑組時，Cpn60伴隨著純化的標的酯酶出現。因此，有必要克服這些新型酯酶基因的表現和純化的問題，如此方能進行酯酶的生物化學性質分析。
關鍵語：活性污泥、酯酶、次選殖
Subcloning and expression of esterase genes from an activated sludge metagenome
R. B. Liaw(1), J. C. Chen(1), C. Y. Lee(2) and M. C. Wu(1)
(1)Livestock Research Institute, Council of Agriculture, Executive Yuan
(2)Department of Agricultural Chemistry, National Taiwan University
The ten putative esterase genes (est1, est2, est3, est4b, est5, est6, est10, est11, est12A, est13) derived from an activated sludge metagenome were successfully cloned into pET-52b(+) 3C/LIC expression vector by PCR. The recombinant plasmids were designated pET52b-Est1, -Est2, -Est3, -Est4B, -Est5, -Est6, -Est10, -Est11, -Est12A, and -Est13, respectively. The ten putative lipolytic genes could be overexpressed in Escherichia coli BL21 Star (DE3), but the expressed enzymes except rEst6 (recombinant Est6) existed in insoluble form. Most of the lipolytic enzymes except Est4B were overexpressed as soluble form when using Escherichia coli ArcticExpress (DE3) as the expression host and cultured at low temperature. However, only purified rEst6 expressed in E. coli BL21 Star (DE3) could be obtained with Strep•Tactin purification kit. Although the putative lipolytic enzymes could be expressed as soluble form in E. coli ArcticExpress (DE3), the problem was that most of the enzymes could not be purified with Strep•Tactin purification kit and His•Bind purification kit. Otherwise, the purified target enzymes accompanied by Cpn60 when using Strep•Tactin purification kit. Therefore, it is necessary to overcome the problems of the expression and purification of these novel lipolytic genes and only then can the biochemical properties of these esterases be characterized in the future.
Key Words: Activated sludge, Esterase, Subcloning