文章詳目資料
中國畜牧學會會誌
加入收藏
下載文章
中文摘要
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。
英文摘要
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 esterase 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 esterases 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 esterases 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 esterase genes and only then can the biochemical properties of these esterases be characterized in the future.