本土液化澱粉芽孢桿菌溶纖酶基因在大腸桿菌及枯草桿菌之選殖與表現

Cloning and expression of a fibrinolytic enzyme (subtilisin BPD2) gene from Bacillus amyloliquefaciens BPD2 in Escherichia coli and Bacillus subtilis

許正忠(Cheng-Chung Hsu)

指導教授 ： 高穗生

朝陽科技大學/理工學院/生物技術研究所/碩士(2009年)

摘要

本研究自150 株本土芽孢桿菌屬菌株 (Bacillus sp.)中，利用脫脂奶與明膠平板，篩選出8 株具有較佳蛋白質水解能力之菌株。再進一步利用纖維蛋白平板，挑選具有最佳血纖維蛋白分解能力之液化澱粉芽孢桿菌分離株BPD2 (Bacillus amyliquefaciens BPD2, Ba-BPD2)，同時選殖其枯草菌素(subtlisin)基因並轉殖到大腸桿菌(Escherichia coli BL21)探討其表現。結果顯示液化澱粉芽孢桿菌BPD2 (Ba-BPD2)菌株枯草菌素基因含有1,165 個核苷酸，381 個胺基酸，蛋白大小約為40 kDa。在37℃環境下以IPTG 誘導重組之枯草菌素蛋白表現，大多數以包含體的形式存在於菌體內。將其包含體經復性處理並以Ni-NTA column 將液化澱粉芽孢桿菌(Ba-BPD2)之枯草菌素純化後，仍無法使其恢復成具有活性的狀態。然而採用低溫環境(16℃)下同樣以IPTG誘導重組之枯草菌素蛋白表現，則大部分會以細胞質內的可溶蛋白質存在，將其破菌並離心的上清液收集後測定血纖維蛋白分解酵素活性單位為18,547 FU/g。另外，將液化澱粉芽孢桿菌(Ba-BPD2)的枯草菌素基因轉殖到枯草桿菌(B. subtilis ISW1214)做胞外分泌系統之探討，能產生具有活性的重組之枯草菌素蛋白，其培養後菌液上清液的酵素活性單位為23,982 FU/g，但是蛋白表現量十分地低。雖然經聚合酶連鎖反應確認在B. subtilis ISW1214 宿主細胞與含有無接合任何基因之穿梭載體pαHY300 的枯草桿菌(B. subtilis ISW1214)中並無帶有枯草菌素的基因，但其以快速蛋白液相層析儀方式純化後的活性染色電泳結果卻發現枯草桿菌(B. subtilis ISW1214)宿主細胞可能會分泌其它具分解血纖維蛋白的成分。

關鍵字

液化澱粉芽孢桿菌BPD2 ； 選殖 ； 表現 ； 枯草桿菌 ； 大腸桿菌 ； 枯草菌素

並列摘要

Among 150 local Bacillus sp. isolates screened, 8 candidates with higher proteolytic activity assayed with skim milk and gelatin plates methods were selected for further testing. B. amyloliquefaciens BPD2 (Ba-BPD2) with the highest fibrinolytic activity by fibrin plate assay was chosen among 8 candidates for gene cloning and expression. The subtilisin gene from Ba-BPD2 was amplified by polymerase chain reaction (PCR) and cloned into expressed vector pET29b. Sequence analysis of the subtilisin BPD2 gene showed an open reading frame of 1,165 nucleotides encoding 381 amino acid residues. The expression plasmid pET29b was transformated into Escherichia coli BL21, the recombination subtilisin E. coli-BPD2 was highly expressed by IPTG induction at 37℃ and accumulated mostly in inclusion body. However, a one-step purification of the insoluble subtilisin E. coli-BPD2 was achieved with Ni-NTA column but no active enzyme can be refolded from the inclusion body. However, the subtilisin E. coli-BPD2 was expressed by IPTG induction at 16℃ and accumulated mostly as cytoplasmic protein and the fibrin degradation unit (FU) by fibrin degradation assay was 18,547 FU/g. The enzyme was also actively expressed by E. coli- B.subtilis shuttle vector pαHY300 in B. subtilis ISW1214 via electroporation and the activity of supernatant was 23,982 FU/g. However, the level of expression was very low. Purification of subtilisin B.s.-BPD2 analysised by FPLC with HiTrap DEAE sepharose FF column, and the collected fractions were analysed on zymography. Although there was no subtilisin BPD2 gene in B. subtilis ISW1214 host cell and B. subtilis ISW1214 with E. coli- B.subtilis shuttle vector pαHY300 by PCR, the result showed the host cell may product another extracellular proteases.

並列關鍵字

Escherichia coli BL21 ； cloning ； subtilisin ； expression ； Bacillus amyliquefaciens BPD2 ； B. subtilis

參考文獻

張靜如，「白殭菌纖溶酶之分離純化與特性研究」，碩士論文，朝陽科技大學生物技術研究所，台中 (2006)。

陳忠民，「石蓮花萃取物對血液凝固之影響分析」，碩士論文，大同大學生物工程學研究所，台北 (2003)。

陳耀霖，「白殭菌纖溶酶之特性分析與其在動物血栓溶解作用之探討」，碩士論文，朝陽科技大學生物技術研究所，台中 (2008)。

Astrup, T., and Mullertz, S., “The fibrin plate metod for estimating fibrinolytic activity.” Arch. Biochem. Biophys., Vol. 40, pp. 346-351 (1952).

Castro, G. R., Andribet, E. P., Ducrey, L. M., Garro, O. A., and Sineriz, F., “Modelling and operation of a turbidity- meter for on-line monitoring of microbial growth in fermenters.” Process Biochem., Vol. 30, pp. 767-772 (1995).