利用大腸桿菌表現重組 subtilisin NAT 之研究

Abstract

納豆激酶 (subtilisin NAT) 是來自於納豆菌中的一種絲胺酸蛋白酶，具有纖維蛋白分解的活性。納豆激酶基因 (aprN) 由三段不同長度的DNA domain所組成 (pre-, pro- and mature)。本研究自一帶有高纖維蛋白分解能力之納豆菌中放大出帶有pro-mature domain 的納豆激酶基因，並將之選殖入大腸桿菌中，以利用IPTG調控其表現 (E. coli M15 / pQE-30 Xa-NK1059)。此外，亦建構帶有C端六組胺酸標誌的菌株以利於後續蛋白質純化 (E. coli JM109 / pQE-30 Xa-NK1059-his)。所選殖的納豆激酶基因與NCBI資料庫的aprN有100% 的相同性。以變性聚丙醯胺膠體電泳及S-2251呈色基質酵素活性測定法確認重組納豆激酶存在於胞溶後的可溶性部分。研究結果顯示，在IPTG誘導8小時後，E. coli M15表現的重組納豆激酶活性較E. coli JM109顯著提升5.9倍 (3.0 SU mL-1 增至 17.8 SU mL-1)。在不同誘導溫度的比較中，37oC誘導12小時可得到最高活性的重組納豆激酶 (40.4 SU mL-1)，然而較長的誘導時間卻使得酵素活性不穩定，而在20oC及25oC誘導均在24小時後測得最高活性 (33.7 及 30.3 SU mL-1)。在放大表現重組納豆激酶的研究中，利用批次饋料策略可獲得最大細胞乾重60.9 mg mL-1，及在誘導20小時後得到最大酵素活性93.5 SU mL-1。此外，C端帶有六組胺酸標誌的重組菌株，在25oC下，誘導24小後可得到最大酵素活性18.5 SU mL-1。然而在以親和層析法純化目標蛋白後發現，帶有酵素活性的分劃與具有酵素表現的分劃並不一致。在本實驗中，我們建構了大腸桿菌M15外源納豆激酶的表現系統，以及帶有C端六組胺酸標誌將可簡化下游納豆激酶之回收步驟。然而，是否存在他種具有同樣酵素活性的蛋白酶仍需進一步研究。

Subtilisin NAT (nattokinase) is an alkaline serine protease produced by Bacillus subtilis natto and well-known for the fibrinolytic activity. Subtilisin NAT is encoded by aprN, which consists of three different domains (pre-, pro- and mature). In this study, the pro-mature sequence of subtilisin gene was amplified from a Bacillus subtilis natto with high amidolytic activity and cloned into Escherichia coli for recombinant subtilisin NAT expression under the control of the inducer, IPTG (E. coli M15 / pQE-30 Xa-NK1059). In addition, a strain with C-terminal hexa-His tag was constructed for a simple process of purification (E. coli JM109 / pQE-30 Xa-NK1059-his). The nucleotide sequence of cloned gene shared 100% identity with the aprN published on NCBI database. The existence and the activity of recombinant subtilisin NAT were detected in the soluble fraction of cell lysate by SDS-PAGE and S-2251 chromogenic enzyme activity assay. After 8-h of IPTG induction, the amidolytic activity of recombinant subtilsin NAT expressed in E. coli M15 was significantly higher by 5.9-fold than it expressed in E. coli JM109 (3.0 SU mL-1 to 17.8 SU mL-1). After 12 h-induction at 37oC, the recombinant E. coli M15 showed the highest amidolytic activity (40.4 SU mL-1); however, the activity was unstable for a longer induction time. While induction at 20oC and 25oC, the activities reached to the maximum after 24h-induction (33.7 and 30.3 SU mL-1, respectively). In scale-up expression of recombinant subtilisin NAT, the maximal biomass accumulated to 60.9 mg mL-1 DCW and the highest amidolytic activity was 93.5 SU mL-1 after 20 h-induction in a fed-batch culture. The C-terminal hexa-His tag containing JM109 strain expressed the highest amidolytic activity of 18.5 SU mL-1 after 24 h-induction at 25oC. However, the amidolytic activities didn’t appear in the fractions containing His-tagged recombinant subtilisin NAT in the affinity chromatography. In this study, we established an E. coli M15 system for exogeneous subtilisin NAT expression. In addition, a simplified downstream recovery process of subtilisin NAT was created by introducing hexa-His tag into the C-terminus. However, further investigations need to be done to confirm the possibility of existence of other kinds of protease possessing the amidolytic activity.