納豆菌液態培養生產枯草桿菌素 NAT 最適條件之探討及其應用於乳酸球菌表現

Abstract

由市面上購得之納豆商品中分離出 34 株納豆菌，並以人工血栓平板篩選出菌株 12-1-2 具有最大血纖維分解活性，利用液態培養方式探討枯草桿菌素 NAT （subtilisin NAT，或稱為 nattokinase，納豆激酶）最適生產條件。本研究並以反應曲面法（response surface methodology, RSM），分別針對不同比例之接種量、葡萄糖與大豆粕添加量進行三因子三階次探討；在前置研究結果顯示，接種量、葡萄糖及大豆粕添加量之中央點分別為 5.0%、1.0% 及 3.0% 之濃度。再由反應曲面圖可預測，當添加 2.93% 之大豆粕、1.75% 之葡萄糖及以 4.00% 之濃度接種，將可獲得最大之酵素活性 13.78 SU/mL。以此最適化條件實際進行培養，測得納豆激酶之最大活性為 13.69 SU/mL，為預測值之 99.3%。遺傳工程操作部份，自納豆菌之染色體 DNA 中選殖表現 subtilisin NAT 之基因 aprN 並設計 3 組不同之引子對，以聚合酶鏈反應（polymerase chain reaction, PCR）增幅不同型式之 aprN，分別為 pro-NK、mature NK 以及 NK-histag，再分別與大腸桿菌表現載體接合後再轉形進入大腸桿菌 E. coli JM109 中。經由核酸定序確認所選殖之 aprN 基因序列正確無誤，得到之質體分別為 pETPNK、pETNK 與 pETNKH。所得到之 pro-NK 及 mature NK 序列進一步接合至大腸桿菌�乳酸菌穿梭載體 (shuttle vector) 中，分別為 pNZPNK 以及 pNZNK，以 NICE 系統（nisin-controlled expression system）於乳酸球菌 Lactococcus lactis NZ9000 中表現 subtilisin NAT。建構完成之 pETNKH 依電轉形方式送入 E. coli BL21 (DE3)，並進行誘導、純化、濃縮以及動物之免疫注射，以製備 NK 之多株抗體，作為乳酸菌表現系統中 subtilisin NAT 表現情形偵測之用。製備完成之抗體，以 NK-histag 測試其靈敏度，結果能以西方墨點法偵測，顯示 anti-NK 抗體製備成功。pNZPNK 以及 pNZNK 同樣以電轉形送入 L. lactis NZ9000 中，以不同濃度之 nisin 誘導，結果發現 nisin 濃度愈高時，菌體之生長愈受到抑制，顯示異源基因在乳酸菌中受 nisin 調控而表現，與 SDS-PAGE 及西方墨點法之結果符合；而帶有 pro-NK 載體之菌株生長較帶有 mature NK 者更加受到抑制，推測原因是 pro-NK 能發揮分子內 chaperone 之功能使蛋白質正確摺疊而發揮酵素活性，使得菌體本身受到蛋白酶傷害。然而胞外及胞內之酵素活性皆未能偵測，推測是由於表現系統缺乏將目標蛋白外泌之訊息胜肽（signal peptide），造成納豆激酶表現後累積在菌體內無法穩定存在，同時影響菌體生長進而減低生產，使得其活性低於偵測極限。未來在乳酸菌表現納豆激酶之研究方向，可朝探討不同啟動子及訊息胜肽等表現元件之修改來進行。

Thirty-four Bacillus subtilis natto strains were isolated from commercial natto, and strain 12-1-2 showed the strongest fibrinolytic activity in the fibrin plate assay. We further studied the optimal production condition for subtilisin NAT of Bacillus subtilis natto 12-1-2 by submerged cultivation and three variables/three levels response surface methodology (RSM) using various inoculum density, glucose concentration and defatted soybean concentration as three variables. The pre-tset showed the central points were 5.0% inoculum density, 1.0% glucose and 3.0% defatted soybean. According to the response surfaces, while culturing by 2.93% defatted soybean, 1.75% glucose and 4.00% inoculum density, we would obtain an activity of 13.78 SU/mL. Processing the experiment with this optimal condition, the activity reached 13.69 SU/mL, which is equal to 99.3% of the predicted value. For genentic engineering, the gene encoding subtilisin NAT with distinct lengths (pro-NK, mature NK and NK-histag) were cloned by polymerase chain reaction (PCR) with various primer pairs, and ligased to Escherichia coli plasmid pET29a. Confirming by DNA sequencing, three plasmids, pETPNK, pETNK and pETNKH, with accurate sequences were constructed succesfully. The pro-NK and mature NK sequences were further ligased to E. coli/LAB shuttle vector pNZ8020 to construct pNZPNK and pNZNK, which express in Lactococcus lactis NZ9000 by nisin controlled-expression system (NICE system). pETNKH was transformed into E. coli BL21 (DE3), followed by induction, purification, and injection to Wisatr rats, to prepare polyclonal antibody for analysis of subtilisin NAT expression. In antibody sensitivity test, the positive result of Western blotting using NK-histag as standard protein indicated that we induced anti-NK antibody successfully. L. lactis NZ9000 harboring various plasmids were cultured under various concentrations of nisin induction, as a result, the cell growth were inhibited by higher nisin concentration. Furthermore, transformants harboring pNZPNK was inhibited more than harboring pNZNK. It was considered that pro-NK possessed its activity after nisin induction and thus damaged cells. These results, correlated with SDS-PAGE and Western blotting, indicated that the heterologous protein was expressed controllably by nisin induction. The function of intermolecular chaperone of pro-NK was supposed to facilitate subtilisin NAT to process correctly in LAB. However, we cannot detect its activity by the fibrin plate assay due to low stability of subtilisin NAT in LAB cells which caused the activity to be lower than detection limit.