血纖維分解酶subtilisin NAT之醱酵生產研究

Abstract

Subtilisin NAT，又稱納豆激酶(Nattokinase)，是一種由Bacillus subtilis natto所分泌，具有分解血纖維活性的絲胺酸蛋白酶。本研究中，我們自市售納豆中，經血纖維平板法分離出29株菌株，其中以no. 6 strain具最高血纖維分解活性。探討培養基中蛋白質種類與濃度，及葡萄糖對no. 6 strain生產subtilisin NAT之影響，結果顯示，最適培養基組成為3%大豆與1%葡萄糖。於5 L醱酵槽進行批次培養，並以H-D-Val-Leu-Lys-pNA為基質測定其amidolytic活性。結果顯示，在培養第38小時，可得到最高活性46.5 SU ml-1，此時生菌數為109 CFU ml-1。我們進一步在E. coli中選殖不同長度的subtilisin NAT基因序列 (pre-pro, pro-, mature type)，進行血纖維分解酶之表現與重組蛋白生產研究，發現只有pro-subtilisin NAT基因可順利表現活性，且活性位於胞內。以5 公升醱酵槽中進行pH-state (7.0)饋料批次培養，並於培養第21個小時添加0.02 mM IPTG，同時將溫度自37℃降至25℃，以進行重組蛋白之誘導生產。結果顯示，於培養第24個小時可得最大菌體量(30.5 g l-1)，並於第36小時可得最高活性91.2 SU ml-1，但此時生菌數降至106 CFU ml-1，推測應是受到所表現之subtilisin NAT之傷害所致。經陽離子交換樹脂CM Sepharose Fast Flow與膠體過濾管柱HiPrep 26/60 SephacrylTM S-100 High Resolution進行醱酵液純化，再經蛋白質N端定序及LC-MS/MS分析及資料庫比對，確認此血纖維分解酵素為subtilisin NAT。

Subtilisin NAT (also designated nattokinase) is a fibrinolytic enzyme secreted by Bacillus subtilis natto during natto fermentation. In this research, 29 strains of B. subtilis natto were isolated from commercial natto, and no. 6 showed the highest fibrinolytic activity. Our results show that a simple medium composed of 3% soybean and 1% glucose was optimal for B. subtilis natto to produce fibrinolytic activity. In 5 L Jar-fermentor batch culture, the highest fibrinolytic activity and viable cells were 46.5 SU ml-1 and 109 CFU ml-1 at the 38th hr culture. Gene of subtilisin NAT with different sequence (mature, pro- and pre-pro- type) was cloned and expressed in E. coli for fibrinolytic assay. Our results indicate that only gene of subtilisin NAT with pro-sequence could produce active subtilisin NAT in recombinant E. coli. Production of recombinant subtilisin NAT was further performed in a 7 L Jar-fermentor with pH-state (7.0) fed-batch culture. The viable cells reached 1010 CFU ml-1 at the 15th hr, and the maximal biomass was 30.5 g l-1 at the 24th hr after inoculation. IPTG was added at the 21st hr for subtilisin NAT induction with temperature changed from 37℃ to 25℃. Fibrinolytic activity was observed after induction and reached the maximum, 91.2 SU ml-1, at the 36th hr. However, the viable cells dramatically decreased to 106 CFU ml-1 because recombinant cells were damaged by subtilisin NAT. Fibrinolytic enzyme was purified with CM Sepharose Fast Flow and HiPrep 26/60 SephacrylTM S-100 High Resolution, and was identified to be subtilisin NAT by N-terminal sequencing and LC-MS/MS.