白鳳豆種子的Bowman-Birk型蛋白酶抑制劑：純化及生化特性分析

Bowman–Birk Type Proteinase Inhibitor from Canavalia ensiformis Seeds: Isolation and Biochemical Properties

本研究以白鳳豆（Canavalia ensiformis）種子為材料，利用硫酸銨分割法進行分離，經透析後發現具胰蛋白酶抑制活性的部分集中於硫酸銨70-95%飽和濃度區間。此部分經DEAE-52纖維素離子交換樹脂與trypsin-Sepharose 4B親和性層析進一步純化，獲得CETI（Canavalia ensiformis trypsin inhibitor）蛋白。純化後的CETI樣品經15% SDS-PAGE分析純度，並使用MALDI-TOF質譜測得其分子量約為9.01 kDa。依據分子量及功能特性，CETI被歸類為Bowman-Birk型胰蛋白酶抑制劑。對此蛋白進行進一步特性分析，結果顯示CETI對熱、酸鹼值及變性劑（SDS）具高耐受性。在100℃處理10分鐘後，CETI仍保有約80%的抑制活性；在pH值2至10範圍內，抑制活性未見顯著變化；而在2%的SDS中反應30分鐘後，其活性仍維持70%-80%。經還原劑DTT處理後，CETI的胰蛋白酶抑制活性顯著下降，顯示其結構穩定性與雙硫鍵密切相關。此外，CETI的N端15個胺基酸序列與其他Bowman-Birk型胰蛋白酶抑制劑序列相似度甚高，其中與雙花扁豆（Dolichosbiflorus）的胰蛋白酶抑制劑序列相同性達87%。以上特性顯示CETI具有穩定且有效的抑制活性，具潛力應用於生物技術或醫藥領域。

In this e×periment, the trypsin inhibitor from Canavalia ensiformis seeds were fractionized using ammonium sulfate and dialyzed, the fractions possessed trypsin inhibiting activity were found at the concentration between 70%–95% of ammonium sulfate. Subsequently, these fractions were collected and passed through a diethylaminoethyl (DEAE-52) cellulose ion e×change resin and trypsin-Sepharose 4B affinity column. A purified Canavalia ensiformis trypsin inhibitor (CETI) was analyzed by15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by a matri×-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer analysis, which determined that its molecular weight was 9.01 kDa. According to its molecular weight, CETI is presumed to be a Bowman-Birk type trypsin inhibitor. A further investigation into the properties of this protein revealed that it possessed e×ceedingly high tolerance to heat and denaturant (sodium dodecyl sulfate; SDS) and remained active in a wide range of pH values. After 10 minutes of treatment at 100℃, the protein retained 80% activity. After treatment in an environment with a pH value of 2-10, its trypsin inhibition activity was not greatly affected. Additionally, CETI maintained 70%-80% of its activity after a 30-minute reaction in 2% SDS. Upon treatment with the reducing agent DTT, CETI's trypsin inhibitory activity significantly decreased, indicating that its structural stability is closely related to the presence of disulfide bonds. Moreover, the N-terminal 15 amino acid sequence of CETI shows high similarity to other Bowman- Birk type trypsin inhibitors, with an 87% sequence identity to the trypsin inhibitor from Dolichos biflorus. These characteristics suggest that CETI is a stable and effective protease inhibitor, with potential applications in biotechnology and the pharmaceutical field.