疊氮化鈉誘變菜豆之α-amylase inhibitor 1純化與其活性之探討

Abstract

菜豆（Phaseolus vulgaris L.）種子內含有澱粉分解酶抑制物 （α-amylase inhibitor 1, α-AI1），能與豬胰臟型澱粉分解酶、人胰臟型澱粉分解酶結合，從而抑制澱粉分解，因此α-AI1可用於改善二型糖尿病的惡化或肥胖症的發生。本研究利用三相分離法（three-phase partitioning, TPP），首先將菜豆粗萃取液pH值調整至5.25後，加入30%飽和硫酸銨以沈澱蛋白，再以1:1 (v/v) 加入第三級丁醇（t-butanol）產生三相，於水層得到純化之α-AI1，其中引種之白腎豆（PI-40）與台灣花豆花莢種（Hwachia），經TPP步驟後可得純化倍數（purification fold）分別為7.6與18倍，而α-AI1總活性仍能維持80與66%。本研究接著以擴增片段長度多型性（amplified fragment length polymorphism, AFLP）分析34個NaN3之菜豆誘變品系，求得各品系間之遺傳變異程度，從中挑選9個菜豆誘變品系與引種之菜豆以TPP系統進行α-AI1之純化，並檢測α-AI1之活性。結果發現菜豆誘變品系SA-05之α-AI1對α-amylase具有較高的抑制活性，可供未來商業化生產α-AI1之參考。

Common bean (Phaseolus vulgaris L.) contain α-amylase inhibitor 1 (α-AI1), which would bind α-amylase and subsequently reduce starch digestion. Thus, α-AI1 can be used to control the deterioration of type II diabetes and obesity. In this study, a three-phase partitioning (TPP) technique was used to extract and purify α-AI1 from common bean. The crud extracts were adjusted to pH 5.25, and 30% ammonium sulfate was added. Then the tert-butanol was added to the mixing solution for obtaining three phases. The results indicated that the α-AI1 proteins were mainly recovered in the aqueous phase. The purified α-AI1 for varieties PI-40 and Hwachia were 7.6 fold and 18 fold, respectively. The recovery of total activity was 81 % and 66 %, respectively, for PI-40 and Hwachia. AFLP (amplified fragment length polymorphism) was subsequently used to assess genetic diversity among Hwachia and it 34 NaN3-induced mutants. Nine NaN3-induced mutants and Hwachia and 2 introduced varieties used for α-AI1 purification by using TPP and then the α-AI1 inhibitor activity was determined. The results showed that the mutant SA-05 had higher inhibitor activity than other NaN3-induced mutants and Hwachia. Thus, mutant SA-05 may be useful in commercial preparation of α-AI1.