甘薯叶片蔗糖酶的分离纯化及其部分性质

Isolation, Purification and Some Properties of Invertase from Sweet Potato

纯化酶经聚丙烯酰胺凝胶电泳显示单一蛋白带，ＳＤＳ－ＰＡＧＥ显示一条蛋自带，其亚基分子量为３９．８ｋＤ。用ＳｅｐｈａｃｒｙｌＳ－２００凝胶过滤测得全酶的分子量为７９．４ｋＤ，该酶由两个相同亚基组成。其表观Ｋｍ为１２ｍｍｏｌ／Ｌ，Ｖｍａｘ为９９．５ｍｇ还原糖ｍｇ－１ｐｒｏｔｅｉｎｈ－１。

Invertuse was isolated and purified from the extract of sweet potato (Ipomeoa batatas Lam. ) leaves by ammonium sulphate precipitation, DEAE-Sepharose column chromatography (Fig. 1), and Sephacryl S-200 gel filtration (Fig. 2 ). It was purified 282. 1-fold with an activity recovery of 22. 7% (Table 1 ). The purified invertase preparation was homogeneous as judged by polyacrylamide gel electrophoresis and SDS-PAGE (Fis. 3 ).The molecular weight of the native invertase was determined by gel filtration to be 79. 4 kD (Fig. 4). The invertase consists of two identical subnuits with an apparent molecular weight of 39. 8kD as determined by SDS-PAGE (Fig.3B). The invertase followed typical Michaelis-Menten kinetics with an apparent Km of 12 mmol/L for sucrose (Fig. 5). The Vmax of the invertase was 99. 5 mg reducing sugar mg-1 protein h-1. The enzymewas shown to be stable between pH 4. 0 and 6. 0 and to have an optimum pH of 5. 0 for its activity (Fig. 6 ). The temperature optimum was 60℃ (Fig. 7).