果菠萝蛋白酶催化功能基团的研究

Studies on Functional Groups of Fruit Bromelain

以菠萝果为材料,应用本实验室开发的工业生产法工艺制备粗酶制品,进一步经DEAE-纤维索(DE-52)和 Sephadex G-75柱层析纯化,获得圆盘电泳单一纯的果酶制剂,测定酶的分子量为29 800,含 19种不同氨基酸,总残基数为286个。化学修饰研究结果表明:巯基、氨基、Trp残基和唯一的His残基是酶活性必需基团,而Ser和羧基与酶活性无关。用邹氏作图法判断 Trp残基的必需基团数,表明酶分子中六个Trp残基仅有一个是酶活性所必需的。

Crude fruit bromelain was prepared from fruit juice of pineapple by means of industrial production .It was further purified by chromatography on DEAE-culluiose (DE-52) and gel filtration on Sephadex G-75. The preparation was shown to be homogeneous on polyacrylsmide gel disc electrophoresis. The molecular weight was estimated to be 2.98x 104by means of Sephadex G-150 gel filtration. The enzyme molecule was found to be consisted of 286 amtno acid residues contributed by 19 different amino acids. The results of the chemical modification of the enzyme demostrated that sulfhydryl-group. amino-group, tryptophan residue and the only one histidine residue are essential, while serine residue and carboxyl-group are not essential for the activity of the enzyme. By the method of C.L. Tsou's plot, the number of essential Trp was found to only one among the six similar residues estimated