福寿螺β-葡萄糖苷酶的分离纯化及性质的初步研究

Priliminary Studies On Isolation, Purification And Some Properties of β Glucosidase From Ampullarium Crossean

以福寿螺内脏为材料，经本实验室开发的工业生产法制备粗酶粉，进一步采用葡聚糖凝胶（ＳｅｐｈａｄｅｘＧ－２００）和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析纯化，获得经ＳＤＳ－聚丙烯酰胺凝胶电泳鉴定为单一纯的β－葡萄糖苷酶制剂．测定该酶的最适反应温度为４３℃，最适反应ｐＨ为５．１；在ｐＨ５．１，４５℃下，该酶催化水杨素水解的Ｋｍ值为３．４０ｍｍｏｌ／Ｌ，活化能为５０．６３ｋＪ／ｍｏｌ．研究几种效应物对酶活力的影响，结果表明：Ｍｎ２＋、Ｃｏ２＋对酶有激活作用，而Ｃｕ２＋、Ｈｇ２＋、Ｐｂ２＋、ＳＤＳ及脲对酶有不同程度的抑制作用，其中Ｃｕ２＋和Ｈｇ２＋对该酶的抑制作用最强。

A β glucosidase (EC 3.2.1.21) from Ampullarium Crossean has been purified by chromatography with Sephadex G 200 gel filtration, DEAE Sephadex A50 ion exchange. The purified enzyme preparation was homogeneous as judged by FPLC and SDS polyacrylamide gel electrophoresis. The specific activity of the enzyme is 7 700 U/mg. The enzyme follows typical Michaelis Menten Kinetics with apparent K m of 3.40mmol/L for Salicin. The activation energy of the enzyme is 50.63 KJ/mol. The optimum temperature is 43℃ (stable to 40℃) and the optimum pH is 5.1 (stable from pH4.0 to 8.0). Mn 2+ and Co 2+ activate the enzyme. The enzyme activity is inhibited in various degree by Cu 2+ 、Hg 2+ 、Pb 2+ 、SDS and urea. Cu 2+ and Hg 2+ strongly inhibit the enzyme activity and the effects are both classified as noncompetitive type.