摘要
本文报道了将Sephadgx G-200与对β-硫酸酯乙砜基苯胺(SESA)首先醚化制备对氨基苯砜乙基交联葡聚糖(ABSE-Sephadex G-200),然后经重氮化固定无花果蛋白酶。固定化酶的活力回收达69％。BANA对该酶固定化过程中的活性变化有保护作用。天然酶与固定化酶都具有良好的耐热性,在69°～70℃,80min固定化酶较天然酶稳定。 用苯甲酰-DL-精氨酰-β-荼胺(BANA)为底物,在半胱氨酸存在下,测定了两种形式酶的动力学性质。在pH7.7的磷酸盐缓冲系统中,37℃,天然无花果蛋白酶的K_m=0.32mol/L;在间歇振摇下固定化酶的表观K′m=1.02mmol/L。最适pH无明显改变,均为pH7.7。
It is reported in this paper that sephadex G-200 and (P-**********b-sulphatoethyl-sulphonyl) aniline (SESA) were first etherizied so as to prepare P-aminobenzene sulphone ethyl (ABSE) -sepadex G-200 and then, ficin was fixed on ABSE-sephadez G-200 by means of diazo-reaction.The recovery of activity the immobilized enzyme was approximatly 69%.Benzoyl-DL-arginine-*********b-naphthyIamine hydrochloride (BANA) was used to protect the enzyme activity during the immobilization process.The native and immobilized enzymes had higher thermal stability.The thermal stability of the immobilized enzyme was higher than that of the native ficin in 69-70**********C for 80 min.When BANA was used as substrate, the kinetic properties of the two types of enzymes were investigated in the presence of cystein.In the system of ph osphate-buffer pH7.7, 37********C, Michaelis constant (km)of the native ficin was 0.32,m mol/L, apparent km of the immobilized enzyme was 1.02mmol/Lwhile being shaken at intervals.The optimal pH of the two enzymes did not chahge, which was pH7.7.
