固定化精氨酸脱亚胺酶的制备与性质研究

Preparation and characterization of immobilized arginine deiminase

从7种大孔型离子交换树脂中筛选出固定化效果最好的弱碱性苯乙烯系阴离子交换树脂D301-G,通过先吸附后交联的方法对精氨酸脱亚胺酶进行固定化条件及固定化酶性质研究。经单因素实验,结果表明,最佳固定化条件为每克树脂加入156 U精氨酸脱亚胺酶液,p H4.0,28℃条件下吸附4 h后,在4℃冷却,加入戊二醛溶液至体系内戊二醛体积分数为0.07%,4℃下交联4 h,最优条件下固定化酶活回收率可达85%以上。固定化酶的最适温度和p H分别为50~60℃和5.0~5.5,较游离酶具有更高的温度稳定性,同时固定化酶的米氏常数Km值比游离酶高。固定化酶在重复使用8次后仍保留57.7%的酶活,表明该固定化酶具有较好的操作稳定性,可为连续生产瓜氨酸提供技术依据。

Arginine deiminase （ADI）was immobilized on 7 kinds of ion-exchange resins, in which D301- G was the best material for the immobilization. And the enzyme was immobilized by cross - linkage of glutaraldehyde. After single - factor experiments,the optimal immobilized conditions were enzyme load of 156 U/g resin,4-hour absorption at 28 ℃ , pH4.0, the glularaldebyde concentralion of 0.07% and 4-hour cross-linking action at 4 ℃. Besides, the activity recovery yield of the immobilized enzyme reached above 85%.The optimal temperature and pH of the immobilized enzyme was 50-60 ℃ and 5.0- 5.5,respectively,and it presented higher thermostability and higher K,o than that of the free enzyme.The immobilized enzyme retained 57.7% of its initial enzyme activity after reuse for eight times. The results showed the enzyme immobilized which provided a technical basis for the application of arginine deminase industrially continuously producing citrulline.