绿豆苯丙氨酸解氨酶的提纯及固相化研究  

Purification and Immobilization of Phenylalanine Ammonia—Lyase from Phaseolus Radiatus L.

作　　者：马克颖 姚侃[1] 

机构地区：[1]兰医生化教研室

出　　处：《兰州医学院学报》1992年第2期89-94,共6页Journal of Lanzhou Medical College

摘　　要：以绿豆芽下胚轴为材料,1.1M 蔗糖中光照24小时,40～60%饱和硫酸铵分段沉淀,Sephadex—G25脱盐,超滤。再经 Sepharose 6B 凝胶过滤,DEAE—SephadexA25离子交换层析,L—苯丙氨酸—Sepharose4B 亲和层析纯化。最后获得的 PAL 纯化241倍,收率为11.3%,经聚丙烯酰胺凝胶浓度梯度电泳鉴定均一。提纯后的 PAL 用三种方法(超声法、脱水再加水法、复乳法)包被于三种脂类组成不同的脂质体中,比较包被结果证明,复乳法的包被率高于其它两种方法,而脱水再加水法制备的脂质体酶活性丧失较少。显微镜及电镜观察表明:三种方法所得脂质体大小均一性不同,制得的脂质体经 Sepharose4B 过滤后更为均一。比较三种不同方法制得的脂质体证明加水脱水法手段温和适于酶的包被。采用两种方法将 PAL 固相化:用碳化二亚胺缩合固相干羧甲基纤维素和用溴化氰反应固相干 Sepharose4B。比较两种固相化的 PAL,从结合率及酶活性二者前一方法均优于后者。L—Phenylalanine ammonia—Lyase(EC 4.3.1.5.)has been pruified from green gram(Phaseolus radiatus L.)hypocotyls which had been exposed to light.After preliminary fractionation by ammonium sulfate and Sephadex G—25 gelfiltration desalting,the enzyme was further purified by(a)gel filtration on Sepharose 6B (b)ion—exchange chromatography on DEAD—Sephadex A—25(c)affinity chromatography on L—Phe Sepharose 4B.The PAL isolated was 241—fold purified with a yield of 11.3%. PAL was entrapped in liposomes with various lipid compositions by different methods:sonication(S); dehydration and rehydration(DR);and double emulsification(DE);The percentages of PAL entrapped in various liposomes and the enzyme activity retained were compared.It shows that the DR method is the most mild technique for enzyme entrapment.The size distribution of liposomes was demonstrated by light micro- graph and transmission electron mierograph. PAL was covalently bound to CM—cellulose by carbodiimide and to Sepharose 4B by cyanogen bromide. Percent of enzyme coupled and the PAL activity of immobitized enzyme were determined.

关 键 词：提纯 脂质体包被酶 解氨酶 

分 类 号：TQ464.8[化学工程—制药化工]