木瓜蛋白酶在金属螯合亲和双水相中分配行为的研究  

Partitioning Behavior of Papain in Metal Chelating Affinity Aqueous Two-phase

作　　者：蒋志国[1] 王伟涛[1] 张海德[1] 王燕华[2] 

机构地区：[1]海南大学食品学院,海口570228 [2]海南大学信息学院,海口570228

出　　处：《食品工业》2016年第1期209-213,共5页The Food Industry

基　　金：国家自然科学基金(31260401)

摘　　要：利用金属螯合双水相亲和分配技术对木瓜蛋白酶的萃取进行了研究。考察了不同无机盐和浓度、PEG的分子量和浓度、亲和配基的加入量、pH、酶添加量对木瓜蛋白酶分配的影响,对不含亲和配基的双水相和含亲和配基的双水相萃取木瓜蛋白酶的效果进行比较。优化的分配条件为:质量分数17%PEG 4 000,1%PEG-IDA-Cu^(2+),18%Na_2SO_4,pH 7.0,酶添加量为2 mg/g,在此条件下,木瓜蛋白酶的酶活性回收率可达到90%左右,蛋白分配比达到5.11。结果表明,PEG 4 000/Na_2SO_4系统比PEG 4 000/(NH_4)_2SO_4系统更有利于木瓜蛋白酶亲和分配;无机盐的浓度和亲和配基的加入量是影响木瓜蛋白酶分配的关键因素;与不含亲和配基的双水相系统相比,亲和配基的加入可以提高木瓜蛋白酶在PEG相的分配,酶活性回收率和蛋白分配比可分别提高10%和2.0左右,表明金属鳌合双水相分配技术分离木瓜蛋白酶是可行的,为该体系的放大试验或规模化生产奠定了相应的试验基础。To improve the selectivity of commercial papain extraction,a simple and inexpensive aqueous two-phase affinity partitioning（ATPAP） system using metal ligands was introduced.The effects of PEG molecular weight,concentration of phase forming components,metal ligand type and concentration,system pH and the commercial papain loading on papain extraction were systematically studied.The optimal conditions for the ATPAP of papain were summarized as,PEG 4 000 17%,sodium sulfate 18%,PEG-IDA-Cu^（2＋） 1%,pH 7.0 and 2 mg/g papain loading.Under such conditions,papain had been efficiently separated and concentrated with enzyme activity recovery rate of 90% and protein distribution ratio of 5.11,respectively.Compared with the PEG 4 000/sodium sulfate system without metal ions ligand,the enzyme activity recovery rate and protein distribution ratio of papain increased respectively by 10% and 2-fold in the system with metal ligand.The results demonstrated that the recovery and purification of the selected enzymes could be dramatically enhanced by using modified ATPS with biospecific ligands attached to PEG molecules that partitioned to the PEG rich phase without significant changes in the system composition.It was shown that affinity aqueous two-phase system（ATPAP） was a promising technique for the purification of commercial papain,and laid the experimental foundation for further amplification experiments or industrial scale production.

关 键 词：亲和分配 双水相 金属配体 木瓜蛋白酶 

分 类 号：TS201.25[轻工技术与工程—食品科学]