海洋金属蛋白酶MP仿生亲和材料纯化条件的优化  

Optimization of purification protocol of biomimetic affinity material for marine metalloprotease MP

作　　者：杨娟[1,2] 李尚勇[1] 马子宾 刘均忠[1] 郝建华[1] 王跃军[1] 孙谧[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,海洋国家实验室海洋药物与生物制品功能实验室,山东青岛266071 [2]上海海洋大学食品学院,上海201306

出　　处：《食品工业科技》2017年第15期146-152,共7页Science and Technology of Food Industry

基　　金：NSFC-山东联合资助海洋研究中心项目(U1406402-5);国际科技合作与交流专项(2014DFG30890);国家实验室-鳌山科技计划(2015ASKJ02-06);国家自然科学基金(41376175);国家863计划(2014AA093516)

摘　　要：目的:建立一种高效的海洋金属蛋白酶MP仿生亲和纯化方法。方法:采用硼酸仿生亲和材料,利用单因素实验,Plackett-Burman实验设计、Box-Behnken实验等响应面优化法,对纯化条件进行优化,利用SDS-PAGE及HPLC分析纯化后样品纯度。结果:利用单因素法和响应面法对纯化条件进行优化和分析,获得硼酸琼脂糖凝胶亲和柱的最佳纯化条件为:粗酶液浓度在100 mg/m L,上样缓冲液为p H8.6的甘氨酸-氢氧化钠,上样速度1 m L/min,洗脱缓冲液为p H5.2的磷酸氢二钠-柠檬酸,洗脱速度2 m L/min,洗脱缓冲液加入75 mmol/L的甘露醇时纯化效果最好,纯化效率提高了11倍,纯化后的样品纯度达到98.8%。Objective： To establish an efficient method for biomimetic affinity purification of marine metalloprotease MP. Methods： Using the biomimetic affinity materials to optimize the purification conditions by response surface optimization method, such as the single factor experiment, Plackett-Burman experiment design, Box-Behnken experiment.And the purity of purified samples by SDS-PAGE and HPLC was analyzed.Results：The purification conditions were optimized by single factor experiment and the response surface design.The study indicated that the best purification conditions of the boronic acid gel were：When enzyme concentration was 100 mg/mL,the optimal loading buffer was glycine-sodium hydroxide of pH8.6 and the sample rate was 1 mL/min.The elution buffer was disodium hydrogen phosphate-citric acid of pHS.2, and elution rate was 2 mL/min, the elution buffer added to mannitol of 75 mmol/L, the purification efficiency was enhanced 11 times and the purity above 98.8%.

关 键 词：金属蛋白酶MP 亲和层析 纯化 

分 类 号：TS201.1[轻工技术与工程—食品科学]