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
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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%。Abstract: 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/m L, the optimal loading buffer was glycine-sodium hydroxide of p H8.6 and the sample rate was 1 m L/min. The elution buffer was disodium hydrogen phosphate-citric acid of p H5.2, and elution rate was2 m L/min, the elution buffer added to mannitol of 75 mmol/L, the purification efficiency was enhanced 11 times and the purity above 98.8%.
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