Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein.
Green synthesis of monolithic enzyme microreactor based on thiol-ene click reaction for enzymatic hydrolysis of protein.
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DOI：
10.1016/j.chroma.2019.460618
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发表时间：
2020-01
期刊：
影响因子：
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通讯作者：
Xue-Ming Zhao;Pei-Ru Fan;Chune Mo;Yanping Huang;Zhaosheng Liu
中科院分区：
文献类型：
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作者：
Xue-Ming Zhao;Pei-Ru Fan;Chune Mo;Yanping Huang;Zhaosheng Liu
本研究采用“硫醇-烯”点击反应固定化胰酶，在毛细管中成功制备了基于绿色合成策略的整体酶反应器。以1-丁基-3-甲基咪唑四氟硼酸盐和氯化胆碱/乙二醇为载体，制备了聚(甲基丙烯酸丁酯-co-α-甲基丙烯酸-乙二醇二甲基丙烯酸酯共聚)聚合物。用“硫醇-烯”反应固定化酶后，用毛细管电泳法测得固定化酶反应器的米氏常数和最大反应速率分别为2.1m ol/L和0.028 m ol/μ。考察了不同实验条件下酶反应器的酶解情况。在S的作用下，牛血清白蛋白可在50小时内实现在线消化，比溶液中消化(12小时)的速度快864倍。BSA能很好地消化，鉴定的多肽数量为73个，覆盖率为82.7%。进一步将该Imer用于大鼠肝脏蛋白质提取物的分析，鉴定出1034个蛋白质组。这些结果表明，这种基于点击反应的Imer在高通量高置信度蛋白质组分析中具有广阔的应用前景。
In this study, a monolithic enzyme reactor based on a strategy of green synthesis was successfully prepared in a capillary with trypsin immobilized by “thiol-ene” click reaction. A polymer of poly(butyl methacrylate-co-α-methacrylic acid-co-ethylene glycol dimethacrylate) was prepared in a mixture of 1-butyl-3-methylimidazolium tetrafluoroborate and choline chloride/ethylene glycol as the support of enzyme reactor. After “thiol-ene” reaction was used for enzyme immobilization, the Michaelis constants and maximum reaction rate of the resulting immobilized enzyme reactors (IMER) were determined by capillary electrophoresis to be 2.1 mmol/L and 0.028 μmol/min, respectively. The enzymatic hydrolysis of the enzyme reactor under different experimental conditions were investigated. A on-line digestion of bovine serum albumin (BSA) on the new IMER can be achieved within 50 s, up to 864 times faster than in-solution digestion (12 h). BSA can be well digested and the numbers of identified peptides were 73 with the coverage rates of 82.7%. The IMER was further used for the analysis of protein extracts from rat liver, and 1034 protein groups were identified. All these results demonstrated that such a click reaction based IMER would be of great prospect in the high throughput analysis for proteome with high confidence.