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›› 2012, Vol. 12 ›› Issue (3): 440-446.

• 生化工程专栏 • 上一篇    下一篇

用石英晶体微天平研究酪蛋白在不同固液界面上的固定化及酶解行为

冯晓毅 秦培勇 葛佳 王平 马光辉 苏志国 高飞 张松平   

  1. 北京化工大学生命科学与技术学院 北京化工大学生命科学与技术学院 中国科学院过程工程研究所,生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室
  • 收稿日期:2012-04-17 修回日期:2012-05-21 出版日期:2012-06-20 发布日期:2012-06-20
  • 通讯作者: 冯晓毅

Casin Immobilization and Enzymolysis Behaviors at Different Solid-Aqueous Interfaces by Quartz Crystal Microbalance

FENG Xiao-yi QIN Pei-yong GE Jia WANG Ping, MA Guang-hui SU Zhi-guo GAO Fei ZHANG Song-ping   

  1. College of Life Science, Beijing University of Chemical Technology College of Life Science, Beijing University of Chemical Technology National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, CAS State Key Lab. Biochem. Eng., Inst. Process Eng., Chinese Academy of Sciences State Key Lab. Biochem. Eng., Inst. Process Eng., Chinese Academy of Sciences State Key Lab. Biochem. Eng., Inst. Process Eng., Chinese Academy of Sciences National Key Laboratory of Biochemical Engingeering, Institute of Process Engineering, Chinese Academy of Sciences National Key Laboratory of Biochemical Engingeering, Institute of Process Engineering, Chinese Academy of Sciences
  • Received:2012-04-17 Revised:2012-05-21 Online:2012-06-20 Published:2012-06-20
  • Contact: FENG Xiao-yi

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摘要： 使用石英晶体微天平(QCM)研究了酪蛋白在聚苯乙烯、Au和SiO2三种芯片表面上的吸附与酶解行为. 结果表明，酪蛋白在聚苯乙烯表面具有较高的非特异性吸附，吸附量为127.9 ng/cm2，而在空白Au和SiO2表面几乎没有非特异性吸附. 通过对Au和SiO2表面进行修饰和活化，酪蛋白可共价固定化在其表面，固定化量分别为178.0和1718.0 ng/cm2. 用胰蛋白酶对吸附的酪蛋白水解后，再用表面活性剂洗涤，可以完全去除聚苯乙烯表面的酪蛋白和其酶解产物，而单独使用表面活性剂仅能洗涤约36%吸附的酪蛋白. 建立了用QCM测定胰蛋白酶在固液界面酶解酪蛋白酶活的方法，对共价固定化在修饰后Au和SiO2表面上的酪蛋白，胰蛋白酶的水解活性分别为0.010和0.157 U/mg，远低于水解游离酪蛋白的17.8 U/mg.

关键词: 石英晶体微天平, 界面吸附, 酶解, 酪蛋白, 胰蛋白酶

Abstract: A quartz crystal microbalance (QCM) with dissipation monitoring was used to characterize the adsorption and subsequent trypsin-catalyzed hydrolysis of casin molecules at several typical interfaces, including Au, SiO2 and polystyrene coated chip surfaces. The results showed that a high non-specific adsorption amount of 127.9 ng/cm2 was observed for casin on polystyrene chip surface, while no apparent adsorption occurred on native Au and SiO2 chips. After activation and modification, casin was covalently immobilized on Au and SiO2 chip surfaces with the amounts of 178 and 1718 ng/cm2, respectively. The subsequent enzymolysis of attached casin by injection of typsin was also monitored using QCM. After trypsin treatment, casin molecules and its hydrolysate peptide adsorbed on polystyrene surface could be efficiently removed with surfactant, without trypsin pre-treatment, however, only 36% of the adsorbed casin was removed by surfactant. A method for measuring activity of trypsin at solid-aqueous interfaces was developed. The measured specific hydrolytic activities of trypsin for immobilized casin on Au and SiO2 chip surfaces are 0.010 and 0.157 U/mg, respectively, which was lower than that for free casin in solution with the specific activity of 17.8 U/mg.

Key words: quartz crystal microbalance, interfacial adsorption, enzymolysis, casin, trypsin

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