Righetti P G, Nembri F, Bossi A, Mortarino M
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Biotechnol Prog. 1997 May-Jun;13(3):258-64. doi: 10.1021/bp970019e.
Among the milk proteins, bovine beta-casein has the peculiarity of containing in its sequence some peptides liable to interfere in mineral nutrition and some peptides with opioid (casomorphines), antihypertensive, and immunomodulatory activities. In this work we propose a novel type of multicompartment enzyme reactor, operating under an electric field, for the continuous hydrolysis of milk proteins such as beta-casein. The enzyme trypsin is trapped, with zwitterionic buffering ions and its substrate beta-casein, in solution between two isoelectric membranes having pI values encompassing the isoelectric point of the enzyme. Additionally, beta-casein is captured inside the same reaction chamber with the aid of sieving membranes, since its pI is too far away from the pI of trypsin. This setup permits the continuous operation at the pH of optimum of activity. The peptides, arising from tryptic hydrolysis of beta-casein, are removed under the influence of the electric field and collected in different chambers in which they are isoelectric and isoionic as well. The purity of the peptides collected is ascertained by capillary zone electrophoresis and their identity confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. This setup allows continuous harvesting of some biologically-active peptides in a pure form. The major advantages of such a reactor system over conventional batch reactors are the great increase in enzyme utilization efficiency and the overall reactor productivity.
在乳蛋白中，牛β-酪蛋白的特点是其序列中含有一些可能干扰矿物质营养的肽段以及一些具有阿片样（酪蛋白吗啡）、抗高血压和免疫调节活性的肽段。在这项工作中，我们提出了一种新型的多隔室酶反应器，它在电场下运行，用于连续水解乳蛋白，如β-酪蛋白。胰蛋白酶与两性离子缓冲离子及其底物β-酪蛋白一起被困在两个等电点膜之间的溶液中，这两个等电点膜的pI值涵盖了该酶的等电点。此外，β-酪蛋白借助筛分膜被捕获在同一反应室内，因为其pI与胰蛋白酶的pI相差太远。这种设置允许在最佳活性pH下连续运行。由β-酪蛋白的胰蛋白酶水解产生的肽段在电场的影响下被去除，并收集在不同的隔室中，在这些隔室中它们也是等电和等离子的。通过毛细管区带电泳确定收集到的肽段的纯度，并通过N端测序和基质辅助激光解吸电离飞行时间质谱法确认其身份。这种设置允许以纯形式连续收获一些生物活性肽。与传统间歇反应器相比，这种反应器系统的主要优点是酶利用效率和整个反应器生产率大幅提高。
