Enzyme immobilization: concepts and effects on proteolysis

In recent years, great advances have been observed in the proteomic area regarding the different approaches adopted as top-down, middle-down and bottom-up. Regarding middle-down and bottom-up approaches, a previous step for protein digestion is necessary for further analysis of the generated peptides. Classical digestion protocols employing proteases are usually carried out in solution, which can lead to a number of disadvantages such as high digestion time, enzymatic autolysis and impossibility to reuse the enzymes. In view of these drawbacks, different approaches have been studied and applied for improving enzymatic efficiency, inhibition of autolysis, reduction of digestion time, and to minimize the number of missed cleavages. In the literature, great efforts have been made in order to achieve maximum efficiency in enzymatic digestion, with emphasis on proteases immobilization. In this review, concepts related to protein digestion, immobilization techniques, as well as the main support used for enzyme immobilization are discussed considering new tools for proteomic analysis.

近年来，蛋白质组学领域在自上而下（top-down）、中分段（middle-down）与自下而上（bottom-up）三类研究策略的应用上取得了显著进展。针对中分段和自下而上这两种研究策略，需先完成蛋白质酶解步骤，方可对产生的肽段开展后续分析。采用蛋白酶的经典酶解方案通常在溶液体系中进行，该方式存在诸多弊端，如酶解耗时较长、蛋白酶易发生自溶，且无法重复利用酶制剂。针对上述缺陷，学界已研究并应用多种策略，旨在提升酶解效率、抑制蛋白酶自溶、缩短酶解时长，并最大限度减少漏切位点数量。现有文献中，科研人员已投入大量精力以实现酶解过程的最高效率，其中尤以蛋白酶固定化技术为研究重点。本综述将结合蛋白质组学分析的新兴工具，对蛋白质酶解相关概念、固定化技术，以及酶固定化常用载体等内容展开探讨。