Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, School of Light Industry , Beijing Technology and Business University , Beijing 100048 , China.
J Agric Food Chem. 2019 Nov 13;67(45):12584-12589. doi: 10.1021/acs.jafc.9b05364. Epub 2019 Nov 1.
Lysozyme has emerged to be a promising alternative to traditional antibiotics to deal with the increasing antibiotic resistance of bacteria. However, its application is hampered by its inferior bactericidal activity against Gram-negative bacteria. To address this problem, a novel "enzyme-cascade fluorescent high-throughput screening (HTS) method" was designed and constructed based on detection of fluorescence resonance energy transfer (FRET) and enzyme-cascade reaction of lysozyme and protease. As a proof of concept, site-saturation mutagenesis libraries targeting at residues of the unstructured stretch at the N-terminus of lysozyme were constructed and screened by the proposed HTS method. The isolated lysozyme variants proved to exhibit higher antibacterial activity against K12, demonstrating the significance of this region for the bactericidal function of lysozyme. The presented cell-based fluorescent HTS method is a new tool for screening lysozyme variants with improved bactericidal efficacy against Gram-negative bacteria and for exploring the sequence-structure-function relationship of lysozyme.
溶菌酶作为一种有前途的替代传统抗生素的方法，可以应对细菌日益增加的抗生素耐药性。然而，其应用受到其对革兰氏阴性菌杀菌活性差的限制。为了解决这个问题，设计并构建了一种新型的“酶级联荧光高通量筛选（HTS）方法”，基于溶菌酶和蛋白酶的荧光共振能量转移（FRET）和酶级联反应的检测。作为概念验证，针对溶菌酶 N 端无规卷曲区域的残基，构建并通过所提出的 HTS 方法筛选了定点饱和突变文库。分离的溶菌酶变体对 K12 的抗菌活性更高，这证明了该区域对溶菌酶杀菌功能的重要性。所提出的基于细胞的荧光 HTS 方法是筛选对革兰氏阴性菌具有更高杀菌效果的溶菌酶变体的新工具，同时也可用于探索溶菌酶的序列-结构-功能关系。
