Abstract：
Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins\' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.
摘要：
抗菌素耐药性构成了重大的全球威胁，达到世界卫生组织报告的危险高水平。新的抗病机制的出现和迅速蔓延，再加上近几十年来缺乏有效的治疗方法，每年导致数千人死于由耐药微生物引起的感染。因此,迫切需要开发能够对抗抗生素抗性细菌的新化合物。具有有效杀菌作用的一类有希望的分子是肽聚糖水解酶。以前,我们克隆并表征了粪肠球菌EnpA（EnpACD）蛋白的M23催化域的生化特性。与M23家族中的其他酶不同，EnpACD表现出广泛的特异性。然而,其活性在低离子强度条件下受到限制。在这项研究中,我们介绍了包含EnpACD与三个不同的SH3b细胞壁结合域融合的三种嵌合酶的工程。这些嵌合体表现出对环境条件的增强的耐受性和在牛和人血清中的持续活性。此外，我们的发现表明，SH3b结构域的添加影响嵌合酶的活性，从而扩大其在对抗抗菌素耐药性方面的潜在应用。这些研究表明，将SH3b结合域添加到EnpACD导致产生对离子强度和pH值具有更广泛耐受性的嵌合体。使他们能够在更广泛的条件下保持活跃。这种方法为获得具有定制特性的抗菌酶提供了一种相对简单的方法，并强调了具有增强功能的蛋白质工程的潜力。有助于有效解决抗菌素耐药性的持续努力。
