Melchor-Moncada Jhon Jairo, Vasquez-Giraldo Santiago, Zuluaga-Vélez Augusto, Orozco Lina Marcela, Veloza Luz Angela, Sepúlveda-Arias Juan Carlos
Grupo Infección e Inmunidad, Departamento de Ciencias Básicas, Facultad de Ciencias de la Salud, Universidad Tecnológica de Pereira, Pereira 660003, Colombia.
Grupo Polifenoles, Facultad de Tecnología, Escuela de Tecnología Química, Universidad Tecnológica de Pereira, Pereira 660003, Colombia.
J Funct Biomater. 2024 Oct 7;15(10):300. doi: 10.3390/jfb15100300.
Antimicrobial resistance (AMR) poses a significant global health threat, necessitating the development of novel antibacterial strategies. Serratiopeptidase (SP), a metalloprotease produced by bacteria such as , has gained attention not only for its anti-inflammatory properties but also for its potential antibacterial activity. However, its protein nature makes it susceptible to pH changes and self-proteolysis, limiting its effectiveness. This study aimed to increase both the enzymatic stability and antibacterial activity of serratiopeptidase through immobilization on titanium oxide nanoparticles (TiO-NPs), leveraging the biocompatibility and stability of these nanomaterials. Commercial TiO-NPs were characterized using TGA/DTG, FT-IR, UV-Vis, and XRD analyses, and their biocompatibility was assessed through cytotoxicity studies. Serratiopeptidase was produced via fermentation using the C8 isolate of obtained from the intestine of L., purified chromatographically, and immobilized on carboxylated nanoparticles via EDC/NHS coupling at various pH conditions. The optimal enzymatic activity was achieved by using pH 5.1 for nanoparticle activation and pH 5.5 for enzyme coupling. The resulting bioconjugate demonstrated stable proteolytic activity at 25 °C for 48 h. Immobilization was confirmed by FT-IR spectroscopy, and the Michaelis-Menten kinetics were determined. Notably, the bioconjugate exhibited two-fold greater antibacterial activity against than the free enzyme or TiO-NPs at 1000 µg/mL. This study successfully developed a serratiopeptidase-TiO bioconjugate with enhanced enzymatic stability and antibacterial properties. The improved antibacterial activity of the immobilized enzyme presents a promising approach for developing new tools to combat antimicrobial resistance, with potential applications in healthcare, food safety, and environmental protection.
抗菌耐药性（AMR）对全球健康构成重大威胁，因此需要开发新的抗菌策略。沙雷氏菌蛋白酶（SP）是一种由诸如沙雷氏菌等细菌产生的金属蛋白酶，不仅因其抗炎特性受到关注，还因其潜在的抗菌活性受到关注。然而，其蛋白质性质使其易受pH值变化和自我降解的影响，从而限制了其有效性。本研究旨在通过固定在二氧化钛纳米颗粒（TiO-NPs）上，利用这些纳米材料的生物相容性和稳定性，提高沙雷氏菌蛋白酶的酶稳定性和抗菌活性。使用热重分析/微商热重分析（TGA/DTG）、傅里叶变换红外光谱（FT-IR）、紫外可见光谱（UV-Vis）和X射线衍射（XRD）分析对商用TiO-NPs进行表征，并通过细胞毒性研究评估其生物相容性。通过使用从L.肠道获得的C8分离株发酵生产沙雷氏菌蛋白酶，通过色谱法纯化，并在不同pH条件下通过1-乙基-3-（3-二甲基氨基丙基）碳二亚胺/ N-羟基琥珀酰亚胺（EDC/NHS）偶联固定在羧化纳米颗粒上。通过使用pH 5.1激活纳米颗粒和pH 5.5进行酶偶联实现了最佳酶活性。所得生物共轭物在25°C下48小时内表现出稳定的蛋白水解活性。通过傅里叶变换红外光谱确认固定化，并测定米氏动力学。值得注意的是，在1000μg/mL时，生物共轭物对[具体细菌名称未给出]的抗菌活性比游离酶或TiO-NPs高两倍。本研究成功开发了一种具有增强酶稳定性和抗菌性能的沙雷氏菌蛋白酶-TiO生物共轭物。固定化酶抗菌活性的提高为开发对抗抗菌耐药性的新工具提供了一种有前景的方法，在医疗保健、食品安全和环境保护方面具有潜在应用。
