Jhon Jairo Melchor-Moncada, Santiago Vasquez-Giraldo, Augusto Zuluaga-Vélez, Lina Marcela Orozco, Luz Angela Veloza, Juan Carlos Sepúlveda-Arias
{"title":"Bioconjugation of Serratiopeptidase with Titanium Oxide Nanoparticles: Improving Stability and Antibacterial Properties.","authors":"Jhon Jairo Melchor-Moncada, Santiago Vasquez-Giraldo, Augusto Zuluaga-Vélez, Lina Marcela Orozco, Luz Angela Veloza, Juan Carlos Sepúlveda-Arias","doi":"10.3390/jfb15100300","DOIUrl":null,"url":null,"abstract":"<p><p>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 <i>Serratia marcescens</i>, 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<sub>2</sub>-NPs), leveraging the biocompatibility and stability of these nanomaterials. Commercial TiO<sub>2</sub>-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 <i>Serratia marcescens</i> obtained from the intestine of <i>Bombyx mori</i> 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 <i>E. coli</i> than the free enzyme or TiO<sub>2</sub>-NPs at 1000 µg/mL. This study successfully developed a serratiopeptidase-TiO<sub>2</sub> 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.</p>","PeriodicalId":15767,"journal":{"name":"Journal of Functional Biomaterials","volume":null,"pages":null},"PeriodicalIF":5.0000,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11508812/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Functional Biomaterials","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.3390/jfb15100300","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ENGINEERING, BIOMEDICAL","Score":null,"Total":0}
引用次数: 0
Abstract
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 Serratia marcescens, 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 (TiO2-NPs), leveraging the biocompatibility and stability of these nanomaterials. Commercial TiO2-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 Serratia marcescens obtained from the intestine of Bombyx mori 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 E. coli than the free enzyme or TiO2-NPs at 1000 µg/mL. This study successfully developed a serratiopeptidase-TiO2 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.
期刊介绍:
Journal of Functional Biomaterials (JFB, ISSN 2079-4983) is an international and interdisciplinary scientific journal that publishes regular research papers (articles), reviews and short communications about applications of materials for biomedical use. JFB covers subjects from chemistry, pharmacy, biology, physics over to engineering. The journal focuses on the preparation, performance and use of functional biomaterials in biomedical devices and their behaviour in physiological environments. Our aim is to encourage scientists to publish their results in as much detail as possible. Therefore, there is no restriction on the length of the papers. The full experimental details must be provided so that the results can be reproduced. Several topical special issues will be published. Scope: adhesion, adsorption, biocompatibility, biohybrid materials, bio-inert materials, biomaterials, biomedical devices, biomimetic materials, bone repair, cardiovascular devices, ceramics, composite materials, dental implants, dental materials, drug delivery systems, functional biopolymers, glasses, hyper branched polymers, molecularly imprinted polymers (MIPs), nanomedicine, nanoparticles, nanotechnology, natural materials, self-assembly smart materials, stimuli responsive materials, surface modification, tissue devices, tissue engineering, tissue-derived materials, urological devices.