Lysozyme antimicrobial activity against Enterococcus Faecalis ­ a pilot study / Atividade antimicrobiana da lisozima contra Enterococcus Faecalis ­ um es-tudo piloto

O preparo químico-mecânico (PQM) do sistema de canais radiculares é essencial para eliminar tecidos infectados e garantir uma desinfecção adequada. O Hidróxido de Cálcio (HC) combinado com o propilenoglicol é frequentemente utilizado como uma pasta intracanal para desinfecção e medicação intraoperatória. No entanto, algumas bactérias, como o Enterococcus faecalis (E. faecalis), podem resistir aos efeitos do hidróxido de cálcio. O Ultracal® é uma medicação de hidróxido de cálcio de alta qualidade e radiopaco usado em procedimentos endodônticos. Já a lisozima é uma substância com propriedades antimicrobianas encontrada em várias partes do corpo humano e tem sido estudada como uma opção promissora para o tratamento de infecções endodônticas. O objetivo do presente estudo foi avaliar e comparar a atividade antimicrobiana do HC com propilenoglicol, Ultracal® e Lisozima contra E. faecalis. Foram realizadas escavações em placas de petri contaminadas com E. faecalis. Após, foi adicionado as medicações intracanais e as placas foram levadas a estufa a 37°C em aerobiose. Os halos de inibição formados foram medidos em 2, 4 e 7 dias. HC apresentou halos de inibição maiores quando comparado as outras medicações e com maior crescimento com o passar dos dias. A lisozima apresentou apenas ação nas primeiras 48 horas, perdendo seu efeito após esse período. Ambas as medicações com hidróxido de cálcio apresentaram valores crescentes. Baseado nos resultados obtidos, conclui-se que as medicações a base de hidróxido de cálcio demonstraram melhor ação contra E. faecalis em ação direta.

The chemical-mechanical preparation (CMP) of root canals system is essential to eliminate infected tissues and ensure adequate disinfection. Calcium hydroxide (CH) combined with propylene glycol is often used as an intracanal medication for intraoperative disinfection and medication. However, some bacteria, such as Enterococcus faecalis (E. faecalis), may resist the effects of calcium hydroxide. Ultracal® is a high-quality radiopaque calcium hydroxide medication used in endodontic procedures. Lysozyme, on the other hand, is a substance with antimicrobial properties found in various parts of the human body and has been studied as a promising option for the treatment of endodontic infections. The aim of this study was to evaluate and compare the antimicrobial activity of CH with propylene glycol, Ultracal®, and Lysozyme against E. faecalis. Petri plates contaminated with E. faecalis were excavated, intracanal medications were added, and the plates were incubated at 37°C in aerobic conditions. The inhibition halos formed were measured at 2, 4, and 7 days. CH showed larger inhibition halos compared to the other medications and exhibited increased growth over the days. Lysozyme showed activity only in the first 48 hours, losing its effect after this period. Both medications with calcium hydroxide showed increasing values. Based on the results obtained, it is concluded that calcium hydroxide-based medications demonstrated better action against E. faecalis in direct action.

Effect of lysozyme combined with hydrothermal pretreatment on excess sludge and anaerobic digestion.

Anaerobic digestion (AD) is widely employed for sludge stabilization and waste reduction. However, the slow hydrolysis process hinders methane production and leads to prolonged sludge issues. In this study, an efficient and eco-friendly lysozyme pre-treatment method was utilized to address these challenges. By optimizing lysozyme dosage, hydrolysis and cell lysis were maximized. Furthermore, lysozyme combined with hydrothermal pretreatment enhanced overall efficiency. Results indicate that: (1) When lysozyme dosage reached 90 mg/g TS after 240 min of pretreatment, SCOD, soluble polysaccharides, and protein content reached their maxima at 855.00, 44.09, and 204.86 mg/L, respectively. This represented an increase of 85.87%, 365.58%, and 259.21% compared to the untreated sludge. Three-dimensional fluorescence spectroscopy revealed the highest fluorescence intensity in the IV region (soluble microbial product), promoting microbial metabolic activity. (2) Lysozyme combined with hydrothermal pretreatment significantly increased SCOD, soluble proteins, and polysaccharide release from sludge, reducing SCOD release time. Orthogonal experiments identified Group 3 as the most effective for SCOD and soluble polysaccharide release, while Group 9 released the most soluble proteins. The significance order of factors influencing SCOD, soluble proteins, and polysaccharide release is hydrothermal temperature > hydrothermal time > enzymatic digestion time.(3) The lysozyme-assisted hydrothermal pretreatment group exhibited the fastest release and the highest SCOD concentration of 8,135.00 mg/L during anaerobic digestion. Maximum SCOD consumption and cumulative gas production increased by 95.89% and 130.58%, respectively, compared to the control group, allowing gas production to conclude 3 days earlier.

Eggshell membrane and its major component lysozyme and ovotransferrin enhance the secretion of decorin as an endogenous antifibrotic mediator from lung fibroblasts and ameliorate bleomycin-induced pulmonary fibrosis.

Aging is a high-risk factor for obstructive and fibrotic lung diseases. Fibrotic lung disease leading to decreased lung function is characterized by interstitial remodeling and tissue scarring (sclerosis), with destruction of alveoli and excess deposition of type I collagen, an extracellular matrix component secreted by fibroblasts. Therefore, regulating transforming growth factor-ß (TGF-ß) as a profibrotic signal is essential to suppress pulmonary fibrosis. In pulmonary fibrosis, TGF-ß signaling is mediated by Smad and YAP/TAZ, and TAZ linked to the pathology of pulmonary function is observed in lung fibroblasts from patients with idiopathic pulmonary fibrosis. Although fibrosis is thought to be irreversible, it is an interventional condition. Decorin (DCN) blocks TGF-ß signaling in pulmonary fibrosis, although there are no cellular pharmacological methods to stimulate DCN secretion. We previously showed that chicken eggshell membrane (ESM, a well-known wound-healing material) promotes dcn gene expression in fibroblasts. In this study, we investigated whether ESM stimulates DCN secretion as an endogenous mediator and ameliorates pulmonary fibrosis. Decorin secretion was significantly enhanced in the WI-38 lung fibroblast culture supernatants supplemented with ESM. This effect was increased with major component lysozyme and maximally promoted in experiments with lysozyme and ovotransferrin (the two main proteins in soluble ESM) at a 16:1 concentration ratio, the ratio in the ESM extract. Decorin secretion by ESM modulates TGF-ß signaling in lung fibroblasts by reducing TAZ and pSmad2 nuclear localization. Decorin siRNA experiments confirmed that nuclear localization of TAZ is DCN-dependent. In a mouse model of bleomycin-induced pulmonary fibrosis, all fibrotic markers of ESM treatment group such as hydroxyproline (a collagen deposition marker), and both evaluation of fibrosis density by automated thresholding of picrosirius red-stained lung tissue scan images and Ashcroft fibrosis scores, and also the nuclear localization of TAZ were reduced after 2 weeks compared with control group. Furthermore, long-term (22 week) ESM consumption by healthy individuals significantly improved vital capacity and the forced expiratory volume in 1 s to forced vital capacity ratio (FEV1/FVC). This study reveals that ESM, a well-established wound-healing material, may be a potential preventive medicine for pulmonary fibrosis.

From sequence to function: Exploring biophysical properties of bacteriophage BFK20 lytic transglycosylase domain from the minor tail protein gp15.

Bacteriophages have evolved different mechanisms of infection and penetration of bacterial cell walls. In Siphoviridae-like viruses, the inner tail proteins have a pivotal role in these processes and often encode lytic protein domains which increase infection efficiency. A soluble lytic transglycosylase (SLT) domain was identified in the minor tail protein gp15 from the BFK20 bacteriophage. Six fragments containing this SLT domain with adjacent regions of different lengths were cloned, expressed and purified. The biophysical properties of the two best expressing fragments were characterized by nanoDSF and CD spectroscopy, which showed that both fragments had a high refolding ability of 90 %. 3D modeling indicated that the bacteriophage BFK20 SLT domain is structurally similar to lysozyme. The degradation activity of these SLT proteins was evaluated using a lysozyme activity assay. BFK20 might use its transglycosylase activity to allow efficient phage DNA entry into the host cell by degrading bacterial peptidoglycan.

Natural agents derived Pickering emulsion enabled by silica nanoparticles with enhanced antibacterial activity against drug-resistant bacteria.

The emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a global health challenge due to the overuse of antibiotics. Natural substances including enzymes and essential oils have shown great potential as alternative treatment options. However, the combinational use of these natural agents remains challenging due to the denaturation of enzymes upon direct contact with oil. In this study, we report the design of a Pickering emulsion containing two natural antibacterial agents, lysozyme and tea tree oil, stabilized by fractal silica nanoparticles. In this design, the enzyme activity is kept and the volatility problem of tea tree oil is mitigated. Due to synergistic bacterial cell wall digestion and membrane disruption functions, potent bactericidal efficacy in vitro against drug-resistant bacteria is achieved. The therapeutic potential is further demonstrated in a wound healing model with drug-resistant bacteria infection, better than a synthetic antibiotic, Ampicillin. This study opens new avenues for the development of natural product-based antimicrobial treatments with promising application potential.

Cronobacter sakazakii lysozyme inhibitor LprI mediated by HmsP and c-di-GMP is essential for biofilm formation and virulence.

Cronobacter sakazakii poses a significant threat, particularly to neonates and infants. Despite its strong pathogenicity, understanding of C. sakazakii biofilms and their role in infections remains limited. This study investigates the roles of HmsP and c-di-GMP in biofilm formation and identifies key genetic and proteomic elements involved. Gene knockout experiments reveal that HmsP and c-di-GMP are linked to biofilm formation in C. sakazakii. Comparative proteomic profiling identifies the lysozyme inhibitor protein LprI, which is downregulated in hmsP knockouts and upregulated in c-di-GMP knockouts, as a potential biofilm formation factor. Further investigation of the lprI knockout strain shows significantly reduced biofilm formation and decreased virulence in a rat infection model. Additionally, LprI is demonstrated to bind extracellular DNA, suggesting a role in anchoring C. sakazakii within the biofilm matrix. These findings enhance our understanding of the molecular mechanisms underlying biofilm formation and virulence in C. sakazakii, offering potential targets for therapeutic intervention and food production settings.IMPORTANCECronobacter sakazakii is a bacterium that poses a severe threat to neonates and infants. This research elucidates the role of the lysozyme inhibitor LprI, modulated by HmsP and c-di-GMP, and uncovers a key factor in biofilm formation and virulence. The findings offer crucial insights into the molecular interactions that enable C. sakazakii to form resilient biofilms and persist in hostile environments, such as those found in food production facilities. These insights not only enhance our understanding of C. sakazakii pathogenesis but also identify potential targets for novel therapeutic interventions to prevent or mitigate infections. This work is particularly relevant to public health and the food industry, where controlling C. sakazakii contamination in powdered infant formula is vital for safeguarding vulnerable populations.

Hydrogel microspheres encapsulating lysozyme/MXene for photothermally enhanced antibacterial activity and infected wound healing.

The high mortality and enormous economic burden of bacterially infected wounds remains a huge challenge for human health. The development of ideal wound dressings with desirable antibacterial and good wound healing properties still remains a major problem affecting the regeneration of bacterially infected wound tissue. Herein, we present novel alginate-based hydrogel microspheres containing lysozyme and MXene (i-Lyso@Alg), in which the positively charged lysozyme is immobilized on the negatively charged MXene by electrostatic interaction. Due to the presence of MXene, i-Lyso@Alg exhibits good thermal effect, drug release behavior and strong antibacterial activity under near-infrared (NIR) irradiation. The synthesized i-Lyso@Alg can realize not only improvement of lysozyme stability but also photothermal responsive up-regulation for biocatalysis of lysozyme. The excellent antibacterial activities of i-Lyso@Alg were attributed to the photothermally enhanced lysozyme activity, assisted by bacterial death caused by local thermal effect of photothermally activated MXene and the physical damage due to the MXene. In addition, in the infected skin wounds of rats, i-Lyso@Alg + NIR significantly accelerates the wound healing process by inhibiting the expression of inflammatory factors and bacterial (Staphylococcus aureus) infection, and inducing the expression of pro-angiogenic factors and tissue remodeling. Overall, the results of this study introduce a pioneering approach by integrating the unique photothermal properties of MXene with the enzymatic action of lysozyme within an alginate-based hydrogel microsphere. This synergistic system not only advances the frontier of antibacterial wound dressings but also represents a significant step towards effective management of infected wounds, which possesses great potential in clinical treatment of infected wounds.

Correlation of Solvent Interaction Analysis Signatures with Thermodynamic Properties and In Silico Calculations of the Structural Effects of Point Mutations in Two Proteins.

The partition behavior of single and double-point mutants of bacteriophage T4 lysozyme (T4 lysozyme) and staphylococcal nuclease A was examined in different aqueous two-phase systems (ATPSs) and studied by Solvent Interaction Analysis (SIA). Additionally, the solvent accessible surface area (SASA) of modeled mutants of both proteins was calculated. The in silico calculations and the in vitro analyses of the staphylococcal nuclease and T4 lysozyme mutants correlate, indicating that the partition analysis in ATPSs provides a valid descriptor (SIA signature) covering various protein features, such as structure, structural dynamics, and conformational stability.

A novel thermophilic lysozyme 4356 from Cohnella sp. A01: Cloning, heterologous expression, biochemical and kinetic characterization.

Lysozymes have gained attention for their antiseptic properties. In silico studies have shown that the enzyme containing lysM can act as an antibacterial agent. Binding of the lysM motif of rSELys to peptidoglycan and molecular dynamics simulations showed that the protein-ligand binding is very stable. rSELys (2016 bp) is a new recombinant glycoside hydrolase from the thermophilic bacterium Cohnella sp. A01 (PTCC number: 1921). Protein expression and purification, a single band with an apparent molecular weight of ∼74 kDa was observed by SDS-PAGE. The kinetic parameters were Km 1.163 mg/ml, Vmax 670.3 U/mg, kcat 1675.75 (S-1), and kcat/Km 1440.88 (M-1S-1). Its optimum temperature was 55 °C and pH 8. Temperature stability also showed that the temperature of 50-60 °C retained more than half of its activity after 90 min. Based on the results, rSELys demonstrated antibacterial effects on both Gram-positive and Gram-negative strains, with inhibition zones of 11 and 9 mm, respectively. SEM analysis confirmed hydrolysis activity, the MIC was determined to be 31.25 µg/ml and 3.9 µg/ml, and MBC 0.97 µg/ml, respectively. CD and fluorescence studies showed that up to a temperature of 85 °C and a pH value of 8-12 no structural changes occur, and thermal stability protein was confirmed.

Clonal Monocytosis of Renal Significance.

Clonal monocytosis reflects a preneoplastic or neoplastic sustained increase in the absolute monocyte count in the absence of reactive causes. Causes of clonal monocytosis include clonal cytopenias with monocytosis and acute and chronic myeloid neoplasms. Chronic myelomonocytic leukemia (CMML) is a prototypical myelodysplastic/myeloproliferative overlap neoplasm in adults, characterized by sustained peripheral blood monocytosis. Renal abnormalities, including acute kidney injury (AKI) and chronic kidney disease (CKD), are frequent in patients with CMML and are predictors of worse outcomes. In addition, AKI/CKD often limits eligibility for allogeneic stem cell transplantation or enrollment in clinical trials. In this review, we highlight clonal monocytosis-related etiologies that give rise to AKI and CKD, with special emphasis on CMML and lysozyme-induced nephropathy (LyN). Monocytes produce lysozyme, which, in excess, can accumulate in and damage the proximal renal tubular epithelium. Early identification of this etiology and a timely reduction in monocyte counts can salvage renal function. Other etiologies of renal injury associated with clonal monocytosis include direct renal infiltration by monocytes, renal extramedullary hematopoiesis, myeloproliferative neoplasm-associated glomerulopathy, auto-immune (membranous nephropathy, minimal change disease) and paraneoplastic manifestations, thrombotic microangiopathy, obstructive nephropathy due to myeloproliferation, and urate nephropathy due to tumor lysis syndrome. We propose to group these mechanistic etiologies of renal injury as clonal monocytosis of renal significance and provide guidance on their diagnosis and management.

How does adulteration of wax foundation affect phenoloxidase and lysozyme activities as selected parameters of immunity in Apis mellifera?

Introduction: The adulteration of wax foundation is, for many reasons, a growing problem of modern beekeeping not only in Europe but also around the world. Wax foundation contaminated with stearin addition leads to a brood die-off, while paraffin addition negatively affects the strength of combs. It is tenable that such adulterated wax foundation reduces bees' immunity. The aim of the study was to determine the activities of two bee immune enzymes, lysozyme and phenoloxidase, in the haemolymph of worker bees which had emerged from combs with wax foundations contaminated with stearin or paraffin. Material and Methods: Combs built with stearin- or paraffin-adulterated wax (both adulterants at concentrations of 10%, 30% or 50%) or pure wax (0% adulterated) foundations were placed in the colonies, one for each adulterant and percentage. The workers were marked upon emergence from these combs and those bees were introduced into one strong colony per adulterant and percentage. Phenoloxidase and lysozyme activities were determined in the haemolymph of 1-, 7- and 14-day-old workers. Results: The higher the concentrations of stearin and paraffin in the wax foundation, the lower the phenoloxidase activities were. These activities increased with the bee age. In contrast, the trends in lysozymes were opposite. Paraffin seems to be less toxic than stearin. Conclusion: Adulteration of wax foundation with even a small amount of stearin or paraffin has negative effects on the functioning of the bee.

Relationship of MicroRNA according to Immune Components of Breast Milk in Korean Lactating Mothers.

Purpose: Human breast milk (HBM) contains immune components that produced and delivered from the mother along with nutrients necessary for the baby. MicroRNA (miRNA) is a small noncoding RNA molecule, that is used as an ideal biomarker for diagnosis and prognosis of various diseases and are more abundant in HBM. We analyzed and compared the immune components and miRNAs of HBM. Methods: HBM were collected from 20 healthy breastfeeding mothers. We measured the amount of lactoferrin, lysozyme, and immunoglobulin A (IgA) and extracted the miRNAs from each breast milk samples. Next, the top 5 and bottom 5 expressed miRNAs were compared and analyzed based on the amounts of the 3 immune components. Results: The mean levels and ranges of lactoferrin, lysozyme, and IgA were 6.33 (2.24-14.77)×106 ng/mL, 9.90 (1.42-17.59)×107 pg/mL, and 6.64 (0.48-20.01)×105 ng/mL, respectively. The miRNAs concentration per 1 mL of skim milk was 40.54 (14.95-110.01) ng/µL. Comparing the bottom 5 and top 5 groups of each immune component, 19 miRNAs were significantly upregulated (6, 9, and 4 targeting lactoferrin, lysozyme, and IgA, respectively) and 21 were significantly downregulated (4, 9, and 8 targeting lactoferrin, lysozyme, and IgA, respectively). There were no miRNAs that were expressed significantly higher or lower in common to all 3 components. However, 2 and 3 miRNAs were commonly overexpressed and underexpressed, in the top 5 groups of lysozyme and IgA concentrations. Conclusion: We identified the immune components and miRNAs in breast milk and found that each individual has different ingredients.

Human milk lactoferrin and lysozyme concentrations vary in response to a dietary intervention.

It is known that human milk (HM)1 antimicrobial protein composition varies during lactation. However, the impact of maternal diet on these antimicrobial proteins, particularly lactoferrin and lysozyme remains unknown. In addition, it is unclear whether daily, circadian, and between breast variations exist for lactoferrin and lysozyme concentrations. We investigated the impact of a low sugar, low fat, high fibre dietary intervention on HM lysozyme and lactoferrin concentrations. HM was sampled across a 3-week period; daily, at different times of day, and from both breasts to measure the level of intraindividual variation. The intervention significantly reduced maternal sugar, total fat, and saturated fat intake. HM lactoferrin concentration declined significantly over the course of the intervention however the effect size was relatively small. In addition, lactoferrin and lysozyme concentrations were variable over time, and differed significantly within and across the day but not between breasts.

Thermal Stabilisation of Lysozyme through Ensilication.

Protein therapeutics, vaccines, and other commercial products are often sensitive to environmental factors, such as temperature and long-term storage. In many cases, long-term protein stability is achieved by refrigeration or freezing. One alternative is the encapsulation of the protein cargo within an inert silica matrix (ensilication) and storage or transport at room temperature as a dry powder. In this paper, we test the effect of three commonly used biological buffers on the ensilication, storage, and desilication of the enzyme lysozyme. We show that ensilication protects lysozyme from heat (100 °C for 1 h) and during storage (18 months at room temperature). The choice of ensilication buffer has little effect on the activity of lysozyme after desilication. Our results provide confidence in the continued pursuit of ensilication as a methodology for protein stabilisation and in its compatibility with biological buffers.

Acid precipitation-hydrothermal synthesis of needle-like hydroxyapatite for protein adsorption from waste phosphogypsum.

In order to promote the high-value utilization of waste phosphogypsum (PG), hydroxyapatite was directly synthesized from PG by acid precipitation-hydrothermal method (PGHAP), which was used for the adsorption of bovine serum albumin (BSA) and lysozyme (LYS). The synthesized PGHAP was characterized by XRD, SEM, FTIR and BET, and the effects of various factors on protein adsorption capacity were studied. The results showed that PGHAP exhibits a clear needle-like morphology, high crystallinity, and an average size of about 200 nm. The pH had the greatest effect on the adsorption of protein, and the highest adsorption capacity was obtained at pH 4.0. In addition, the adsorption mechanism of protein on PGHAP was explored by adsorption kinetics and adsorption isotherm. The adsorption of protein on PGHAP conforms to the Intra-particle diffusion model kinetic model, the maximum adsorption capacity of protein on PGHAP can reach 31 mg/g, which is comparable to other adsorbents in this field. In addition, the adsorption behaviour of PGHAP on protein is more appropriately described by Langmuir isotherm model, which indicates that the binding site with uniform energy on the surface of PGHAP realizes the monolayer adsorption of protein. The main adsorption mechanisms are ion exchange, co-precipitation, complexation reaction and so on. Therefore, the needle-like PGHAP synthesized from waste PG is a protein adsorbent with industrial application potential.

Capillary Flow-Based One-Minute Quantification of Amyloid Proteolysis.

Quantifying the formation and decomposition of amyloid is a crucial issue in the development of new drugs and therapies for treating amyloidosis. The current technologies for grasping amyloid formation and decomposition include fluorescence analysis using thioflavin-T, secondary structure analysis using circular dichroism, and image analysis using atomic force microscopy or transmission electron microscopy. These technologies typically require spectroscopic devices or expensive nanoscale imaging equipment and involve lengthy analysis, which limits the rapid screening of amyloid-degrading drugs. In this study, we introduce a technology for rapidly assessing amyloid decomposition using capillary flow-based paper (CFP). Amyloid solutions exhibit gel-like physical properties due to insoluble denatured polymers, resulting in a shorter flow distance on CFP compared to pure water. Experimental conditions were established to consistently control the flow distance based on a hen-egg-white lysozyme amyloid solution. It was confirmed that as amyloid is decomposed by trypsin, the flow distance increases on the CFP. Our method is highly useful for detecting changes in the gel properties of amyloid solutions within a minute, and we anticipate its use in the rapid, large-scale screening of anti-amyloid agents in the future.

Separation of Lysozyme-Ovotransferrin Complexes and the Cooperative Role of Their Components in Egg White.

A complex of ovotransferrin and lysozyme was directly isolated from egg white using an anti-transferrin antibody-immobilized membrane after antiserum proteins were separated by non-denaturing two-dimensional electrophoresis and transferred onto a membrane. The complex retained lysozyme activity that catalyzes the breakdown of peptidoglycans in the bacterial cell wall at the ß1-4 bond between N-acetylmuramic acid and N-acetylglucosamine residues. The activity of the purified lysozyme was suppressed to 6.4% in the presence of 1 µmol Fe2+, whereas that of the mixture of the purified lysozyme and ovotransferrin was maintained at 58%. The activity of the purified lysozyme was suppressed to 35% in the presence of 10 nmol Fe3+, whereas that of the mixture of the purified lysozyme and ovotransferrin was maintained at 66%. Furthermore, the bacteriolytic activity against Bacillus subtilis of egg white with reduced glycoproteins such as ovotransferrin was assessed, and the bacteriolytic activity was found to be suppressed in the presence of Fe2+ and Fe3+. This suppression was ions, thereby alleviating the inhibition of lysozyme activity by iron ions. A complex of ovotransferrin and lysozyme is efficient because ovotransferrin effectively captures iron ions near lysozyme. Thus, protein complexes containing enzymes can be applied to control their activity.

Zinc oxide-Ulva lactuca nanocomposite is a robust dietary immunostimulant in the red swamp crayfish (Procambarus clarkii).

Aquaculture industry suffers significant limitations such as low resistance to diseases and expensive feed. This study investigated the antibacterial and immunostimulatory activities of ZnO-Ulva lactuca nanocomposite (ZnO-Ul NC) in the Procambarus clarkii. Zinc oxide nanoparticles (ZnO NPs) and ZnO-Ul NC were synthetized and characterized by electron microscopies as well as Fourier transform infrared spectroscopy. ZnO NPs and ZnO-Ul NC inhibited the growth of the isolated species Citrobacter freundii and Enterobacter hormaechei. For immunostimulatory evaluation, six crayfish groups (control, U. lactuca, ZnO L, ZnO H, ZnO-Ul L, and ZnO-Ul H) were fed on commercial diet, Ulva lactuca powder, and low or high dose of ZnO NPs or ZnO-Ul NCs, respectively for 90 days. The highest levels of total hemocyte count, granular cells%, phenoloxidase (PO) activity in hemolymph, and NO, superoxide dismutase (SOD), and GSH in hepatopancreas were all reported in the ZnO-Ul groups. The expression of proPO, SOD, and lysozyme exhibited the highest upregulation in the ZnO-Ul H group. Taken together, dietary ZnO-Ul NC significantly improved the non-specific immunity and antioxidant milieu of the crayfish at the genomic and proteomic levels. ZnO-Ul NC is cost effective, easily synthesized, and a promising immunostimulant for Procambarus clarkii that could be used in the aquaculture.

Covalent Lysozyme Immobilization on Enzymatic Cellulose Nanocrystals.

Nanostructured materials represent promising substrates for biocatalysts immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme-based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on the enzymatic CNCs and on their TEMPO oxidized derivatives (TO-CNCs), comparing immobilization yields, materials properties, and enzymatic activities. The materials were characterized by X-ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR-FTIR), bicinchoninic acid (BCA) assay, field-emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO-CNCs, based on the success of covalent bonding and on the stability of the isolated biocojugates.

Characterization and application of LysSGF2 and HolSGF2 as potential biocontrol agents against planktonic and biofilm cells of common pathogenic bacteria.

Antimicrobial resistance represents a global health emergency, necessitating the introduction of novel antimicrobial agents. In the present study, lysozyme and holin from Shigella flexneri 1.1868 phage SGF2, named LysSGF2 and HolSGF2, respectively, were cloned, expressed, and characterized. LysSGF2 and HolSGF2 showed lytic activities against S. flexneri 1.1868 cells at 4-55 °C and pH 3.1-10.3. LysSGF2 exhibited antimicrobial activity against five gram-negative and two gram-positive bacteria. HolSGF2 showed antimicrobial activity against four gram-negative and one gram-positive species. The antibacterial activities of LysSGF2 and HolSGF2 were determined in liquid beverages, including bottled water and milk. The relative lytic activity of LysSGF2 combined with HolSGF2 against the tested bacteria was approximately 46-77 % in water. Furthermore, the combination markedly decreased the viable counts of tested bacteria by approximately 3-5 log CFU/mL. LysSGF2 and HolSGF2 could efficiently remove biofilms on polystyrene, glass, and stainless-steel. The efficacy of the LysSGF2 and HolSGF2 combination against the tested bacteria on polystyrene was 58-71 %. Combination treatment effectively killed biofilm cells formed on stainless-steel and glass by 1-4 log CFU/mL. ese results indicate that LysSGF2 and HolSGF2 can successfully control both the planktonic and biofilm cells of common pathogenic bacteria, suggesting that the combined or single use of LysSGF2 and HolSGF2 may be of great value in food processing.