Characterization Of Lysozyme Research Articles

Fabrication and characterization of lysozyme fibrils/Zein complexes for resveratrol encapsulation: Improving stability, antioxidant and antibacterial activities

Resveratrol (Res), a naturally occurring hydrophobic polyphenol, boasts numerous health-promoting bio-functionalities. However, its limited water solubility and stability impede further applications in the food industry. This study aims to address these challenges by fabricating stable Res-loaded lysozyme fibrils/zein (Ly-F/Z) complexes. The complexes were prepared using an antisolvent precipitation method. The interaction mechanism between Ly-F and zein was elucidated through dynamic light scattering, Fourier-transform infrared spectroscopy and dissociative experiments, revealing the involvement of hydrogen bonding, electrostatic forces and hydrophobic interactions in complex formation. The Ly-F/Z complexes were utilized to encapsulate Res, resulting in an encapsulation efficiency of 82.58 %. X-ray diffraction analysis confirmed the successful encapsulation of Res within Ly-F/Z complexes, presenting an amorphous state. The Ly-F/Z-Res complexes exhibited a “fruit tree” morphology with dense fruit, showcasing remarkable stability, antioxidant and antibacterial activities. Consequently, the Ly-F/Z complexes can serve as promising delivery systems for Res in functional foods.

Extraction and Characterization of Lysozyme from Salted Duck Egg White.

Salted duck egg white (SDEW), as the main by-product in the production process of salted egg yolk, has not been effectively used as a food resource because of its high salt concentration. This study creatively used isoelectric point precipitation, ultrafiltration, and cation exchange to separate and purify lysozyme from SDEW and preliminarily explored the enzymatic properties of lysozyme. The results showed that the relative molecular weight of lysozyme was about 14 KDa, and the specific activity of lysozyme reached 18,300 U/mg. Lysozyme had good stability in the temperature range of 30 °C to 60 °C and pH of 4 to 7. Metal ions, Fe2+, Cu2+, and Zn2+, strongly inhibited lysozyme activity. Different surfactants showed certain inhibition effects on lysozyme from SDEW, among which glycerin had the strongest inhibitory effect. This study aimed to provide a theoretical reference for industrial purification and production of lysozyme from SDEW.

Purification and characterization of lysozyme from Chinese Lueyang black-bone Silky fowl egg white

Lysozyme, an important antibacterial protein, is an enzyme that cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan in cell walls. The novel lysozyme was purified and characterized from Chinese Lueyang black-bone silky fowl (CBSF) egg white, and its N-terminal amino acid sequence, enzymatic properties, and antibacterial activity were investigated. The CBSF lysozyme was purified using adsorption chromatography, ammonium sulfate precipitation, ion exchange chromatography, and size-exclusion chromatography. The purification fold and yield were 3.28 and 14.69%, respectively. The purified lysozyme was revealed as a single protein band with SDS–PAGE and had a MALDI-TOF/TOF molecular weight of 14305.57 Da and a final specific activity of 3.49 × 105 U/mg protein using Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of the lysozyme were 50 °C and 6.0, respectively. The 20 N-terminal amino acid residues of the purified lysozyme were determined to be KVFGRCELAAAMKRHGLDNY, showing some homology to the N-terminus of the odontophoridae egg white lysozyme. The purified lysozyme exerted a potent antimicrobial activity toward indicator microorganisms, including Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. However, its inhibition of gram-negative activity was weaker than that of the Gram-positive bacteria.

Purification and characterization of lysozyme in Persian sturgeon, Acipenser persicus (Borodin, 1897) from the Southwest Caspian Sea

Lysozyme (N-acetylmuramide glyconohydrolase, (EC 3.2.1.17)) is a unique enzyme which cleaves the β-1,4 linkages of N-acetylmuramic and N-acetylglucosamine of the peptidoglycan, which leads to the lysis of the bacterial cell wall. Lysozyme, as a self-defense enzyme, is produced in many organs of vertebrates. The present study describes purification and characterization of lysozyme from Acipenser persicus (Borodin, 1897). After the extraction process, ion exchange chromatography was utilized to purify the enzyme. The SDS-PAGE analysis confirmed that the molecular weight was about 14 kDa. Moreover, some of the biochemical properties such as optimum temperature, pH and the effect of metal ions on the activity of purified enzyme were investigated. Based on the results the optimum activity and pH were obtained at 50 °C and 6.5 respectively. The purified lysozyme was active in the presence of different salts including NaCl (0–0.125 M), KCl (0.075–0.125 M), MgCl2, and CaCl2 (0.005 M). Kinetic parameters were also calculated.

AURAMINE O AS POTENTIAL AMYLOID MARKER: FLUORESCENCE AND MOLECULAR DOCKING STUDIES

The applicability of Auramine O to the detection and characterization of lysozyme and serum albumin amyloid fibrils has been assessed using the fluorimetric titration and molecular docking. The parameters of the dye binding to native and fibrillar proteins were estimated in terms of the Langmuir adsorption model. It was found that Auramine O displays the high affinity for amyloid fibrils, being of the same order of magnitude as that of the classical amyloid markers. The dye also showed greater fluorescence response to lysozyme fibrils and lower sensitivity to the native protein, than Thioflavin T. Furthermore, unlike Thioflavin T, Auramine O was able to detect the morphological differences between lysozyme and albumin fibrils due to the shifts in the position of the emission maxima of the fibril-incorporated fluorophore. The molecular docking studies revealed that Auramine O and Thioflavin T form the most stable complexes with the G54_L56/S60_W62 groove of lysozyme fibrils, running parallel to the fibril axis. The results obtained suggest the contribution of both hydrophobic and electrostatic interactions to the stabilization of the dye complexes with amyloid fibrils.

Characterization of Lysozyme from Brown Eared Pheasant Egg White

Characterization of Lysozyme from Brown Eared Pheasant Egg White

Purification and Characterization of Lysozyme from the Black-Lined Limpet <I>Cellana nigrolineata</I>

Purification and Characterization of Lysozyme from the Black-Lined Limpet <I>Cellana nigrolineata</I>

Isolation and characterization of a c-type lysozyme from the nurse shark

Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein.

Purification and characterization of lysozyme from filipino venus, Ruditapes philippinarum

Lysozyme from Filipino venus (Ruditapes philippinarum) was purified by ion-exchange and gel filtration chromatography. The purification fold and yield were 3,402 and 32.4%, respectively. The molecular weight was determined to be 13.4 kDa by SDS-PAGE. The specific activity of lysozyme was 3.76×105 units/mg protein with Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of lysozyme were 75°C and 5.5, respectively. Lysozyme activity was decreased with about 45% after heat treatment for 30 min at 80°C, and completely inactivated at 100°C. It was activated by NaCl (10–70 mM), MgCl2, and CaCl2 (2–5 mM) whereas it was inhibited by ZnCl2 (2–30 mM).

The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis

A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41–79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 °C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms.

Molecular Cloning, Recombinant Expression, and Characterization of Lysozyme from Chinese Shrimp Fenneropenaeus chinensis

Lysozyme catalyzes the hydrolysis of bacterial cell walls and acts as an innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named it as Fc-lysozyme ( FcLyz in short). The full length of the gene is of 709 bp, and the open reading frame (477 bp) encodes 158 amino acids. The predicted protein has a signal peptide (–1∼–18 residue) and the molecular weight of the mature protein (residue 1–140) is of 16.2 kD. A Lyz 1 domain (residue 1–130) in the FcLyz was identified by SMART analysis. The results of semi-quantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-β-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria and the MIC reached 3.43 μM, while it showed relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important effector molecules against pathogens in innate immunity of the shrimp.

Conformational and Quantitative Characterization of Lysozyme Extracted from Galyfilcon and Senofilcon Silicone Hydrogel Contact Lenses

Purpose: To compare two solvents for retrieval of lysozyme deposited on a silicone hydrogel (SH) contact lens material galyfilcon A (GA; Acuvue Advance). Methods: Two buffers used were 50:50 acetonitrile/0.02% trifluoroacetic acid (buffer 1) and 50:50 acetonitrile/50 mM NaOH (buffer 2). Results: Extraction efficiency from GA lenses was 74% (buffer 1) and 83% (buffer 2). Buffer 2 decreased lysozyme activity > buffer 1. Ex vivo GA lenses showed total protein deposition of 2–16 μ g/lens with total lysozyme deposition of 0.3–3.9 μg/lens. Conclusions: We have developed a low acid strength extraction buffer that can be used to efficiently extract active lysozyme protein from novel siloxane-based contact lens materials.

Purification and characterization of lysozyme from purple washington clam Saxidomus purpurata

Lysozyme was purified from purple washington clam Saxidomus purpurata by sequential procedures using Chitopearl Basic BL-01 affinity and TSKgel ODS-120T column chromatographies. Molecular mass of the purified enzyme was estimated to be 12 kDa by SDS-PAGE. Optimum pH of the enzyme was 5.2 toward Micrococcus lysodeikticus cells. The optimum temperature was 50°C. The enzyme was stable in the range of pH 4.8–6.8 and 20–90°C. Further, the N-terminal amino acid sequence of the enzyme showed similarity to lysozymes from invertebrates. However, the specific activity of the enzyme toward M. lysodeikticus cells and p-nitrophenyl penta-N-acetyl-β-chitopentaoside was 143 times and 12 times higher than that of hen egg white lysozyme, respectively.

Purification and characterization of lysozyme from plasma of the eastern oyster ( Crassostrea virginica)

Lysozyme was purified from the plasma of eastern oysters ( Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.

Flocculation and characterization of protein by anionic copolymer containing reactive functional groups

Two types of anionic copolymers with reactive functional groups of amido, carboxyl and sulfonate were prepared and used to study the flocculation and characterization of lysozyme in aqueous solution. Initial increase of the ionic strength promotes flocculation, but no flocculation occurs with further increase of ionic strength. High ionic strength in solution also makes the aggregates of protein–copolymer sensitive to shear. The two types of flocculants require high pH to achieve high charge density for interacting with lysozyme effectively. Adsorption of lysozyme to the anionic copolymers was influenced strongly by pH and ionic strength. The results reveal an understanding of the types of zeta ( ζ) potential when lysozyme aggregates with copolymers. The performances of the anionic copolymers are related to their compositions when they are used as flocculants. The results confirm the mechanism of charge neutralization that prevails for highly charged polymers.

T8 1:30 Purification and characterization of lysozyme(s) from the Eastern oyster, Crassostrea virginica: A preliminary study

T8 1:30 Purification and characterization of lysozyme(s) from the Eastern oyster, Crassostrea virginica: A preliminary study

Purification and Characterization of Lysozyme from Hemolymph of Heliothis virescens Larvae

Lysozyme is an important antibacterial protein in the insect defense system. Lysozyme was isolated from hemolymph of Heliothis virescens larvae using gel filtration and ion-exchange chromatography. Heliothis lysozyme had a molecular mass of 16,000 daltons by SDS-PAGE. Using acid gel electrophoresis, Heliothis lysozyme migrated faster than egg white lysozyme. The pI of Heliothis lysozyme was estimated as greater than 9.5. Heliothis lysozyme had specific bactericidal activity against three Gram-positive bacteria but no activity against Escherichia coli. The bactericidal activity was stable at 100°C at pH 3.0 after 60 min incubation, but was labile at 100°C at pH 6.8 after 60 min incubation. Heliothis lysozyme was an inducible protein that increased 9 times when comparing unvaccinated with vaccinated larvae. Lysozyme from H. virescens was more similar in molecular mass, heat sensitivity and pH sensitivity to lysozyme isolated from Galleria mellonella and Bombyx mori than to lysozyme isolated from Hyalophora cecropia.

Evidence and characterization of lysozyme in six species of freshwater crayfishes from astacidae and cambaridae families

1. 1. A lysozyme activity was found in four Astacidae and two Cambaridae species. 2. 2. Tested on Micrococcus luteus cell wall, the reaction was stable when heated in acidic buffer but heat labile in an alkaline one. 3. 3. The activity is present in serum, hemocyte lysate and plasma. 4. 4. In serum, the lysis occurred within the first 15 min; the less active was the plasma. 5. 5. In the six species, the kinetics followed the same pattern. 6. 6. Different animals of the same species exhibited a great variability in lysozyme activity: 32–53.8% of lysis after 1 hr in serum, 7–31.7% in plasma, and 20–50.3% in hemocyte lysate. 7. 7. Different stresses weakening the animals lowered the response to less than 25% of lysis after 1 hr instead of 60–86% in serum or 38–72% in hemocyte lysate. 8. 8. The lysozyme molecules seemed to be located in hemocytes and released during experimental cell lysate or in vitro coagulation. 9. 9. Plasma reaction could represent the background of the hemolymph activity occurring in natural conditions.

The isolation and characterization of lysozyme from human foetal membranes: A comparison with the enzyme from other sources

1. 1. Lysozyme (muramidase) was isolated from an acidic extract of human foetal membranes by adsorption and elution from octadecyl silica. It was further purified by gel-filtration and ion-exchange. 2. 2. The final product was homogeneous by high performance liquid chromatography (h.p.l.c.) and electrophoresis. It was indistinguishable from human milk lysozyme by all criteria investigated, including amino acid composition, electrophoretic mobility, retention time on h.p.l.c. and sequence of the first nine residues. 3. 3. Human uterine decidual tissue was shown to contain a similar concentration of lysozyme to foetal membranes. The enzyme was also present at lower concentrations in amnion, placenta and amniotic fluid.