Lysozyme

BACKGROUND: In recent years the use of sulfur dioxide, a commonly used additive in winemaking, has been questioned because of its toxic effects on human health. Studies have been conducted to find alternatives that can effectively substitute for this additive in all its various technological functions. In previous work, lysozyme and oenological tannins were found as possible substitutes in controlling bacterial undesirable fermentations and phenolic oxidation. However, data on the volatile composition of wines obtained by that protocol are lacking. In this work, the effects on volatile composition of white wines by the substitution of SO2 during fermentation with lysozyme and tannin were studied. At the same time, the technological performance of two strains of yeast that produce low amounts of SO2 were evaluated.RESULTS: The results showed that both SO2 and lysozyme prevented the development of undesirable bacterial fermentations. The study of volatile compounds shows differences in the alcohol, acid and ester contents among the final products: wines fermented with strain 1042 and lysozyme had higher total alcohol concentration, while the addition of SO2 promoted higher production of 3-methyl-1-butanol, 3-methylthio-1-propanol, phenylethyl alcohol and 4-hydroxy-benzenethanol. Esters, as a total, were influenced by the different strain and tannins addition, while amounts of medium-chain fatty acid ethyl esters and their corresponding fatty acids were found in higher amounts in wines coming from fermentations with lysozyme. The sensory analysis revealed a preference for wines to which lysozyme and tannins had been added.CONCLUSION: The data suggest that the addition of lysozyme and oenological tannins during alcoholic fermentation could represent a promising alternative to the use of SO2 and for the production of wines with reduced content of SO2. The composition of the volatiles in the final wines was affected by the different vinification protocols (mainly with regards to alcohols and ethyl esters). Copyright © 2009 Society of Chemical Industry

This study aims to evaluate the impact of various applications (Ozone, sonication, and shellac and also chitosan‐lysozyme) on stored table eggs for 6‐weeks at ambient temperature. In this study, the functional properties and color characteristics have been monitored. Functional characterization as assayed by foaming capacity (FC) values of eggs (albumin & whole) significantly decreased from 1175 ‐ 741 (initial) at 0 weeks to 241.6 ‐ 200.3 (control), 758.3 ‐ 466.2 (shellac), 560 ‐ 333.3 (chitosan‐lysozyme), 466.6 ‐ 275 (sonication), 458.3 ‐ 291.6 (ozone) at the end of storage respectively. Eggshell surface L* values were 93.77±0.61 (control), 92.84±0.61 (shellac), 94.17±0.51 (chitosan‐lysozyme), 94.62±0.52 (sonication), and 93.99±0.52 (ozone) immediately after the treatments but no major significant changes were observed during storage. The lightness (L*) of egg yolk decreased significantly (p<0.05) during storage from 54.95±1.90 to 52.51±2.04 that indicates degradation of carotenoids in the yolk and development of brownish lipid amine‐aldehydes with protein discolouration. Chitosan‐lysozyme, sonication, and ozone treatments significantly enhanced the surface color of the eggs. However, the shellac coating maintained the highest functionality (FC) by sealing porous among all other treatments during the storage.

The growth of single crystals of tetragonal HEW lysozyme and thaumatin I into glass capillaries was monitored by time lapse video-microscopy. The crystals were obtained by unidirectional transport of the precipitating agent through capillaries of internal diameter ranging from 0.2 to 1.2 mm, using the gel acupuncture technique. For crystals growing from true protein solutions, the measured average growth rates varies with capillary diameter from 1.7 to 3.7 Å/s for thaumatin and from 2.8 to 22 Å/s for lysozyme. The measured average growth rates for crystals growing into gelled protein solutions were 1.8 Å/s for thaumatin and 2.5 Å/s for lysozyme. The trend in the variation of the growth rate with time is similar and suggests that, for capillaries with internal radius lower than 0.8 mm, diffusion dominates the global mass transport control. However, the existence of convection rolls near the crystal-solution interface and close to zones with high density gradient cannot be discarded.

We present a study on lysozyme dissolved in mixtures of water and urea, which is ubiquitously used as a protein denaturant. Despite the wide use of urea, the basic molecular mechanisms inducing protein unfolding are not still clarified. Small-angle neutron scattering (SANS) experiments have been performed using little amounts of denaturant in solutions in order to investigate the urea effect on lysozyme preceding the unfolding
process. A global fit strategy, applied to analyze SANS experiments, provides an estimation of the average composition of the solvent in the close vicinity of the protein surface and the change of the protein-protein interactions due to the presence of urea. In particular, the thermodynamic equilibrium constant responsible for cosolvent balancing between the bulk and solvation layer has been determined. It turns out that urea is
preferentially driven to the protein surface, confirming literature results at infinite dilute conditions. SANS data also reveal a possible variation of the protein net charge as a function of urea concentration, opening new perspectives and questions about the protein surface architecture at the first stages of unfolding processes.

In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial
pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial
peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising
alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type
lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The
identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG
was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained
a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression
analysis revealed that CsLyzG was distributed in major immune organs with highest expression in
head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal
(Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two
antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed
that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen
which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow
cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning
electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively,
CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber
to be used as an ecofriendly antibacterial substance in aquaculture.

Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.

Healthcare-associated infections (HAIs) are the infections that patients get while receiving medical treatment in a medical facility with bacterial HAIs being the most common. Silver and gold nanoparticles (NPs) have been successfully employed as antibacterial motifs; however, NPs leaching in addition to poor dispersion and overall reproducibility are major hurdles to further product development. In this study, the authors design and fabricate a smart antibacterial mixed-matrix membrane coating comprising colloidal lysozyme-templated gold nanoclusters as nanofillers in poly(ethylene oxide)/ poly(butylene terephthalate) amphiphilic polymer matrix. Mesoporous silica nanoparticles–lysozyme functionalized gold nanoclusters disperse homogenously within the polymer matrix with no phase separation and zero NPs leaching. This mixed-matrix coating can successfully sense and inhibit bacterial contamination via a controlled release mechanism that is only triggered by bacteria. The system is coated on a common radiographic dental imaging device (photostimulable phosphor plate) that is prone to oral bacteria contamination. Variation and eventually disappearance of the red fluorescence surface under UV light signals bacterial infection. Kanamycin, an antimicrobial agent, is controllably released to instantly inhibit bacterial growth. Interestingly, the quality of the images obtained with these coated surfaces is the same as uncoated surfaces and thus the safe application of such smart coatings can be expanded to include other medical devices without compromising their utility.

Small-angle X-ray scattering (SAXS) and elastic and quasi-elastic neutron scattering techniques were used to investigate the high-pressure-induced changes on interactions, the low-resolution structure and the dynamics of lysozyme in solution. SAXS data, analysed using a global-fit procedure based on a new approach for hydrated protein form factor description, indicate that lysozyme completely maintains its globular structure up to 1500 bar, but significant modifications in the protein-protein interaction potential occur at approximately 600-1000 bar. Moreover, the mass density of the protein hydration water shows a clear discontinuity within this pressure range. Neutron scattering experiments indicate that the global and the local lysozyme dynamics change at a similar threshold pressure. A clear evolution of the internal protein dynamics from diffusing to more localized motions has also been probed. Protein structure and dynamics results have then been discussed in the context of protein-water interface and hydration water dynamics. According to SAXS results, the new configuration of water in the first hydration layer induced by pressure is suggested to be at the origin of the observed local mobility changes.

Low-frequency collective vibrational modes in proteins have been proposed as being responsible for efficiently directing biochemical reactions and biological energy transport. However, evidence of the existence of delocalized vibrational modes is scarce and proof of their involvement in biological function absent. Here we apply extremely sensitive femtosecond optical Kerr-effect spectroscopy to study the depolarized Raman spectra of lysozyme and its complex with the inhibitor triacetylchitotriose in solution. Underdamped delocalized vibrational modes in the terahertz frequency domain are identified and shown to blue-shift and strengthen upon inhibitor binding. This demonstrates that the ligand-binding coordinate in proteins is underdamped and not simply solvent-controlled as previously assumed. The presence of such underdamped delocalized modes in proteins may have significant implications for the understanding of the efficiency of ligand binding and protein–molecule interactions, and has wider implications for biochemical reactivity and biological function.

Packing interactions in bacteriophage T4 lysozyme were explored by determining the structural and thermodynamic effects of substitutions for Ala98 and neighboring residues. Ala98 is buried in the core of T4 lysozyme in the interface between two alpha-helices. The Ala98 to Val (A98V) replacement is a temperature-sensitive lesion that lowers the denaturation temperature of the protein by 15 degrees C (pH 3.0, delta delta G = -4.9 kcal/mol) and causes atoms within the two helices to move apart by up to 0.7 A. Additional structural shifts also occur throughout the C-terminal domain. In an attempt to compensate for the A98V replacement, substitutions were made for Val149 and Thr152, which make contact with residue 98. Site-directed mutagenesis was used to construct the multiple mutants A98V/T152S, A98V/V149C/T152S and the control mutants T152S, V149C and A98V/V149I/T152S. These proteins were crystallized, and their high-resolution X-ray crystal structures were determined. None of the second-site substitutions completely alleviates the destabilization or the structural changes caused by A98V. The changes in stability caused by the different mutations are not additive, reflecting both direct interactions between the sites and structural differences among the mutants. As an example, when Thr152 in wild-type lysozyme is replaced with serine, the protein is destabilized by 2.6 kcal/mol. Except for a small movement of Val94 toward the cavity created by removal of the methyl group, the structure of the T152S mutant is very similar to wild-type T4 lysozyme. In contrast, the same Thr152 to Ser replacement in the A98V background causes almost no change in stability. Although the structure of A98V/T152S remains similar to A98V, the combination of T152S with A98V allows relaxation of some of the strain introduced by the Ala98 to Val replacement. These studies show that removal of methyl groups by mutation can be stabilizing (Val98----Ala), neutral (Thr152----Ser in A98V) or destabilizing (Val149----Cys, Thr152----Ser). Such diverse thermodynamic effects are not accounted for by changes in buried surface area or free energies of transfer of wild-type and mutant side-chains. In general, the changes in protein stability caused by a mutation depend not only on changes in the free energy of transfer associated with the substitution, but also on the structural context within which the mutation occurs and on the ability of the surrounding structure to relax in response to the substitution.(ABSTRACT TRUNCATED AT 400 WORDS)

YUMURTA BİYOAKTİF BİLEŞENLERİ (2): LİZOZİM

EGG BIOACTIVE COMPONENTS (2): Lysozyme

Özet
Gıda sanayisinde özellikle doğal kaynaklı antimikrobiyal komponentlerin kullanıldığı biyo-koruma metotlarına olan ilginin gün geçtikçe artmasıyla birlikte, doğal enzimlerin (lizozim) gıdalarda kullanım potansiyeli de artış göstermiştir. Lizozim ticari olarak değerli bir enzim olup, günümüzde çeşitli gıdalarda doğal bir antimikrobiyal ajan olarak, peynir, bira ve şarap üretiminde koruyucu ya da mikrobiyal prosesleri kontrol etmek amacıyla kullanılmaktadır. Lizozim, yumurta beyazında (toplam yumurta beyazı proteinlerinin %3,5'i) oldukça fazladır ve tavuk yumurtası albumini günümüzde bu enzimin temel ticari kaynağıdır.
Derleme çalışması ile lizozimin önemi, etki mekanizması, ticari önemi ve ayrımına dair güncel literatür değerlendirilerek okuyucuların istifadesine sunulmuştur.

Anahtar kelimeler: yumurta, biyoaktif bileşen, katma değer, antimikrobiyal aaktivite, lizozim.

Abstract:
The potential of using natural enzymes (lysozyme) in foods has also increased, with the increasing interest in bio-preservation methods, especially in the food industry, in which natural origin antimicrobial components are used. Lysozyme is a commercially valuable enzyme and is used today as a natural antimicrobial agent in various foods, to control preservative or microbial processes in the production of cheese, beer and wine. Lysozyme is very abundant in egg white (3.5% of total egg white protein), and chicken egg albumin is today the main commercial source of this enzyme.
The current literature on the importance, mechanism of action, commercial importance and distinction of lysozyme has been evaluated and presented to the benefit of the readers.

Key words: egg, bioactive component, added value, antimicrobial aactivity, lysozyme.

Fe3O4 magnetic nanoparticles modified with tetraethyl orthosilicate and bovine serum albumin (Fe3O4@TEOS@BSA) were synthesized and efficiently used to separate lysozyme from egg white. Glutaraldehyde was used to crosslink the bovine serum albumine molecules around the nanoparticles. The surface modifications were attested by transmission electron microscopy, infrared spectroscopy, thermogravimetry analysis, and zeta potential. The material was thermally stable, and its surface charge was pH-dependent. The best lysozyme adsorption and desorption were obtained at pHs 10.0 and 5.0, respectively. The pseudo-second-order model fitted well into the lysozyme adsorption kinetic data and the time for the equilibrium was 15 min. The adsorption equilibrium results were best described by the Freundlich model. Fe3O4@TEOS@BSA particles were very efficient to extract lysozyme from chicken egg, according to the SDS-PAGE analyses. The extracted molecules maintained their enzymatic activity in about 90%. Fe3O4@TEOS@BSA particles were easily recycled, with their reuse being possible 5 times with the same performance.

The present study aimed to determine the effects of four experimental diets on growth, immunological parameters, and coloration of the white Oscar, Astronotus ocellatus (Agassiz in Spix and Agassiz, 1831).  So that, 120 individuals, with an average weight of 7.5 ± 0.5 g, were randomly distributed in 12 aquaria (10 fish in each aquarium). The fish were fed with 4 diets as follows: pigment-free (as the control), astaxanthin, turmeric powder, and spirulina powder (100 mg kg-1) for eight weeks. At the end of the experiment, growth and immunological indices, as well as coloration were measured. There was a significant difference between control and the astaxanthin treatment in all growth indices except for condition factor (p<0.05). There were also significant differences between control and the spirulina treatment in the survival rate and growth rate; between control and the turmeric treatment in the increased length; and between control and turmeric and spirulina treatments in food conversion ratio (p<0.05). The results also indicated that there was a significant difference between the astaxanthin treatment and other treatments in lysozyme activity as well as between control and the spirulina treatment in terms of IgM level (p<0.05). However, such a significant difference was not observed in total immunoglobulin and total carotenoids (p>0.05). There was a significant difference between some treatments in the intensity of primary and secondary colors (p<0.05). The findings exhibited that all three types of pigments employed in this study could improve the growth factors, immunological parameters, and coloration of white Oscar; however, astaxanthin outperformed the others.

Sirotkin, V.A. Lysozyme in water-acetone mixtures: Residual enzyme activity and preferential solvation. / V.A. Sirotkin, A.A. Kuchierskaya // In: Kozlowska, I., Walczak, K., (eds.) Acetone: Biochemistry, Production and Uses. - Nova Science Publishers, Inc., Hauppauge, NY, 2018. - P.47-72.

ABSTRACT
Numerous benefits of employing enzymes in non-aqueous organic liquids exist, including catalysis of industrially-important synthetic reactions, suppression of undesirable side reactions caused by water, and high solubility of hydrophobic reagents. Residual enzyme activity was investigated to monitor stabilization/destabilization of hen egg-white lysozyme at low, intermediate, and high water content in acetone at 25 o C. The advantages of our methods are as follows: 1. Residual enzyme activity curves can be determined in the entire range of water content in organic liquids. 2. Enzyme activity values can be measured at fixed reaction conditions.

Background: Sodium or potassium nitrite is widely used as a curing agent in sausages and other cured meat products. Nitrite has strong antimicrobial and antioxidant effects and generates cured meat color. Nitrite, however, can react with secondary or tertiary amines in meat to form carcinogenic, teratogenic and mutagenic N-nitroso compounds. Several findings have been suggested that high consumption of processed meat may increase the risk of cancer, and emphasized that dietary nitrosamines are positively associated with cancer. Lysozyme is one of the major egg proteins that have antimicrobial and antioxidant characteristics. Therefore, lysozyme can be used in meat processing to prevent microbial growth and oxidative degradation in meat products during storage. This study is focused on evaluating the antimicrobial and antioxidant effects of lysozyme extracted from egg white as a replacer of nitrite in a cooked Italian-type chicken sausage.

Methods: Four curing treatments including 100% nitrite (control), 100% lysozyme (treatment 1), 25% nitrite + 75% lysozyme (treatment 2) and 50% nitrite + 50% lysozyme (treatment 3) were used to prepare Italian-type chicken sausage samples. Recipe was developed with 64%  (w/w) meat, 17% (w/w) binder (bread crumble), 12% (w/w) ice, 4%  (w/w) vegetable oil, 2%  (w/w) salt, 1% (w/w) spices (chili, black pepper, cardamom). Prepared samples were cooked in an 80 °C smoke house to a core temperature of 65 °C and cooled in cold water to 20-25 °C subsequently packed in polyethylene and stored in a freezer (-18 °C). The antimicrobial effect lysozyme was tested using Escherichia coli and Salmonella. The growth of these pathogens at 0, 3 and 5 days of storage of spore inoculation was determined. The antioxidant activity of lysozyme was determined using the TBARS value during the 25 d storage period. The redness (a*), lightness (L*), and yellowness (b*) of sausages were analyzed using a Minolta color meter (CR 410, Konica Minolta Inc., Japan). The proximate composition (AOAC, 2002) of frozen (-18 °C) sausage samples and sensory properties of cooked samples were determined.

Results: 50% nitrite + 50% lysozyme (treatment 3) was as effective as control (100% nitrite) in suppressing the growth of Escherichia coli, Salmonella and limiting lipid oxidation in the Italian-type chicken sausage. Treatment 3 was not significantly different from the control, for lightness (L*), redness (a*) and yellowness (b*) values (P > 0.05) but showed the best sensory characteristics among the treatments (p < 0.05). Moisture content of control sample was significantly higher (p < 0.05) than other treatments while crude protein, crude fat, crude fiber and ash content were not significantly differ each. In term of the cost, both treatment 3 and control have shown approximately equal values.

Conclusion: This study demonstrated that lysozyme can be used as an effective nitrite replacer in the Italian-type chicken sausage. Replacing 50% of nitrate salt with 50% lysozyme did not show any negative effects in controlling microbial growth, preventing lipid oxidation, and color changes but improved the sensory characteristics.

Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F7 (from papain–trypsin hydrolysate), F8 (from papain hydrolysate) and F3 (from trypsin hydrolysate) fractions. The IC50 values were 0.03, 0.155 and 0.23 mg/ml for F7, F8 and F3, respectively. The F7 fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-ThrAsn-Arg (MW: 1428.6 Da). Lineweaver–Burk plots suggest that the F7 peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (Km, Vmax, and Ki) for the F7 peptide were measured and compared to the control.

Keywords:
lysozyme;chitosan;coating;shell eggs;egg quality;functional properties
Abstract
BACKGROUND
The interior quality, shell impact strength and functional characteristics of eggs coated with chitosan and lysozyme–chitosan combinations were evaluated for enhancing egg freshness during storage. A 10% (w/w) lysozyme solution was incorporated into 1% (w/w) chitosan film-forming solution at ratios of 0, 10, 20 and 60% (w/w).

RESULTS
Storage time and coating had significant effects on Haugh unit, yolk index, weight loss, albumen pH, dry matter, relative whipping capacity (RWC) and albumen viscosity. Uncoated eggs had higher albumen pH and weight loss and lower albumen viscosity. All coated eggshells showed greater puncture strength than uncoated eggshells, resulting in extended shelf life. The 20 and 60% lysozyme–chitosan coatings were more effective in maintaining the internal quality of eggs (e.g. pH, dry matter and RWC). Attributes such as pH, dry matter and RWC were better after the 20% lysozyme–chitosan treatment than after the other treatments.

Conclusion
The 10, 20 and 60% lysozyme–chitosan coatings, considered active packaging, showed promising attributes. They could be a viable alternative to existing techniques for maintaining the internal quality of fresh eggs during long-term storage. Chitosan coatings also improved shell strength. This study also confirms that measurements of albumen quality (pH, dry matter, viscosity and RWC) are excellent indicators of egg freshness. © 2013 Society of Chemical Industry