Molecular Characterization and Antibacterial Activity of a G-Type Lysozyme in Yellow Drum(Nibea albiflora)

Lysozyme(EC3.2.1.17)plays an important role in the immune response;as a nonspecific immune factor,it can resist causative agents.Lysozyme can be divided into c-type and g-type in fish.In a previous study,through genome-wide association analysis,the g-type lysozyme gene,which is named NaLyg in yellow drum(Nibea albiflora),was found to be a key candidate gene for disease resistance in response to Vibrio harveyi infection.The cDNA of NaLyg was 1025 bp,including four exons and three introns,and its open reading frame(ORF)had a full-length of 582 bp,encoding 193 amino acids.NaLyg was found to be conserved during evolution through bioinformatic analyses.The NaLyg protein possessed a sugar binding domain and three catalytic sites,including Glu71,Asp84 and Asp101.Quantitative qRT-PCR results confirmed that NaLyg gene mRNA was visibly increased after V.harveyi infection.The NaLyg protein purified by prokaryotic expression killed some gram-negative bacterial pathogens by inducing cell wall destruction,including V.harveyi,Aeromonas hydrophila and Edwardsiella tarda.Moreover,the NaLyg protein killed two gram-positive bacteria,Bacillus subtilis and Staphylococcus aureus.Taken together,the experimental results suggested that the NaLyg protein of N.albiflora played an important role in fighting bacterial infections.

Concentration-Dependent Effect of Nickel Ions on Amyloid Fibril Formation Kinetics of Hen Egg White Lysozyme:a Raman Spectroscopy Study

Nickel,an important transi-tion metal element,is one of the trace elements for hu-man body and has a crucial impact on life and health.Some evidences show the excess exposure to metal ions might be associated with neurological diseases.Herein,we applied Raman spectroscopy to study the Ni(II)ion effect on kinetics of amyloid fibrillation of hen egg white lysozyme(HEWL)in thermal and acidic conditions.Using the well-known Raman indicators for protein tertiary and secondary structures,we monitored and analyzed the concentration effect of Ni(II)ions on the unfolding of tertiary structures and the transformation of sec-ondary structures.The experimental evidence validates the accelerator role of the metal ion in the kinetics.Notably,the additional analysis of the amide I band profile,combined with thioflavin-T fluorescence assays,clearly indicates the inhibitory effect of Ni(II)ions on the formation of amyloid fibrils with organizedβ-sheets structures.Instead,a more significant promotion influence is affirmed on the assembly into other aggregates with disordered struc-tures.The present results provide rich information about the specific metal-mediated protein fibrillation.

Coordination and Photoisomerization of Azobenzene-Amino Acid Schiff Base Copper(II) Complexes to Lysozyme

In this study, we exhibited an amino acid (arginine and threonine) derivative Schiff base copper(II) complexes incorporating an azobenzene moiety as a photoresponsive site and conjugated it to egg white lysozyme, a well-known protein, to change ligand conformation under binding to lysozyme. Among several spectroscopic investigations, ESR clearly showed that the nitrogen atom of the amino acid residue of lysozyme was bound to the paramagnetic copper(II) ion of the complex, and UV light irradiation confirmed photoisomerization of the azobenzene moiety of the ligand to cis-form. The binding mode was considered by means of spectroscopic as well as computational methods, whereas complete crystallographic verification was still a preliminary stage.

家蚕抗菌蛋白Cecropin D和Lysozyme的分离纯化及性质鉴定

经大肠杆菌E．coli K12D31诱导后的家蚕幼虫，其免疫血淋巴经CM-Sepharose CL-6B离子交换层析、Sephadex G-100凝胶过滤层析及HPLC分离后，得到2种抗菌蛋白，经液相色谱-电喷雾质谱（ESI-MS）分析鉴定，纯化的2种抗菌蛋白分别是家蚕抗菌肽eeeropin D和溶菌酶lysozyme酸性电泳（A-PAGE）测活免疫血淋巴得到抗菌蛋白活性谱：cecropin D在8h无显著表达，12h有较强抗菌活性，30h达到最高，以后逐渐降低；lysozyme在8h后检测到表达，12h到达高峰，之后逐渐下降，48h的血淋巴中未检测到明显的表达．研究认为抗菌蛋白lysozyme和cecropin D是家蚕幼虫感染细菌8h后大量表达的抗菌蛋白，主要参与细菌感染12～30h的血淋巴对细菌的清除，是参与家蚕幼虫抗菌免疫的重要蛋白．

Hemozoin triggers tumor necrosis factor alpha-mediated release of lysozyme by human adherent monocytes:new evidences on leukocyte degranulation in P.falciparum malaria

Objective:Avidly phagocytosed hemozoin(malarial pigment) alters several functions of human monocytes and stimulates generation of several cytokines.Recently,we showed that phagocytosis of hemozoin by human monocytes increases expression and activity of matrix metalloproteinase-9,a proteolytic enzyme available in specific gelatinase granules,which contain several enzymes including lysozyme.Present work investigated active lysozyme release after phagocytosis of hemozoin and its dependence on production of tumor necrosis factor alpha. Methods:After phagocytosis of hemozoin,hemozoin-containing trophozoites or control meals(opsonized nonparasitized red blood cells and latex particles),monocyte supematants were monitored for 2 hours,in presence of blocking anti-human tumor necrosis factor alpha antibodies or recombinant human tumor necrosis factor alpha cytokine in selected experiments.Lysozyme release was evaluated by a specific spectrometric assay measuring lysozyme activity after coincubation of cell supematants with suspensions of Mycrococcus Lysodeikticus,while levels of soluble tumor necrosis factor alpha were analyzed by specific enzyme-linked immunodsorbent assay. Results:Levels of lysozyme activity and soluble tumor necrosis factor alpha protein were increased in hemozoin in-or trophozoites-laden monocytes supematants.Phagocytosis per se(control meals) also increased lysozyme release,but levels were significantly lower than those obtained after phagocytosis of hemozoin or trophozoites. Interestingly,all effects on lysozyme release observed after phagocytosis were abrogated by blocking anti-human tumor necrosis factor alpha antibodies,while they were mimicked by recombinant human tumor necrosis factor alpha cytokine.Conclusions:Present work shows that phagocytosis of hemozoin promotes monocyte degranulation and enhances active lysozyme release.The effect requires tumor necrosis factor alpha mediation.

Corrosion Inhibition by Co-Immobilized Lysozyme and Lipase in Circulating Cooling Water System

The corrosion inhibition performance of co-immobilized lysozyme and lipase was investigated in a recirculating cooling water system. Four methods were carried out in co-immobilization, and the operating parameters were optimized by using the respond surface methodology(RSM). The corrosion inhibition performance of co-immobilized lipase and lysozyme was evaluated by weight loss measurements and electrochemical measurements. The results revealed that the optimal co-immobilization method should be the sequential immobilization of lysozyme and then lipase. The inhibition efficiency was 86.10% under the optimal co-immobilized conditions. Electrochemical data showed that co-immobilized lysozyme and lipase was a mixed-type inhibitor and the corrosion inhibition efficiency was 81%.

Exploring the Performance of Magnetic Immobilized Lysozyme on Sludge Hydrolysis and Mechanism of Improving Dewaterability of Excess Sludge

The performance of the immobilized lysozyme and the native lysozyme on enhancing the excess sludge dewaterability was investigated.The results indicated that the specific resistance to filtration(SRF)decreased by 62.8%for native lysozyme and 53.6%for immobilized lysozyme at the enzyme dosage of 9 mg/g(dry sludge).Correlation analysis was carried out to explore the role of different extracellular polymeric substance(EPS)fractions on excess sludge dewaterability.The results illustrated that the SRF negatively correlated with protein,polysaccharide from soluble EPS(S-EPS)and loosely bound EPS(LB-EPS)and positively correlated with that from tightly bound EPS(TB-EPS).Three-dimensional excitation emission matrix(3D-EEM)fluorescence analysis combined with the scanning electron microscope(SEM)images,revealed that sludge floc structure and microbial cells were destroyed by enzymatic treatment,and that the enzymatic hydrolysis could help to improve the transformation of hydrophilic groups from TB-EPS and the performance of the excess sludge dewatering process.The assessment of hydrolysis using the immobilized enzyme provided a new insight for the safe disposal of the sludge.

Molecular characterization and functional analysis of two new lysozyme genes from soybean cyst nematode(Heterodera glycines)

Soybean cyst nematode(SCN, Heterodera glycines(I.)) is one of the most important soil-borne pathogens for soybeans. In plant parasitic nematodes, including SCN, lysozyme plays important roles in the innate defense system. In this study, two new lysozyme genes(Hg-lys1 and Hg-lys2) from SCN were cloned and characterized. The in situ hybridization analyses indicated that the transcripts of both Hg-lys1 and Hg-lys2 accumulated in the intestine of SCN. The q RT-PCR analyses showed that both Hg-lys1 and Hg-lys2 were upregulated after SCN second stage juveniles(J2 s) were exposed to the Grampositive bacteria Bacillus thuringiensis, Bacillus subtilis or Staphylococcus aureus. Knockdown of the identified lysozyme genes by in vitro RNA interference caused a significant decrease in the survival rate of SCN. All of the obtained results indicate that lysozyme is very important in the defense system and survival of SCN.

Combined Use of Co-immobilized Lysozyme and Lipase and Chemical Inhibitors in Circulating Cooling Water System

In order to improve the stability and corrosion inhibition performance of bioenzyme, lipase and lsozyme were co-immobilized on the mesoporous molecular sieve MCM-41 by the adsorption method. Then the immobilized enzymes were combined with amino trimethylene phosphonic acid and polyaspartic acid to prevent corrosion caused by circulating cooling water. The weight-loss method and electrochemical techniques were used to evaluate the performance of composite inhibitors. The co-immobilized lysozyme and lipase achieved good inhibition effects. After they were combined with amino trimethylene phosphonic acid and polyaspartic acid, the corrosion inhibition properties were further improved. The inhibition efficiency was promoted to 94.4%. During the corrosion inhibition process, the immobilized enzymes played an important role. The addition of corrosion inhibitor could inhibit the anodic dissolution and cathodic hydrogen evolution process of carbon steel at the same time. The adsorption of co-immobilized lysozyme and lipase composite inhibitor on the steel surface was a joint action involving physical adsorption and chemical adsorption.

The Effect of pH Difference Between Two Phases on the Partition of Lysozyme in Aqueous Two-Phase System

In the investigation of effect of KSCN on the partitioning of lysozyme in PEG2000/arnmonium sulfate aqueous two-phase system, it was found that the KSCN could alter the PH difference between the two phases, and thus affect the partition of lysozyme. The relationship between partition coefficients of lysozyme and pH differences between two phases was discussed.

Therapeutical effect of marine lysozyme suppository on bacterial vaginitis caused by S. aureus and E. coli

Aim To research therapeutical effect of marine lysozyme suppository on bacterial vaginitis caused by S. aureus and E. coll. Methods Lysozyme obtained from concha ostreae which were used to preparate marine lyso- zyme suppository. The identification and test of suppository was in line with the standards stated in Chinese pharma- coperia（2010 edition）. After determined its quality control, we studied its therapeutical effect on bacterial vaginitis which caused by S. aureus and E. coli, by vaginitis model in rats infection of S. aureus bacteria and E. coli. Results The preparation technology of marine lysozyme suppository was simple, convenient and clinically effective. The marine lysozyme suppository was delivered by dose 0.5, 0.25, 0. 125 g ~ kg-1, the cure rates of S. aureus infec- tion were 80% , 50% and 30% , respectively, the cure rates of E. coli infection were 90% , 60% and 30% re- spectively, the cure rates of mixed infection were 92.9% , 82. 1% and 92.9% respectively. Marine lysozyme sup- pository had an outstanding therapeutical effect on bacterial vaginitis which caused by S. aureus and E. coll. Con- clusion The preparative process of marine lysozyme suppository was practicable, and it had a good therapeutic effect on bacterial vaginitis caused by S. aureus and E. coll.

STRUCTURAL STUDIES ON PROTEINS IN MICROEMULSION I．FTIR STUDY ON LYSOZYME IN D_2O／SURFACTANT／n－HEPTANE SYSTEM

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Time-resolved ESI-MS Investigation on Reduction and Alkylation in Unfolding Process of Lysozyme

The kinetic procedure of the unfolding of lysozyme induced by the reduction of disulfide was monitored by the time-resolved ESI-MS with a sheath liquid assistant electrospray interface. It was found that the reduction process for the eight disulfides had a less difference in the reaction time after denatured treatment. In addition, the alkylation of the reduced free thiols was much slower than the reduction procedure. An artifact peak produced by the CID fragmentation in the mass spectra was identified and the possible mechanism of the Hofmann elimination reaction was proposed.

Layer-by-layer nanostructured protein loaded nanoparticles: A feasibility study using lysozyme as model protein and chitosan as coating material

Stabilization of proteins in delivery devices and design of appropriate protein carriers are major research issues due to the extreme sensitivity of proteins.Previously,negatively charged nanoparticles,consisting of poly(lactic-co-glycolic acid)(PLGA)and poly(styrene-co-4–styrene-sulfonate)(PSS),showed considerably high loading capacity for positively charged model protein lysozyme depending on the surface charge density of nanoparticles.

Choice of Force Fields and Water Models for Sampling Solution Conformations of Bacteriophage T4 Lysozyme

A protein may exist as an ensem-ble of di erent conformations in solution,which cannot be repre-sented by a single static structure.Molecular dy-namics(MD)simulation has become a useful tool for sampling protein conformations in solution,but force elds and water models are important issues.This work presents a case study of the bacteriophage T4 lysozyme(T4L).We have found that MD simulations using a classic AMBER99SB force eld and TIP4P water model cannot well describe hinge-bending domain motion of the wild-type T4L at the timescale of one microsecond.Other combinations,such as a residue-speci c force eld called RSFF2+and a dispersion-corrected water model TIP4P-D,are able to sample reasonable solution conformations of T4L,which are in good agreement with experimental data.This primary study may provide candidates of force elds and water models for further investigating conformational transition of T4L.

Concentration-Dependent Inuence of Silver Nanoparticles on Amyloid Fibrillation Kinetics of Hen Egg-White Lysozyme

Understanding the inuence of nanoparticles on the formation of protein amyloid brillation is cru-cial to extend their application in related biological diagnosis and nanomedicines.In this work,Ra-man spectroscopy was used to probe the amyloid brillation of hen egg-white lysozyme in the pres-ence of silver nanoparticles(Ag-NPs)at di erent concentrations,combined with atomic force mi-croscopy and thioavin T(ThT)uorescence assays.Four representative Raman indicators were utilized to monitor transformation of the protein tertiary and secondary structures at the molecular level:the Trp doublet bands at 1340 and 1360 cm^(-1),the disul de stretching vibrational peak at 507 cm^(-1),the N-Cα-C stretching vibration at 933 cm^(-1),and the amide I band.All experimental results con rmed the concentration-dependent inuence of AgNPs on the hen egg-white lysozyme amyloid brillation kinetics.In the presence of AgNPs at low concentration(17μg/mL),electrostatic interaction of the nanoparticles stabilizes disul de bonds,and protects the Trp residues from exposure to hydrophilic environment,thus leading to formation of amorphous aggregates rather than brils.However,with the action of AgNPs at high concentration(1700μg/mL),the native disul de bonds of hen egg-white lysozyme are broken to form Ag-S bonds owing to the competition of electrostatic interaction from a great deal of nanoparticles.As for providing functional surfaces for protein to interact with,AgNPs play a bridge role in direct transformation from-helices to organized-sheets.The present investigation sheds light on the controversial e ects of AgNPs on the kinetics of hen egg-white lysozyme amyloid brillation.

Maternal lysozyme concentrations in the eggs of the Great Cormorant (Phalacrocorax carbo) in relation to breeding density and laying order

Background: Females can differentially deposit the immune factor lysozyme into eggs based on conditions of local breeding density and laying order.Materials: We collected 80 eggs from Great Cormorants(Phalacrocorax carbo) and then analyzed whether the level of lysozymes in the eggs is related to breeding density and laying order.Results: Between clutches,the level of lysozyme in eggs is positively related to breeding density; while within a clutch,the level of lysozyme is positively related to the laying order.Conclusion: When parents breed under conditions of high density,they allocate more lysozymes to their offspring,a trait adaptive to the local environment.That the increase in the level of lysozymes is a function of the laying order seems a necessary condition to mitigate the hierarchy among siblings for improving the survival of the entire clutch.

THE LONGITUDINAL OBSERVATION OF THE EFFECT OF ANTIBIOTICS ON HUMAN TEAR LYSOZYME ACTIVITY

It is well known that lysozyme, which catalyzes the hydrolysis of bacterial cell walls, is one of the important substances protecting the eye surface from bacterial infections. It was reported that the aminoglycoside antibiotics strongly inhibited the tear lysozyme activity. The lysozyme activity of tears which were added to different con- centrations of two aminoglycoside antibiotics and three other antibiotics was determined longitudinally. No influence of any antibiotics on tear lysozyme activity was found. Therefore it is unnecessary to worry about that the use of aminoglycosida antibic tics may weaken the defence function of the eye.

Forward-Light-Scattering Characterization of Pre-Crystalline Aggregates in Crystallizing Lysozyme Solutions

We present a method to characterize lysozyme pre-crystalline aggregates using a forward-light-scattering technique, which is highly sensitive to protein aggregates. The static light scattering properties at small angles of crystallizing lysozyme aggregates are discussed, and related to the crystallization conditions based on the concentration of added precipitant NaCl. Lysozyme solutions that started to crystallize because of precipitant exhibited profiles of forward light scattering that could be fitted by non-integer power laws, which indicated fractal aggregations of lysozyme had formed. Pre-crystalline solutions, in which lysozyme crystals later grew, had dense structural fractal clusters with fractal dimensions of D > 1.5. In contrast, solutions with aggregates in which crystals did not grow, had forward-light-scattering profiles that deviated from a power law or had lower power values.

Immobilization of Lysozyme on Biomass Charcoal Powder Derived from Plant Biomass Wastes

Biomass charcoal powder (BCP) was used as a carrier matrix for immobilization of chicken egg white lysozyme. BCP was derived from plant biomass wastes such as dumped adzuki beans by pyrolysis without combustion under nitrogen atmosphere and grinding with a jet mill. The amount of lysozyme immobilized on BCP of adzuki beans by adsorption was 11 μmol/g (0.16 g/g) at pH 7.0. The optimum pH values for free and immobilized lysozyme activities were 6.8 and 7.2, respectively. The optimum temperature for both free and immobilized lysozyme activities was 25℃. The half-life of immobilized lysozyme exhibited 1.8-fold compared to that of free lysozyme at 5℃. Moreover, the half life of immobilized lysozyme was 7 times greater than that of lysozyme at 90℃.