Lysozyme Substrate Research Articles

Learning to Read and Write in the Language of Proteins

Learning to Read and Write in the Language of Proteins

An improved lysozyme substrate preparation method for the biochemistry kinetics laboratory

The mutation and growth of Bacteriophage T4 Lysozyme (T4L) is easily accomplished in an undergraduate biochemistry laboratory and allows students to visualize how mutations affect enzyme stability and kinetics. However, full Michaelis-Menten parameters have been difficult to observe due to lack of a readily available, small molecule substrate. A FITC-tagged peptidoglycan has been used previously, but it is not commercially available and its preparation method was not appropriate for the undergraduate laboratory. Here, we describe a cost-effective, non-toxic, and reproducible means of generating FITC-tagged peptidoglycan fragments. Fluorescence assays monitoring substrate digestion using HEWL and T4L produced signals sufficient for reliable analysis of lysozyme kinetics.

Electrical monitoring of infection biomarkers in chronic wounds using nanochannels

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients’ life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20–100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids.Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.

Network of Entamoeba histolytica HSP18.5 dimers formed by two overlapping [IV]-X-[IV] motifs.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with low molecular weight that prevent the aggregation of proteins during stress conditions and maintain protein homeostasis in the cell. sHSPs exist in dynamic equilibrium as a mixture of oligomers of various sizes with a constant exchange of subunits between them. Many sHSPs form cage-like assemblies that may dissociate into smaller oligomers during stress conditions. We carried out the functional and structural characterization of a small heat shock protein, HSP18.5, from Entamoeba histolytica (EhHSP18.5). It showed a pH-dependent change in its oligomeric state, which varied from a tetramer to larger than 48-mer. EhHSP18.5 protected Nde I and lysozyme substrates from temperature and chemical stresses, respectively. The crystal structure of EhHSP18.5 was determined at a resolution of 3.28 Å in C2221 cell with four subunits in the asymmetric unit forming two non-metazoan sHSP-type dimers. Unlike the reported cage-like structures, EhHSP18.5 formed a network of linear chains of molecules in the crystal. Instead of a single [IV]-X-[IV] motif, EhHSP18.5 has two overlapping I/V-X-I/V sequences at the C-terminus giving rise to novel interactions between the dimers. Negative staining Electron Microscopy images of EhHSP18.5 showed the presence of multiple oligomers: closed structures of various sizes and long tube-like structures.

Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature.

Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins’ sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25–35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

Lipopolysaccharide increases lysozyme adhesion to chitin

Background. To study the effect of lipopolysaccharide on the adsorption of lysozyme on chitin.Methods. Egg white lysozyme, lipopolysaccharide and chitin were used. The lyophilized cells of M. lyzodeikticus were used as a lysozyme substrate. Lysozyme activity was determined by the bacteriolytic method using different concentrations (8-512 μg/ml). Lysozyme adsorption on chitin was determined by the loss of lysozyme from solution after shaking with chitin. The effect of lipopolysaccharide (0.6-40 μg/ml) on lysozyme adsorption was evaluated by the loss of lysozyme from the solution after shaking with 50 mg of chitin.Results. In determining the activity of lysozyme by the bacteriolytic method, the linear nature of the dependence of activity on the concentration of the enzyme is observed only at low concentrations (8-100 μg / ml). Lipopolysaccharide does not affect the bacteriolytic activity of lysozyme, however, dose-dependently increases the adsorption of lysozyme on chitin.Conclusion. Lipopolysaccharide increases the adsorption of lysozyme on chitin.

Effects of heavy metals and albumin on lysozyme activity

Lysozyme is an antibacterial enzyme that is found in most of body fluids. Lysozyme in tears plays a primary role in protecting eye from harmful environments; if lysozyme is degraded or inhibited, eyes are likely to be more vulnerable to bacterial infection. In this study, lysozyme activity was evaluated according to varying concentrations of heavy metals, copper, zinc, cobalt and manganese and light metal, calcium that are frequently found in airborne particulate matters and was assayed using a dyequenching lysozyme substrate, Micrococcus lysodeikticus. Less fluorescence intensity was observed with increasing amounts of copper, zinc, manganese and cobalt but not with calcium suggesting that these metals have some affinity with lysozyme and inhibit lysozyme activity. When albumin, the second most common protein in tears, was added on the reaction of lysozyme and metals, lysozyme activity was partially restored. This finding suggests that the albumin might protect damage caused by metals on lysozyme. To identify whether the decrease in enzymatic activity was related to structural changes of lysozyme, SDS-PAGE was conducted and only with copper did lysozyme show marked smearing bands on the SDS-gel, meaning that copper degraded lysozyme consistent with the sharpest activity decrease.

Surface Plasmon Resonance based sensing of lysozyme in serum on Micrococcus lysodeikticus-modified graphene oxide surfaces

Lysozyme is an enzyme found in biological fluids, which is upregulated in leukemia, renal diseases as well as in a number of inflammatory gastrointestinal diseases. We present here the development of a novel lysozyme sensing concept based on the use of Micrococcus lysodeikticus whole cells adsorbed on graphene oxide (GO)-coated Surface Plasmon Resonance (SPR) interfaces. M. lysodeikticus is a typical enzymatic substrate for lysozyme. Unlike previously reported sensors which are based on the detection of lysozyme through bioaffinity interactions, the bioactivity of lysozyme will be used here for sensing purposes. Upon exposure to lysozyme containing serum, the integrity of the bacterial cell wall is affected and the cells detach from the GO based interfaces, causing a characteristic decrease in the SPR signal. This allows sensing the presence of clinically relevant concentrations of lysozyme in undiluted serum samples.

Lysozyme as sensitive reporter for fluorometric and PCR based detection of E. coli and S. aureus using magnetic microbeads

We describe the use of lysozyme as a sensitive reporter for detection of bacteria. The assay comprises the following steps: First, specific primers are used for targeting the rDNA of E. coli and S. aureus, respectively, and the polymerase chain reaction is applied to amplify bacterial DNA. Then, the target DNA binds to the capture probe immobilized on magnetic beads, with lysozyme acting as signal reporter that catalyzes the hydrolysis of an enzyme substrate to form the blue fluorescent product 4-methylumbelliferone. Coupled with multiple lysozyme substrates, this fluorometric detecting method has a detection limit of 50 CFUa <...mL(-1) bacteria. The optical changes may also be detected by colorimetry and monitored with bare eyes. The method was selective over other bacteria and was successfully applied to the determination of E. coli and S. aureus in human serum, which provides a perspective for lysozyme application and species-specific pathogenic bacteria detection.

Comparison of bacteriolytic activity of human interleukin-2 and chicken egg lysozyme on Lactobacillus plantarum and Escherichia coli cells

The article continues studies of the recently discovered bacteriolytic activity of interleukin-2. It was detected earlier that interleukin (IL-2) possesses greater substrate specificity in comparison with chicken egg lysozyme. IL-2 disrupted the cell wall of Escherichia coli but did not lyse lysozyme substrates such as the cell walls of Micrococcus luteus and Bacillus subtilis. In the present study it is demonstrated for the first time that both IL-2 and chicken egg lysozyme are capable of lysing Lactobacillus plantarum. The effects of IL-2 and chicken egg lysozyme on Lactobacillus plantarum are compared with those on Escherichia coli. The dependences of the rate of lysis on the concentration of bacteriolytic factors and pH are studied.

Properties of lysozyme/low methoxyl (LM) pectin complexes for antimicrobial edible food packaging

The objective of this study was to determine the properties of lysozyme/low methoxyl (LM) pectin complexes in order to develop an edible antimicrobial film. At a constant lysozyme concentration (0.714g/L), the gradual LM pectin addition (0–2g/L) induced an increase of turbidity up to a threshold LM pectin concentration above which turbidity decreased. These results were interpreted in terms of lysozyme/LM pectin complexes formation followed by a change in the apparent particle size distribution and the electrophoretic mobility (ζ-potential). This complex formation was shown to considerably decrease the lysozyme antimicrobial activity probably due to lysozyme substrate diffusional limitation and/or a decrease of the enzyme mobility. The formed complexes were then used to manufacture an edible antimicrobial film able to control the lysozyme release. The presence of pectinases in the release medium enhanced the lysozyme release confirming that the developed edible antimicrobial film can be used to protect foods against lysozyme sensitive microorganisms and particularly those producing pectinolytic enzymes.

Characterization of two resuscitation promoting factors of Listeria monocytogenes

In actinobacteria, resuscitation promoting factor (Rpf) proteins have been described as having the ability to increase the viable count of dormant cultures and stimulate growth of vegetative cells through lag phase reduction. Recently, it was suggested that proteins Lmo0186 and Lmo2522 of Listeria monocytogenes are equivalent to Rpf proteins based on their genomic context and conserved domain architecture. It was proposed that they have evolved through non-orthologous displacement of the Rpf domain found in actinobacteria. Here we present biological and biochemical data supporting a function of Lmo0186 and Lmo2522 as Rpfs. These proteins are collectively dispensable for growth but a lmo0186 lmo2522 double mutant exhibits an extended lag phase when diluted in minimal medium. This phenotype could be partially complemented by medium supplementation with fM to nM concentrations of purified hexahistidine-tagged versions of Lmo0186 and Lmo2522, showing that these proteins can stimulate growth. Gel filtration analysis and cross-linking experiments suggest that the recombinant proteins in solution are elongated monomers. Both proteins display muralytic activity against crude cell wall preparations and are active against an artificial lysozyme substrate. Our study thus supports the hypothesis that Lmo0186 and Lmo2522 are functional equivalents of actinobacteria Rpf proteins and represents the first characterization of two Rpf homologues from firmicutes.

Lysozyme Immobilized on Micro-Sized Magnetic Particles: Kinetic Parameters at Wine pH

In order to use lysozyme as an anti-microbial agent during the winemaking process, hen egg-white lysozyme (LYZ) was covalently immobilized on two different micro-size magnetic particles (tosyl-activated and carboxylated, TSA and CA, respectively). A cell suspension of Oenococcus oeni, an oenological strain involved in the winemaking process, was utilized as LYZ substrate. Both a kinetic study and a study of the stability of free and immobilized LYZ were performed in McIlvane buffer at pH3.2, that is the average minimum pH value in wine. The activity and kinetic parameters measured for the free LYZ at pH3.2 are lower than those reported at the optimum pH (4.5); however the residual activity at pH3.2 is sufficient to be of interest for further immobilization and applications in winemaking. All kinetic parameters of both biocatalysts (LYZ-CA and LYZ-TSA) are altered after immobilization, probably due to the structural modifications in the active site caused by covalent attachment to the supports. The half-life calculated at 25°C was 39h for free LYZ, while it increased to 280 and 134h for LYZ-TSA and LYZ-CA, respectively. This result indicates that immobilization improves the enzyme stability and that LYZ can be utilized in wine applications in its immobilized forms. In addition, LYZ-TSA seems to be the best biocatalyst for further applications in winemaking.

Newly folded substrates inside the molecular cage of the HtrA chaperone DegQ

The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA proteins have been analyzed in detail, their chaperone activity remains poorly characterized. Here we describe cryo-electron microscopic structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates, providing a structural model of HtrA proteins in their chaperone mode. Up to six lysozyme substrates bind inside the DegQ 12-mer cage and are visualized in a close-to-native state. An asymmetric reconstruction reveals the binding of a well-ordered lysozyme to four DegQ protomers. DegQ PDZ domains are located adjacent to substrate density and their presence is required for chaperone activity. The substrate-interacting regions appear conserved in 12- and 24-mer cages, suggesting a common mechanism of chaperone function.

72 A plasma etching and gravimetric monitoring system (PEGMS) for lipid and protein location studies

Purpose: In the tear film lysozyme makes up approximately 35% of the total protein detected and is widely implicated in contact lens spoilation. It functions primarily as a bacteriolytic enzyme in the innate defense of the ocular surfaces. Recently interest in this protein has focused on its ability to function effectively in the presence of contact lens and care solution products. Method: The classic methods based on its interaction with, and the lysis of, the Grampositive bacterial strainMicrococcus lysodeicticus,measured by turbidity at 450nm, were used to investigate lysozyme antimicrobial activity. In addition a fluorescence-based assay which measures the digestion products from a DQTM lysozyme substrate, with excitation maxima at 494nm and fluorescence emission maxima at 518nm, was assessed. Results: Disadvantages of turbidity based assayswere apparent. Additionally many studies use egg white lysozymes as a cheaper assay alternative to human sourced protein, but in addition to compositional variation, human and chicken lysozymes differ in enzymatic activity with a 4:1 ratio respectively. Batch to batch variation in lysozyme preparations was also observed. The kinetic fluorescencebased assaywhich was able to detect activity down to 20U/ml was established and was used to assess the effects that extraction techniques and sample preparation have on the outcome of the activity data. Conclusions: An investigation into the true nature and extent of lysozyme denaturation, which is essential to determine the importance of this phenomenon and its impact on tear film anti-microbial function, was facilitated by more sensitive and varied analytical techniques.

Enzymes of SPZ7 phage: Isolation and properties

Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysinfrom phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.

Protein–protein recognition and interaction hot spots in an antigen–antibody complex: Free energy decomposition identifies “efficient amino acids”

The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method was applied to the study of the protein-protein complex between a camelid single chain variable domain (cAb-Lys3) and hen egg white lysozyme (HEL), and between cAb-Lys3 and turkey egg white lysozyme (TEL). The electrostatic energy was estimated by solving the linear Poisson-Boltzmann equation. A free energy decomposition scheme was developed to determine binding energy hot spots of each complex. The calculations identified amino acids of the antibody that make important contributions to the interaction with lysozyme. They further showed the influence of small structural variations on the energetics of binding and they showed that the antibody amino acids that make up the hot spots are organized in such a way as to mimic the lysozyme substrate. Through further analysis of the results, we define the concept of "efficient amino acids," which can provide an assessment of the binding potential of a particular hot spot interaction. This information, in turn, can be useful in the rational design of small molecules that mimic the antibody. The implications of using free energy decomposition to identify regions of a protein-protein complex that could be targeted by small molecules inhibitors are discussed.

Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.

Effect of N-acyl substitution at glucosamine residues on lysozyme-catalyzed hydrolysis of cell-wall peptidoglycan and its oligosaccharides.

The effect of N-substitution with various acyl groups at glucosamine residues on lysozyme-catalyzed hydrolysis of cell wall peptidoglycan and its oligosaccharides was studied. The lysozyme-resistant cell wall peptidoglycan from Bacillus cereus, N-unsubstituted at most of its glucosamine residues, was N-acylated and used as substrate for lysozyme. The N-acetylated, N-formylated, N-propionylated and N-butyrylated preparations of the peptidoglycan were hydrolyzed by lysozyme with relative maximum rates of 1.00, 0.05, 0.38 and 0.10 respectively, and with apparent Km values, expressed in disaccharide unit concentration, of 0.1, 0.6, 2.4 and 10 mM respectively, whereas the N-succinylated preparation was unsusceptible to this enzyme. Peptidoglycan oligosaccharides, GlcN-MurAc-GlcNAc-MurAc, GlcNAc-MurAc-GlcN-MurAc-GlcNAc-MurAc and GlcN-MurAc-GlcN-MurAc-GlcNAc-MurAc, were also N-acylated to be examined as substrates for lysozyme. The N-acetylated, N-formylated, N-propionylated and N-butyrylated preparations from the tetrasaccharide were hydrolyzed at 0.5 mM with relative rates of 1.00, 0.04, 0.1 and 0.01 respectively, but the N-succinylated tetrasaccharide was unaffected by lysozyme. The N-formylated, N-propionylated, N-butyrylated and N-succinylated preparations from the former hexasaccharide were hydrolyzed with relative rates of 1.5, 22, 1.6 and 0.8 respectively, as compared to the value of about 2000 for the N-acetylated hexasaccharide. The N-acylated preparations from the latter hexasaccharide, except the N-succinylated one, were hydrolyzed at rates similar to those of hydrolysis of the corresponding preparations from the former hexasaccharide. The above results seem to be accounted for by the predominant importance of the alkyl moieties of the N-acyl substituents on the glucosamine residues located in subsite C in the enzyme-substrate complexes.

A glycosyl hydrolase activity of mammalian reovirus σ1 protein can contribute to viral infection through a mucus layer

The mammalian reovirus σ1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between σ1 and chicken-type lysozymes prompted us to investigate additional functions of the σ1 protein. Expression in Pichia pastoris yeast cells showed that σ1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in σ1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in σ1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified σ1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of σ1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.