Commercial Lysozyme Research Articles

Dietary supplementation of lysozyme can improve growth rate, laying performance, blood biochemistry, and mRNA levels of some related genes in different plumage-colored quails

The impact of dietary lysozyme (LZ) supplementation on the growth and laying performance was investigated over 4 weeks of growing and 6 weeks of laying periods in two different plumage color (white and brown-feathered) Japanese quail varieties. For each variety, 240 birds were randomly assigned into four groups with four replicates for each group. The first group (control) was fed a basal non-supplemented diet (BD). Whereas the 2nd, 3rd, and 4th received the BD supplemented with commercial LZ (CLZ) at 100 mg/kg diet, and natural LZ (NLZ) at 100 and 200 mg/kg diet, respectively. The main findings included significant increases in body weights and gains in the white-feathered quails supplemented with NLZ1 compared to the control and NLZ2. However, there were no significant differences in the case of brown-feathered quails in all LZ supplementations. Moreover, the different dietary LZ lowered FI in both quails with the lowest intake observed in the brown-feathered quails. Accordingly, enhanced FCR was reported in the CLZ groups for both quail varieties and in NLZ1 and NLZ2 for the white-feathered and brown-feathered quails, respectively. In both quail varieties, the NLZ2 significantly lowered serum creatinine and urea and increased albumen and globulin levels compared with other groups. Histologically, the best hepatic histological features were found in both quail’ varieties fed the NLZ1-supplemented diet. Accompanying LZ-induced modulations in the expression levels of GHR, IGF-1, leptin, CCK, FAS, and ACC genes in both quail varieties were reported. Besides, both quail varieties in NLZ1& NLZ2 supplementation exhibited significant increases in hen day egg production, egg weight, egg mass, and hatchability percentages along with differences in external and internal egg qualities compared with LZ-free diet or CLZ. Therefore, NLZ could be used as an effective feed supplement to enhance the growth and egg performance of Japanese quail with caution being drawn to the supplementation dose about quail variety.

Lysozyme impacts gel properties of egg white protein via electrostatic interactions, polarity differences, local pH regulation, or as a filler

The effects of lysozyme on egg white gel properties and their underlying causes were investigated under comparison between lysozyme removed with ion exchange resin and three levels of commercial lysozyme powder (1/2, 2/2, 3/2 the natural concentration in egg white) re-added in the lysozyme-removed system. Results showed that a lysozyme-removed gel obtained the best water holding capacity (61.61 %), lowest cooking loss (11.85 %), and enhanced textural properties (hardness, 638.04 g; resilience, 0.57; and gumminess), which was attributed to lysozyme promoting protein aggregation and weakening electrostatic repulsion by charge neutralization and competition for water, and this could be eliminated by removing lysozyme. Besides, the stronger intermolecular interactions (enhanced ionic bonds, hydrogen bonds and inhibited hydrophobic interactions), the shorter transverse relaxation time (T21 and T22), as well as more uniform microstructure formed in the lysozyme-removed gel, allowing the gels to bind more water molecules. With return of lysozyme, the gel properties were weakened to varying degrees, which was also ascribed to the filling of lysozyme in gel matrix narrowed interspace for binding and storage of water. In sum, adjustment on the content of lysozyme can regulate the gel properties of egg white, so as to obtain gels with regulable gel quality and processing characteristics.

Design and characterization of a novel lytic protein against Clostridium difficile.

Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall–binding domains, proven to be involved in bacterial cell wall remodeling and cell division. Although autolysins in C. difficile have been reported, the autolysins have failed to yield impressive results when used as exogenous lytic agents. In this study, we expressed and characterized the binding domains (Cwp19-BD and Acd-BD) and catalytic domains (Cwp19-CD, Acd-CD, and Cwl-CD) of C. difficile autolysins, and the domains with the best binding specificity and lytic activity were selected towards C. difficile to design a novel lytic protein Cwl-CWB2. Cwl-CWB2 showed good biosafety with significantly low hemolysis and without cytotoxicity. The results of fluorescence analysis and lytic assay demonstrated that Cwl-CWB2 has higher binding specificity and stronger lytic activity with a minimum inhibitory concentration at 13.39 ± 5.80 μg/mL against living C. difficile cells, which is significantly stronger than commercial lysozyme (3333.33 ± 1443.37 μg/mL) and other reported C. difficile autolysins. Besides, Cwl-CWB2 exhibited good stability as about 75% of the lytic activity was still retained when incubated at 37 °C for 96 h, which is considered to be a potential antimicrobial agent to combat C. difficile.Key points • Several binding domains and catalytic domains, deriving from several Clostridium difficile autolysins, were expressed, purified, and functionally characterized. • A novel C. difficile lytic protein Cwl-CWB2 was designed from C. difficile autolysins. • The binding specificity and lytic activity of Cwl-CWB2 against C. difficile showed advantages compared with other reported C. difficile autolysins. • Cwl-CWB2 exhibited significantly low hemolysis and cytotoxicity against normal-derived colon mucosa 460 cell. Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-12010-0.

Covalent organic frameworks as crystalline sponges for enzyme extraction and production from natural biosystems

Biomacromolecules, as represented by enzymes have played critical parts in various fields, especially in biomanufacture and biomedicinal applications. However, their purification from complex biological systems is of great significance but very challenging. Herein, we develop an approach using high-performance covalent organic frameworks (COFs) as crystalline sponges to extract and produce enzymes directly from the complex natural biosystem. For instance, lysozyme with high purity and activity is successfully extracted from chicken egg white through a simple adsorption and elution process. Attributed to the well-tuned pore microenvironment of COFs, both high uptake and selectivity can be realized by this high-performance crystalline sponges. Notably, the encapsulated lysozyme can be smartly and conveniently released by pH adjustment of the eluent. The harvested lysozyme demonstrates ultrahigh purity and improved activity, surpassing the commercial lysozyme (activity boosted up to 344%). The mild operation condition and good protection effect of COFs contribute to the high activity of the obtained enzyme. Moreover, compared with conventional separation media, COFs sponges demonstrate superior selectivity, efficiency, and reusability. One gram of COFs can produce ∼ 0.69 g of enzyme product per batch, and the COFs can be reused for at least 5 times. This study opens up a new avenue to extract enzymes and provides valuable guidance for the design of crystalline sponges for highly efficient bioseperation.

Mapping out the Degree of Freedom of Hosted Enzymes in Confined Spatial Environments

Summary The integration of enzymes with solid materials is crucial for promoting their industrialization. Understanding the enzyme behavior upon association within a confined space, though of fundamental importance for biocomposite development, remains a persistent, unresolved challenge. Here, we present a comprehensive elucidation of the spatial environment’s impact on hosted enzymes’ degree of freedom and, consequently, their accompanying reactivity. Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy allows the direct detection of host-guest interactions at atomic resolution, while the tailorable synthesis of covalent organic frameworks (COFs) enables an evaluation of factors affecting such interactions. Specifically, lysozyme is found to be more constrained and less active along with increasing hydrophilicity of the COFs. These results support the establishment of a connection between the hydrophilicity of the spatial environment and the resulting biocomposites’ reactivity, enabling the prediction of the performance of unknown biocomposites. This study provides a unique insight into the mechanistic pathways underpinning biocatalysis.

Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn2+. When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5–9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.

Different Analytical Approaches in Assessing Antibacterial Activity and the Purity of Commercial Lysozyme Preparations for Dairy Application

Hen egg-white lysozyme (LSZ) is currently used in the food industry to limit the proliferation of lactic acid bacteria spoilage in the production of wine and beer, and to inhibit butyric acid fermentation in hard and extra hard cheeses (late blowing) caused by the outgrowth of clostridial spores. The aim of this work was to evaluate how the enzyme activity in commercial preparations correlates to the enzyme concentration and can be affected by the presence of process-related impurities. Different analytical approaches, including turbidimetric assay, SDS-PAGE and HPLC were used to analyse 17 commercial preparations of LSZ marketed in different countries. The HPLC method adopted by ISO allowed the true LSZ concentration to be determined with accuracy. The turbidimetric assay was the most suitable method to evaluate LSZ activity, whereas SDS-PAGE allowed the presence of other egg proteins, which are potential allergens, to be detected. The analytical results showed that the purity of commercially available enzyme preparations can vary significantly, and evidenced the effectiveness of combining different analytical approaches in this type of control.

Effects of a Forced Solution Flow on the Step Advancement on {110} Faces of Tetragonal Lysozyme Crystals: Direct Visualization of Individual Steps under a Forced Solution Flow

To grow protein crystals of better quality, there still exists an open question whether a solution flow should be suppressed or intentionally introduced. To obtain a comprehensive understanding of the effects of a solution flow, we directly measured the velocities of individual elementary steps (on {110} faces of tetragonal lysozyme crystals) under a forced solution flow, for the first time, by laser confocal microscopy combined with differential interference contrast microscopy. When we used crystals grown by a two-dimensional (2D) nucleation growth mechanism in a solution of commercial lysozyme (98.5% purity, from Seikagaku Co.), while increasing the solution flow rate, the step velocity decreased monotonically. We confirmed that this decrease in the step velocity with flow rate was due to the enhancement of the mass transfer of impurity (mainly covalently bonded dimer), by the observation using a lysozyme further purified (dimer was removed). In contrast, when we used crystals grown by a spiral growth mechanism in a commercial lysozyme solution, with increasing the flow rate, the step velocity increased and had the maximum at the flow rate of 10 μm/s, and then decreased monotonically. Also, the step velocity was 2–4 times higher than in the case of the 2D nucleation growth. These results demonstrate that the growth of spiral steps is less affected by impurities because the density of spiral steps is much higher than that of 2D island ones.

Rapid and selective determination of urinary lysozyme based on magnetic molecularly imprinted polymers extraction followed by chemiluminescence detection

A rapid, low cost and selective chemiluminescence method coupled with magnetic molecularly imprinted polymers extraction was developed to detect lysozyme in human urine samples. Compared with traditional solid-phase extraction, this method could achieve selective extraction for the lysozyme, avoid the time consuming elution from a column or centrifugation steps, and then showed great potential in the high-throughput screening of clinical samples. The parameters affecting the performance of extraction and chemiluminescence were investigated. Under optimal conditions, the whole analytical procedure was completed within 12 min and spiked recovery ranged from 90.1% to 103.7% (R.S.D. ≤ 6.7%). The limit of quantitation was 5 ng mL −1. Furthermore, the results obtained by the proposed method were linearly correlated to those by commercial lysozyme detection kit ( r = 0.9595). Finally, the validated method was used to measure the urinary lysozyme of renal disease patients and healthy controls. The results confirmed the reliability and practicality of the protocol and revealed a good perspective of this method for biological sample analysis.

Affinity Purification of Egg-White Allergens for Improved Component-Resolved Diagnostics

Background: Egg is a common cause of food-allergic reactions, especially among young children. Some egg-allergic patients do, however, tolerate heated egg products and component-resolved diagnostics (CRD) may facilitate prediction of different disease manifestations. Commercially available preparations of the egg-white allergens, ovomucoid, ovalbumin, conalbumin and lysozyme, have been reported to contain impurities which interfere with accurate CRD. Methods: Commercial preparations of the 4 egg-white allergens were characterized using allergen-specific monoclonal chimeric human/mouse IgE antibodies in experimental ImmunoCAP® tests. Further purification of commercial ovomucoid, ovalbumin and conalbumin preparations was performed by chromatography based on affinity to monoclonal antibodies. Purity was monitored by size exclusion chromatography, SDS-PAGE, Western blotting and experimental ImmunoCAP tests using allergen-specific chimeric IgE antibodies. IgE reactivity to the highly purified egg components was analyzed in 83 samples from egg white-sensitized individuals. Results: Preparations of commercially available ovomucoid, ovalbumin and conalbumin were found to contain other egg allergens which were removed by chromatographic purification. No impurities were detected in the commercial lysozyme preparation. Previously unknown complexes between the target allergens and contaminating allergens were detected and removed by affinity chromatography. IgE reactivity to ovalbumin was most common in the analyzed samples (87%), followed by ovomucoid (72%), conalbumin (69%) and lysozyme (58%). Conclusions: In this study we demonstrate the advantage of using monoclonal antibodies for purification, and monoclonal chimeric IgE antibodies for characterization, of egg allergens intended for CRD. Our study also established that ovalbumin, ovomucoid, conalbumin and lysozyme are all major allergens.

Highly efficient and low-cost purification of lysozyme: A novel tris(hydroxymethyl)aminomethane immobilized affinity column

A highly efficient and low-cost affinity chromatography strategy for lysozyme (LZM) purification is reported. Using tris(hydroxymethyl)aminomethane (Tris) as ligand and macroporous silica spheres as matrix, a novel affinity column was prepared. The high specificity, stability and repeatability of this Tris immobilized affinity column were proved by LZM separations from protein mixture solutions for 20 circles and 6 months test. LZM purified from chicken egg white on the Tris affinity column had even higher purity than the commercial standard and well-maintained activity of 8287 U/mg (activity of commercial LZM was 8171 U/mg). The efficient affinity process avoiding expensive or fragile ligand would bring advantages to the routine production of LZM from chicken egg white.

Impurity effects on orientation of lysozyme crystals nucleated on fatty acid thin films

Commercially available lysozyme samples that have different lot numbers (E02Z04 and E05802) were crystallized on fatty acid thin films. The orientation of lysozyme crystals nucleated on the films was investigated by atomic force microscopy and optical microscopy. The numbers of lysozyme crystals with specific planes parallel to the films are different. In other words, the impurities contained in commercial lysozyme significantly affect the orientation of lysozyme crystals. Detailed analysis of the orientation distribution of the lysozyme crystals nucleated from the purified sample showed that acetic acid is one of the substances promote the epitaxy.

Direct AFM observations of impurity effects on a lysozyme crystal

Impurity effects on the growth of tetragonal lysozyme crystals have been studied using in situ atomic force microscopy. Commercially available hen egg white lysozyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining, and purified by re-crystallization and successive high pressure liquid chromatography. On the (1 1 0) crystal surface, there was no significant difference in morphology between crystals grown in commercial and in purified solutions. On the (1 0 1) surface, however, a large number of small particles were found when the crystal was grown in the commercial solution, while the surface grown in the purified solution was quite smooth. Among the typical residual impurities contained in commercial lysozyme, only covalently bound lysozyme dimer yielded such particles. From measurements of particle separation and an estimate of the critical nucleation size, we infer that the particles reduced the step velocity according to the mechanism described by Cabrera et al. [N. Cabrera, D.A. Vermilyea, in: R.H. Doremus et al. (Eds.), Growth and Perfection of Crystals, 1958, P. 393].

Effects of purification on the crystallization of lysozyme

We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal ↔ orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

塩化リゾチーム製剤の品質試験および塩化リゾチームシロップの配合変化

Protein content in syrup preparation of commercial lysozyme chloride could not be determined by Lowry method or spectrophotometric measuring at 280 nm due to the presence of additives in syrup which interfered with the measurements. However, highly accurate protein content could be obtained by a filter paper technique for the microdetermination of protein based on Lowry method where protein content in the syrup obtained by this method corresponded to potency. Specific activity of lysozyme in the syrup determined by use of Micrococcus lysodeikticus in suspension as lysozyme substrate was the same activity as purified lysozyme. Therefore, it seems that lysozyme highly purified was used for the syrup preparation.The compatibility of lysozyme syrup with some commercial syrup preparations was studied. When Inolin syrup was mixed with the lysozyme syrup, 50% of the lysozyme activity was lost after 7 days at room temperature, whereas significant loss was not observed at 4°C. However, other syrups showed good compatibility with the lysozyme syrup under the same condition. Consequently, lysozyme chloride syrup seemed to be a stable preparation and to be mixed with other syrups.

Comparative Quality Test of Lysozyme Chloride Preparations

Fourteen lots of tablets, capsules and granules of commercial lysozyme chloride preparations, manufactured by six differerent companies, were evaluated for their pharmaceutical qualities. Significant difference of in vitro dissolution rate was noted between these products, when tested with 1st and 2nd fluids in the disintegration test of J. P. IX as dissolution media in the rotation method of U. S. P. XIX. The dissolution rate may have relationship with clinical effect of the products. By disc electrophoresis, impure protein was found to be contained in the preparations of all but one manufacturer. New quantitative analytical method by means of the area of bacteriolysis on gel-plate was developed in this study. The new method proved to produce results same as those obtained by the conventional method on the basis of the change in turbidity depending on bacteriolysis.

The effect of proteolytic activity on plasma renin activity assay

Carrier proteins are used in the radioimmunoassay of angiotensin I. It has been shown that the proteolytic activity found in commercial lysozyme will falsely elevate the observed plasma renin activity. This effect is probably due to the fact that human plasma contains proteinase inhibitors which inhibit this activity during radioimmunoassay of angiotensin I. This proteolytic activity can be inhibited by diisopropyl fluorophosphate or by heating lysozyme.

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl −, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl − present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D 2O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k 2, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.

Autolytic activity associated with competent group H streptococci.

Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05 m tris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02 m 2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells of Hydrogenomonas eutropha, a group A Streptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.