Chitinolytic enzymes: catalysis,substrate binding,and their application

After the epoch-making report on X-ray crystal structure of a lysozyme-N-acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 +) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate-assisted mechanism. Hen egg white lysozyme has the binding cleft represented by (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)(-1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application.     

HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.     

Inhibition of Human Immunodeficiency Type 1 Virus (HIV-1) Life Cycle by Different Egg White Lysozymes

Lysozyme is a relatively small enzyme with different biological activities, which is found in tears, saliva, egg white, and human milk. In the study, the anti-HIV-1 activity of lysozymes purified from quail, Meleagris, and hen egg white has been determined. For this end, a time-of-drug-addition assay was performed to identify the target of anti-HIV-1 agents and for determination of probable anti HIV-1 mechanism of the studied lysozyme, the binding affinity of the lysozymes to the human CD4 receptor was studied by molecular docking method. To define structural differences between studied lysozymes, structural motifs of them were predicted by MEME tool. Quail, hen, and Meleagris lysozymes showed potent anti-HIV-1 activity with EC₅₀ of 7.5, 10, and 55 nM, respectively. The time-of-drug-addition study demonstrated that the inhibitory effect of all purified lysozymes is before HIV-1 infection. The frequency and intensity of CD4 expression in PBMCs decreased in the presence of all mentioned lysozymes. Also, the expression level of C-C chemokine receptor type 5 (CCR5) and chemokine receptor type 4 (CXCR4) on CD4+ T cells was not changed in cells treated with these lysozymes. The results of in silico study confirmed that the binding energy of quail lysozyme with CD4 was more than that of other studied lysozymes. The results revealed that these lysozymes restrict HIV-1 attachment to host cell CD4.     

Human milk lysozyme: unpublished data concerning the establishment of the complete primary structure; comparison with lysozymes of various origins

Details concerning the establishment of the complete primary structure of human milk lysozyme (previously published in a preliminary note) are presented. The chymotryptic peptides obtained from the reduced alkylated enzyme were purified and their amino acid sequences determined chiefly by the ‘Edman-dansylation’; procedure, and in two cases by partial acid or peptic hydrolyses. The tryptic peptides are alined into a single chain containing 129 amino acid residues, on the basis of overlapping peptides. Two labile glutamine residues easily converted into glutamic acid residues were characterized. Human milk lysozyme is compared with other human lysozymes (from normal and leuchaemic individuals) prepared by our group. The structure proposed is identical with the sequence of human leuchaemia lysozyme (from the urine of a patient with chronic monocytic leuchaemia) reported by Canfield. Human milk lysozyme is also near by related to several bird egg-white lysozymes (and bovine α-lactalbumin): Identical positions of Cys and Trp residues and of the residues essential for the catalytic activity or involved in some hydrogen bonds; several identical regions, especially in the β-sheet region; between 71 and 77 identical amino acid residues. It is suggested that by an insertion and a deletion in the sequence of human milk lysozyme, sequences homologous to those of bird lysozymes can be obtained.     

New data concerning lysozyme from goose egg-white: tryptophan content, stability and sensitivity

New data concerning the tryptophan content, the stability, and the specificity of goose egg-white lysozyme are reported. This enzyme occupies a special place among the mammelian lysozymes already studied and the general definition of a lysozyme had to be modified. The mechanism of action is discussed.     

Release of lysozyme from the branched polyelectrolyte-lysozyme complexation

On the basis of the discretely charged sphere model of lysozyme, the release behavior of lysozyme from the branched polyelectrolyte-lysozyme complexation is investigated by adding salt and changing the pH values of the solution. It is found that, with the increase of the salt ionic strength of the solution, the lysozymes are gradually released from the oppositely charged polyelectrolyte as a result of the screening of electrostatic attraction between the two ionic species by adding the salt. Interestingly, there exists a critical salt ionic strength at which all proteins are released from the branched polyelectrolyte, and the polyelectrolyte-protein complexation is broken completely. Beyond the critical value, the increase of the salt ionic strength causes self-association of the proteins released from the branched polyelectrolyte-protein complexation. The self-association of the protein is detrimental in biological systems. By calculating the second virial coefficient, we found that the optimal salt content for the dispersion of proteins coincides with the critical ionic strength, because the second virial coefficient reaches its maximum at the critical ionic strength. Similarly, increasing the pH value of the solution can also release the lysozymes from the polyelectrolyte, because the increase of pH value of the solution changes the charge distribution and net charge of the lysozyme, weakens the attraction between lysozymes mediated by polyelectrolyte, and finally leads to the dissolution of the complexation of branched polyelectrolyte with lysozymes in strong alkaline solution. In addition, by exploring the effect of architecture of the polyelectrolyte on the release behavior of proteins, we found that it is more difficult to release proteins from the branched polyelectrolyte than from the linear polyelectrolyte.     

The primary chemical structure of human milk lysozyme: establishment of a temporary formula

The detailed primary sequences of the N-terminal moiety (72 amino acid residues) and of the C-terminal end (23 amino acid residues) of human milk lysozyme (129 residues) are reported. A tentative complete structure of the enzyme is compared to hen and duck egg-white lysozymes which are very near related proteins despite many amino acid replacements (around 50), the insertion of an additional residue in the N-terminal and a deletion in the C-terminal moiety.     

用蛋白电泳和高效凝胶排阻色谱法分析还原脲变性蛋白溶菌酶稀释复性过程的集聚体

本文利用蛋白电泳和高效凝胶排阻层析法分析了还原脲变性蛋白溶菌酶稀释复性过程中的集聚体。当用复性液稀释复性还原脲变性蛋白溶菌酶时,会迅速产生可观量的沉淀。沉淀和上清液的不连续十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻层析分析结果表明,还原脲变性蛋白溶菌酶在稀释复性过程中除了能够复性成天然态蛋白溶菌酶分子外,还会形成可溶的蛋白溶菌酶分子二聚体和三聚体,二聚体和三聚体主要是靠分子间二硫键的错配连接而成的;可溶的蛋白溶菌酶分子二聚体之间通过非共价键相互作用而形成集聚体沉淀,而可溶的三聚体溶菌酶分子则仍处于复性液上清液中。     

The adsorption of lysozymes: A model system

Hen egg white lysozyme was adsorbed onto clean borosilicate glass and n-pentyl silane-treated glass surfaces. Both modified (reductively methylated) and native lysozyme were studied. Variable angle X-ray photoelectron spectroscopy (VA-XPS) suggested differences in the nature of the adsorbed layer depending on substrate properties, as well as on degree of methylation of the protein. Adsorbed film thickness (as measured in the dehydrated state by XPS) ranged from 14 Å on hydrophilic glass to 25 Å on the hydrophobic surface. Degree of surface coverage ranged from 45% on the hydrophobic to 69% on the hydrophilic surface. The results suggest that lysozyme unfolds to a greater extent and covers more surface on the hydrophilic glass, possibly due to strong electrostatic interactions at the pH 7.4 conditions used in the study. An analysis of the surface structure of native hen lysozyme by molecular graphics has also been performed, suggesting that adsorption on hydrophobic surfaces should occur via the hydrophobic patch opposite the enzyme active site cleft. A comparison with human lysozyme has also been made using total internal reflection fluorescence (TIRF) spectroscopy to measure protein adsorption on model surfaces. The two proteins have significantly different interfacial properties.     

Studies on Aggregation Interaction between Reduced Denatured Egg White Lysozymes during Refolding Procedure in Urea Solution

The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

Conformational changes of lysozymes with different numbers of disulfide bridges in sodium dodecyl sulfate solutions

Four disulfide bridges of hen egg-white lysozyme were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and 0SS lysozymes, respectively). The 3SS lysozyme maintained the native conformation at pH 7.0 and 3.0. Even upon the reduction of two disulfide bridges, the protein conformation still remained unchanged at pH 7.0. Upon the reduction of all four disulfide bridges, the helicity, [θ]₂₇₀, and tryptophan fluorescence changed at pH 3.0 as well as at pH 7.0. The helicity of each derivative increased in a solution of sodium dodecyl sulfate (SDS). The SDS-induced helicity of the 0SS lysozyme was lower at pH 7.0 and higher at pH 3.0 than that of the intact lysozyme with four disulfide bridges. The helix formation appears to occur in originally nonhelical parts in each derivative at pH 7.0. In the cases of the 2SS and 0SS lysozymes at pH 3.0, however, some of the helices appear to be reformed also at moieties where the original helices are disrupted upon the cleavage of disulfide bridges. Received: 17 September 2000/Accepted: 24 March 2000     

脲或盐酸胍溶液中变性溶菌酶复性过程中集聚体的研究

建立在蛋白质变性-复性三态模型的基础上, 给出了一个描述在变性液中变性蛋白质复性时蛋白质浓度和其复性率的关系式. 通过这个关系式, 可以获得两个重要的描述蛋白质变性-复性体系特征的参数, 一个是包含在一个集聚体分子中的变性蛋白质的分子数目n, 另一个是蛋白质从原始态到形成集聚体过程中的表观集聚平衡常数K. 以三种溶菌酶在脲和盐酸胍溶液中的变性-复性过程对此方程进行了验证, 结果表明所给出的方程能够很好地描述三种溶菌酶在这两种变性液中的复性结果, 三种溶菌酶在两种变性液中有形成二分子集聚体的趋势. 变性溶菌酶在复性过程中的电泳和高效凝胶排阻色谱也同时能够监测到复性过程中集聚体的形成, 并且监测结果与上述方程所得的结果一致.     

Amino Acid Functionalized Superparamagnetic Nanoparticles Inhibit Lysozyme Amyloid Fibrillization

Nanoparticles have great potential to be used in various biomedical applications, including therapy or diagnosis of amyloid-related diseases. The physical and chemical properties of iron oxide superparamagnetic nanoparticles (MNPs) functionalized with different amino acids (AAs), namely, with lysine (Lys), glycine (Gly), or tryptophan (Trp), have been characterized. The cytotoxicity of nanoparticles and their effect on amyloid fibrillization of lysozymes in vitro was also verified. The AA-MNPs under study are nontoxic to human SHSY5Y neuroblastoma cells. Moreover, the AA-MNPs were able to significantly inhibit lysozyme amyloid fibrillization and destroy amyloid fibrils. Kinetic studies revealed that the presence of AA-MNPs affected lysozyme fibrillization, namely, the lag phase and steady-state phase of the growth curves. The most effective activities were observed for Trp-MNPs, which revealed the importance of aromatic rings in the structure of AAs used as coating agents. The obtained results indicate the possible application of these AA-MNPs in the treatment of amyloid diseases associated with lysozyme or other amyloidogenic proteins.     

Lysozyme photo-oxidation by singlet oxygen: properties of the partially inactivated enzyme

This work studies the behaviour of partially inactivated lysozyme formed by the effect of singlet oxygen, which was obtained through the irradiation of the native enzyme solution with polychromatic visible light using Methylene Blue as a sensitizer. The polyacrylamide gel analysis of the partially inactivated lysozyme solution shows the presence of different protein fractions. One of them, which corresponds to 53% of the original enzyme, has the same migration as the native enzyme. The others are a mixture of fractions (47%) that show slower migration to the cathode. When this experiment is carried out in the presence of sodium dodecyl sulfate, only one fraction is obtained, which rules out the presence of covalently aggregated forms of lysozyme. The partially inactivated lysozyme has lost 74% of the fluorescence emission of the tryptophan (Trp) residues. By using the anionic quencher iodide, it is determined that 45 and 36% of the fluorescence emission arising from the native and partially inactivated enzyme, respectively, are due to Trp residues exposed to the solvent. Michaelis-Menten constants (K(in)) of 0.296 and 0.511 (mg/ml) and maximum initial rates (Vmax) of 0.295 and 0.190 (mg/ml min) are determined for the native and the partially inactivated enzyme solutions, respectively. The same inactivation profile is found when the denaturing effect of increasing urea concentration on both the native and partially inactivated lysozyme is studied. It is postulated that the partially inactivated lysozyme solution is composed of one protein fraction with enzymatic activity similar to that of the native enzyme and also of a mixture of fractions (47% of the total enzyme) with very low activity and characterized by a high tryptophan photo-oxidation.     

Controllable immobilization of proteins in quenched spherical polyelectrolyte brushes as observed by fluorescence spectroscopy and small angle X-ray scattering

The immobilization of lysozymes (pI = 11) onto anionic spherical polyelectrolyte brushes (SPB) which consist of a solid polystyrene core and a densely grafted poly(styrene sulfonate) (PSS) shell was systematically studied by fluorescence spectroscopy and small angle X-ray scattering. Results show that the capture of lysozyme by PSS brush is a dynamic process, which involves a quick agglomeration stage and a slow rearrangement one. And lysozyme inclines to immobilize in the inner layer of the brush, and saturation of lysozyme adsorption onto the SPB is gradually reached as the protein concentration increases, proceeding from the inside to the outside of the brush layers. As increasing the pH and ionic strength, the lysozyme previously adsorbed will be partially released and migrate from the inner to the outer layer of SPB. Last competitive adsorption tests between lysozyme and BSA or β-glucosidase were performed, indicating that besides electrostatic interaction counterion release force also plays an important role in protein adsorption. SPB was proved to be ideal candidate for controllable immobilization of protein, which can be extended into various applications, such as drug delivery and protein separation. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 1577–1588     

Prediction of the orientations of adsorbed protein using an empirical energy function with implicit solvation

When simulating protein adsorption behavior, decisions must first be made regarding how the protein should be oriented on the surface. To address this problem, we have developed a molecular simulation program that combines an empirical adsorption free energy function with an efficient configurational search method to calculate orientation-dependent adsorption free energies between proteins and functionalized surfaces. The configuration space is searched systematically using a quaternion rotation technique, and the adsorption free energy is evaluated using an empirical energy function with an efficient grid-based calculational method. In this paper, the developed method is applied to analyze the preferred orientations of a model protein, lysozyme, on various functionalized alkanethiol self-assembled monolayer (SAM) surfaces by the generation of contour graphs that relate adsorption free energy to adsorbed orientation, and the results are compared with experimental observations. As anticipated, the adsorbed orientation of lysozyme is predicted to be dependent on the discrete organization of the functional groups presented by the surface. Lysozyme, which is a positively charged protein, is predicted to adsorb on its 'side' on both hydrophobic and negatively charged surfaces. On surfaces with discrete positively charged sites, attractive interaction energies can also be obtained due to the presence of discrete local negative charges present on the lysozyme surface. In this case, 'end-on' orientations are preferred. Additionally, SAM surface models with mixed functionality suggest that the interactions between lysozyme and surfaces could be greatly enhanced if individual surface functional groups are able to access the catalytic cleft region of lysozyme, similar to ligand-receptor interactions. The contour graphs generated by this method can be used to identify low-energy orientations that can then be used as starting points for further simulations to investigate conformational changes induced in protein structure following initial adsorption.     

A g-Type Lysozyme from Deep-Sea Hydrothermal Vent Shrimp Kills Selectively Gram-Negative Bacteria

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.     

Interaction of tylophorinidine with lysozyme

The interaction of tylophorinidine (TPD) with lysozyme—a model protein—with biological activity, was investigated by determining its fluorescence and by assessing its activity under various conditions. The results indicated that TPD associated with lysozyme at pH 9.2 efficiently with an association constant K_a of 3.3 X 10⁴ M^–1 at 26°C. K_a increased with the increasing temperature in the range 26 to 55°C; the calculated enthalpy change ΔH was found to be 2.3 kcal/mol. Under the same conditions as above TPD also associated with the free amino acid tryptophan with a K_a of 1.7 X 10⁴ M^–1 indicating half the efficiency of its association with protein lysozyme. TPD associated lysozyme was less active than the uncomplexed enzyme in the above temperature range although beyond 45°C the inhibition was more significant. The results imply that TPD binds lysozyme outside the cleft region in the temperature range studied here. However, with increasing temperature the cleft region is gradually widened and/or the whole molecule is expanded such that the accommodation of whole or part of the TPD molecule is facilitated leading to the blockage of lytic activity.