Lysozyme Adsorption onto Different Supports: A Comparative Study

The interaction between proteins and solid surfaces has been investigated. The aim of this work is to compare three different materials (hydroxyapatite, polystyrene with core-shell structure (PE-CS) and a functionalized styrene divinylbenzene copolymer) to be used as adsorbents for lysozyme, known as a “hard” protein. Tests were performed according to an experimental design in order to compare the effects of pH, lysozyme and phosphate buffer concentration onto the adsorbed amount of protein. A 2³ factorial design and a cross design, which was performed in triplicate, were used to distinguish the most important variables and to infer about the interaction between them. Hydroxyapatite showed the best performance—higher adsorbed amount of lysozyme and smaller dispersion (72.2 ± 0.9 mg/g). However, PE-CS can be regarded as a promising support as high amounts of lysozyme are adsorbed onto this material with relatively small dispersion.     

壳聚糖的溶菌酶降解

用粘度法和还原糖法研究了溶菌酶降解壳聚糖过程中温度、pH值、时间、酶浓度、底物浓度以及金属离子对降解速度的影响.比较合适的降解条件是:温度45℃,pH值5.0,适当增大酶浓度和底物浓度能够加速壳聚糖的降解,在一定的底物浓度下溶菌酶降解壳聚糖的反应不遵循简单的一级反应动力学.一定浓度的Cu2+、Zn2+、Ba2+、Cr3+能够促进壳聚糖在溶菌酶作用下的降解,而K+、Ca2+和高浓度的Zn2+会抑制酶活力的发挥.     

Investigation of Lysozyme-Chitobioside Interactions Using Synchronous Luminescence and Lifetime Measurements

Beside their ability to disrupt the outer membranes of some microorganisms, lysozymes also experience interactions with chitins or their fluorescent analogs. It has been well established that chitins bind to the cleft of lysozymes and the subsites of the location of the different N-acetylglucosamines that are parts of chitins have been identified. Moreover, it has been well documented that a 1,4--bond must be located between subsite D and subsite E to be cleaved. Nevertheless, a better understanding of the biophysical and biochemical processes is needed.In this paper, pulsed fluorescence was used to further investigate the mechanism by which the binding of fluorescent analogs of chitin (4-methylumbelliferyl chitobiose and 4-methylumbelliferyl chitotriose) to hen egg-white lysozyme results in an increase of their fluorescence intensity. Although such an increase is not observed when these chitobiosides bind to turkey egg-white lysozyme, synchronous fluorescence techniques show that this binding induces a quenching of the native fluorescence of both these proteins.The findings of this study, associated with previously published cyrstallographic data allow us to suggest that the system lysozyme-chitobioside partitions in two three-dimensional conformational states: an enzymatic active conformation and a storage conformation. These states are separated by an energy barrier, with the storage conformation being more populated than the enzymatic active conformation below 45°C.     

Using the fast fourier transform in binding free energy calculations

According to implicit ligand theory, the standard binding free energy is an exponential average of the binding potential of mean force (BPMF), an exponential average of the interaction energy between the unbound ligand ensemble and a rigid receptor. Here, we use the fast Fourier transform (FFT) to efficiently evaluate BPMFs by calculating interaction energies when rigid ligand configurations from the unbound ensemble are discretely translated across rigid receptor conformations. Results for standard binding free energies between T4 lysozyme and 141 small organic molecules are in good agreement with previous alchemical calculations based on (1) a flexible complex ( for 24 systems) and (2) flexible ligand with multiple rigid receptor configurations ( for 141 systems). While the FFT is routinely used for molecular docking, to our knowledge this is the first time that the algorithm has been used for rigorous binding free energy calculations. © 2017 Wiley Periodicals, Inc.     

EFFECTS OF HIGH HYDROSTATIC PRESSURE AND OTHER FACTORS ON BACILLUS STEAROTHERMOPHILUS *

研究了高静压协同水分活性、热处理工艺和溶菌酶等方法对嗜热脂肪芽孢杆菌的灭菌作用．结果表明,介质的水分活性明显影响芽孢对压力的敏感性,水分活性越高,芽孢残存率越低．采用不同的热处理工艺来增强压力的灭菌作用,发现高压处理前的预热处理（４５℃,２０ｍｉｎ）比高压处理后的热处理有更高的灭菌效果;与其他预处理温度（４５℃,８５℃）相比,６５℃预热处理获得的芽孢残存数更低．微波预热处理比传统热处理有更好的协同灭菌效果．溶菌酶的存在有助于提高芽孢对压力处理的敏感性．观测了压力与溶菌酶对嗜热脂肪芽孢杆菌的协同作用效果．当介质中溶菌酶浓度为３３３４×１０－２ｍｏｌ／（ｓ·Ｌ）时,在３００ＭＰａ下处理１５ｍｉｎ,芽孢残留量大约可下降５个数量级     

Acylation of amino functions of proteins with monomethoxypoly (ethylene glycol)-N-succinimide carbonate

Monomethoxypoly(ethylene glycol)-N-succinimide carbonate (SC-PEG) was used to prepare PEG-lysozyme, PEG-papaya proteinase III, PEG-catalase, and PEG-lactoperoxidase conjugates. SC-PEG produced extensively modified enzymes under mild conditions (pH 7.0; 25°C) within a couple of hours. PEG-enzyme conjugates showed equal or even greater specific activity provided that low-molecularweight substrates were used to evaluate the biological activities. However, papaya proteinase III and lysozyme lost their proteolytic and bacteriolytic activities, respectively, on conjugation with PEG. This was most probably because of steric factors, since no drastic conformational changes could be detected after conjugation of these enzymes with PEG chains. Unlike these enzymes, the secondary structures of the two hemoproteins were somewhat affected by the covalent attachment of PEG chains as shown by FTIR experiments. These results confirmed the potential usefulness of SC-PEG, for which a novel route of synthesis making use ofN,N′-disuccinimidyl carbonate was described.     

Calorimetric and volumetric data of salting of hen egg lysozyme using NaCl solution at pH 8.8

Heat effects and densities of hen egg lysozyme in the phosphate buffer at pH 8.8 and various NaCl concentrations were determined at 25°C by LKB 10700-2 microcalorimeter and an Anton Paar 60/602 densimeter. The relation between the changes of the enthalpy and apparent molar volumes vs. molality of NaCl were determined. The data are discussed together with the data obtained previously for hen egg lysozyme solutions with NaCl salt in Na-acetate buffer pH 4.2.     

Constant pH molecular dynamics in generalized Born implicit solvent

A new method is proposed for constant pH molecular dynamics (MD), employing generalized Born (GB) electrostatics. Protonation states are modeled with different charge sets, and titrating residues sample a Boltzmann distribution of protonation states as the simulation progresses, using Monte Carlo sampling based on GB-derived energies. The method is applied to four different crystal structures of hen egg-white lysozyme (HEWL). pK(a) predictions derived from the simulations have root-mean-square (RMS) error of 0.82 relative to experimental values. Similarity of results between the four crystal structures shows the method to be independent of starting crystal structure; this is in contrast to most electrostatics-only models. A strong correlation between conformation and protonation state is noted and quantitatively analyzed, emphasizing the importance of sampling protonation states in conjunction with dynamics.     

Microcalorimetric determination of displacement adsorption enthalpies of protein refolding on a moderately hydrophobic surface at 308 K

Both microcalorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of guanidine – denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ΔH values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L^–1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L^–1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.     

Sequential adsorption of bacteriophage T4 lysozyme stability variants at solid–water interfaces

The sequential adsorption of the wild type T4 lysozyme and one of its structural stability variants was studied, using ellipsometry and ¹²⁵I radioisotope labeling techniques. The mutant lysozyme was produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability. The mutant protein was more resistant to surfactant-mediated elution, and apparently adsorbed at the interfaces with a greater interfacial area/molecule than the wild typeT4 lysozyme. However, the results of each type of experiment suggested that sequential adsorption and exchange of proteins occurred only in the case of the less stable mutant followed by the wild type. This suggests that, in these exchange reactions, properties of the adsorbing protein (e.g. its ability to adsorb when a relatively small amount of unoccupied area is present) were more important than the apparent binding strength of the adsorbed protein molecules.