聚苯乙烯为致孔剂的分子印迹膜对溶菌酶吸附性能的研究

探索聚苯乙烯微球的添加对于分子印迹膜对溶菌酶(Lysozyme, Lyz)吸附性能的影响.以溶菌酶为模板分子,丙烯酰胺、N,N'-亚甲基双丙烯酰胺为功能单体、聚苯乙烯(Polystyrene,PS)微球为致孔剂,制备聚苯乙烯为致孔剂的分子印迹聚合物膜(PS-MIP).PS-MIP的吸附平衡时间为30 min,短于未添加聚苯乙烯微球的分子印迹聚合物膜(MIP);PS-MIP与MIP对于Lyz的吸附能力均明显好于聚苯乙烯为致孔剂的非分子印迹聚合物膜(PS-NIP)与未添加聚苯乙烯微球的非分子印迹聚合物膜(NIP);PS-MIP对Lyz的选择性要好于MIP,而卵清蛋白和牛血清白蛋白在PS-MIP和MIP上的荧光信号没有差别.实验结果表明,在分子印迹膜聚合过程中添加聚苯乙烯微球,有利于缩短吸附时间,增大溶菌酶的吸附量并提升选择性.     

噬菌体T4溶菌酶溶液构象模拟中分子力场和水模型的选择

蛋白质在溶液中可能以不同构象的集合形式存在,不能用单一的静态结构来表示. 分子动力学模拟已成为对溶液中蛋白质构象进行采样的有用工具,但分子力场和水模型的选择是关键问题. 这项工作介绍了噬菌体T4溶菌酶的个例研究. 本文发现,使用经典的AMBER99SB力场和TIP4P水模型,分子动力学模拟不能很好地描述野生型噬菌体T4溶菌酶在微秒时间尺度上的铰链弯曲结构域运动. 其它新型力场和水模型的组合,如被称为RSFF2+的残基特异性力场和离散校正的水模型TIP4P-D,能够对噬菌体T4溶菌酶溶液构象进行合理的采样,与实验数据有良好的一致性. 这项工作为进一步研究噬菌体T4溶菌酶的溶液构象转变提供了分子力场和水模型的参考.     

Ultrasensitive SERS Detection of Lysozyme by a Target‐Triggering Multiple Cycle Amplification Strategy Based on a Gold Substrate

An ultrasensitive surface enhanced Raman scattering (SERS) method has been designed to selectively and sensitively detect lysozyme. The gold chip as the detection substrate, the aptamer‐based target‐triggering cascade multiple cycle amplification, and gold nanoparticles (AuNPs) bio‐barcode Raman probe enhancement on the gold substrate are employed to enhance the SERS signals. The cascade amplification process consists of the nicking enzyme signaling amplification (NESA), the strand displacement amplification (SDA), and the circular‐hairpin‐assisted exponential amplification reaction (HA‐EXPAR). With the involvement of an aptamer‐based probe, two amplification reaction templates, and a Raman probe, the whole circle amplification process is triggered by the target recognition of lysozyme. The products of the upstream cycle (NESA) could act as the “DNA trigger” of the downstream cycle (SDA and circular HA‐EXPAR) to generate further signal amplification, resulting in the immobility of abundant AuNPs Raman probes on the gold substrate. “Hot spots” are produced between the Raman probe and the gold film, leading to significant SERS enhancement. This detection method exhibits excellent specificity and sensitivity towards lysozyme with a detection limit of 1.0×10^?15 M . Moreover, the practical determination of lysozyme in human serum demonstrates the feasibility of this SERS approach in the analysis of a variety of biological specimens.     

Penetration Process of a Hydrated Deep Eutectic Solvent Through the Stratum Corneum and its Application as a Protein Penetration Enhancer

The penetration mechanism of choline chloride-glycerol deep eutectic solvent (DES) through the stratum corneum (SC) as a potential solvent for a novel enhancer of protein penetration into the skin was investigated in a wide and small angle X-ray diffraction study. We found that DES penetrated through intercellular lipids but not the corneocytes. DES seemed to extract a portion of lipids of the short lamellae in the SC. Hydrated DES with a DES to water weight ratio of 9 to 1 (9DES-1H₂O) showed the strongest interaction with the lipids in the SC compared with water, DES, and hydrated DESs with another weight ratio of DES to water (DES : water=8 : 2). In a skin penetration test with a fluorescently labelled lysozyme, 9DES-1H₂O increased the amount of penetration through the SC by two-fold compared with HEPES buffer.     

Structural Effect and Composition of Anionic Copolymer on Protein Flocculation

Flocculation of lysozyme with anionic copolymers of acrylamide, acrylic acid, and sodium styrene sulfonate used as flocculants was performed in pursuit of high flocculation efficiency. Two major factors, pH and ionic strength, are used to investigate the relationship of the flocculation behavior of protein by copolymers and the functional group (–COOH, –NH₂, –SO₃H) compositions of these copolymers. The protein flocculation can be controlled by adjusting pH. In addition, the various copolymers exhibit differing effects on ionic strength induced protein flocculation. FT-Raman spectroscopy was applied to investigate the mechanism of interaction between protein and copolymer. An attempt was made to understand how the pH and ionic strength change the surface chemical characteristics of protein and copolymers, as well as the relationship between the structure of copolymer and the protein flocculation process.     

Quenching interactions and nonexponential decay: tryptophan 138 of bacteriophage T4 lysozyme

Site-directed mutagenesis has been used to prepare variants of bacteriophage T4 lysozyme that contain only one tryptophan residue at position 138 and to change the residues in the immediate environment of this buried residue. Replacement of glutamine-105 by alanine results in a 2.7-fold increase in fluoresence quantum yield and converts the fluorescence decay from a highly nonexponential form to a single-exponential decay. This is atributed to electron transfer quenching of tryptophan-138 fluorescence by glutamine-105. Replacemeent of alanine-146 by threonine results in a 1.6-fold decrease in fluorescence intensity, indicating enhanced quenching by glutamine-105; replacement of glutamine-105 by alanine in this species results in a 5-fold in crease in fluorescence intensity. The interpretation of the nonexponential decay of the glutamine-105-containing species is discussed in terms of reversibility of the quenching process.     

Preferential solvation of lysozyme by dimethyl sulfoxide in binary solutions of water and dimethyl sulfoxide

To reveal the denaturation mechanism of lysozyme by dimethyl sulfoxide (DMSO), thermal stability of lysozyme and its preferential solvation by DMSO in binary solutions of water and DMSO was studied by differential scanning calorimetry (DSC) and using densities of ternary solutions of water (1), DMSO (2) and lysozyme (3) at 298.15 K. A significant endothermic peak was observed in binary solutions of water and DMSO except for a solution with a mole fraction of DMSO (x ₂) of 0.4. As x ₂ was increased, the thermal denaturation temperature T _m decreased, but significant increases in changes in enthalpy and heat capacity for denaturation, ΔH _cal and ΔC _p, were observed at low x ₂ before decreasing. The obtained amount of preferential solvation of lysozyme by DMSO (∂g ₂/∂g ₃) was about 0.09 g g⁻¹ at low x ₂, indicating that DMSO molecules preferentially solvate lysozyme at low x ₂. In solutions with high x ₂, the amount of preferential solvation (∂g ₂/∂g ₃) decreased to negative values when lysozyme was denatured. These results indicated that DMSO molecules do not interact directly with lysozyme as denaturants such as guanidine hydrochloride and urea do. The DMSO molecules interact indirectly with lysozyme leading to denaturation, probably due to a strong interaction between water and DMSO molecules.     

溶菌酶降解壳聚糖条件的研究

研究了溶茵酶对壳聚糖的降解条件,结果表明：脱乙酰度约70％的壳聚糖较易被溶菌酶水解;水解反应的最适温度为55℃、pH值为4．0,低速摇床振荡对水解有利;在壳聚糖水解初期,溶液中还原糖浓度迅速增加,0．5h后水解速率逐渐减慢,至8h后还原糖浓度的增加已很缓慢;酶解液中还原糖的生成量瞳壳聚糖及溶茵酶浓度的增加而增大,当壳聚糖溶液浓度为20mg／mL、溶茵酶浓度为2．48mg／mL时,水解6h后,还原糖的含量可达6．758mmol／L。水解6h后酶解液中还原糖浓度与壳聚糖的浓度呈线性关系。     

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.     

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.