A Novel β-1, 4-N, 6-O-Diacetylmuramidase from Streptomyces griseus and its Chemical Modification

A novel lysozyme namedβ-1, 4-N, 6-O-diacetylmuramidase R2 was purified and characterized from Streptomyces griseus. The molecular weight of the enzyme was determined by MALDI-TOF-MS as 23.5 kDa. The N-terminal amino acid sequence was DTSGVQGIDVS-HWQG.Chemical modification ofβ-1, 4-N, 6-O-diacetylmuramidase R2 indicated that sulfhydry1 group and carbamidine of arginine residues are not essential for the activity of the enzyme, but lysine residues and imidazole of histidine residues are essential for the activity. The number of essential tryptophan and carboxyl groups was found that only one tryptophan residue and three carboxyl groups in the active site.     

硝基羟乙唑与溶菌酶反应机制的荧光光谱研究

分别在298,310,318 K温度下,利用荧光光谱法研究了pH=7.40生理条件下硝基羟乙唑(TRI)与溶菌酶(LYSO)的相互作用机理。结果表明,TRI与LYSO间通过静态猝灭方式相互作用。测定了LYSO与TRI反应的结合常数、结合位点数。利用反应过程的热力学参数,确定了LYSO-TRI体系的作用力类型;由Hill系数得出了LYSO或TRI的协同性;根据非辐射能量转移理论,确定了TRI到LYSO的结合距离,同时采用同步光谱法考察了TRI对LYSO构象的影响。     

牛血清白蛋白-脲醛树脂-二氧化锆固相萃取剂的制备及评价

合成了3μm无孔脲醛树脂-二氧化锆(UF-ZrO2)复合微球,并用扫描电子显微镜对其进行表征.利用基体表面上的酰胺键与丙烯酸甲酯和乙二胺进行三次马氏加成反应和胺化反应,形成末端为胺基的聚胺基酰胺星型树状间隔臂.再用咪唑法将牛血清白蛋白(BSA)对其表面进行修饰,制成BSA-UF-ZrO2固相萃取剂.以溶菌酶和卵清蛋白作为测试蛋白,分别考察了时间,pH值,洗脱试剂及其浓度对BSA-UF-ZrO2固相萃取剂吸附量的影响.结果表明,溶菌酶和卵清蛋白最佳吸附时间是4 h,缓冲溶液的pH值是6.0和5.0,最佳洗脱试剂是0.0625 mol.mL-1的KSCN(溶菌酶)和0.1250 mol.mL-1的KSCN(卵清蛋白).     

密码子优化的人溶菌酶基因在毕赤酵母中的分泌表达及抗菌活性分析

人溶菌酶（human lysozyme,hLYZ）是一种天然活性蛋白质,具有抗菌性,目前主要应用于食品添加剂、动物饲料、药物治疗领域。根据毕赤酵母表达偏爱密码子特点,通过优化hlyz基因密码子,人工合成目的基因,构建了分泌表达载体pPIC9K-hlyz。通过电击转化法将pPIC9K-hly质粒重组至毕赤酵母GS115染色体中,经筛选得到重组毕赤酵母GS115-pPIC9K-hlyz。在28 ℃、pH 6.0、转速240 r·min^-1、1%甲醇诱导下表达120 h,发酵液上清酶活达到最高,为（2 414.9±108.1）U·mL^-1,蛋白浓度为166.7 μg·mL^-1。发酵液上清经过Sephadex G50纯化,获得较纯的hLYZ,经SDS-PAGE分析,在近14.7 kDa处显示单一条带。抗菌实验结果表明,重组hLYZ对革兰氏阳性菌的抗菌效果大于革兰氏阴性菌;重组hLYZ的抗菌效果是hLYZ标准品的10~20倍。     

Proteins, lipids, and water in the gas phase

Evidence from mass‐spectrometry experiments and molecular dynamics simulations suggests that it is possible to transfer proteins, or in general biomolecular aggregates, from solution to the gas‐phase without grave impact on the structure. If correct, this allows interpretation of such experiments as a probe of physiological behavior. Here, we survey recent experimental results from mass spectrometry and ion‐mobility spectroscopy and combine this with observations based on molecular dynamics simulation, in order to give a comprehensive overview of the state of the art in gas‐phase studies. We introduce a new concept in protein structure analysis by determining the fraction of the theoretical possible numbers of hydrogen bonds that are formed in solution and in the gas‐phase. In solution on average 43% of the hydrogen bonds is realized, while in vacuo this fraction increases to 56%. The hydrogen bonds stabilizing the secondary structure (α‐helices, β‐sheets) are maintained to a large degree, with additional hydrogen bonds occurring when side chains make new hydrogen bonds to rest of the protein rather than to solvent. This indicates that proteins that are transported to the gas phase in a native‐like manner in many cases will be kinetically trapped in near‐physiological structures. Simulation results for lipid‐ and detergent‐aggregates and lipid‐coated (membrane) proteins in the gas phase are discussed, which in general point to the conclusion that encapsulating proteins in “something” aids in the conservation of native‐like structure. Isolated solvated micelles of cetyl‐tetraammonium bromide quickly turn into reverse micelles whereas dodecyl phosphocholine micelles undergo much slower conversions, and do not quite reach a reverse micelle conformation within 100 ns.

     

Theoretical study of ground and excited state potential energy surfaces for the Ca+-H2 complex

The potential energy surfaces of the Ca⁺-H₂ complex are calculated using the internally contracted multireference CI method (ICMR CI) and complete active space SCF (CAS SCF) reference wave functions. The calculations involve both the ground and the excited states correlating to (3d)²D and (4p)²P Ca⁺ terms and are carried out for C_∞v and C_2v configurations. Anisotropy of the potential surfaces has also been analysed by computing the interaction energy for some representative points as a function of the angle between the H₂ molecular axis and the Ca⁺—centre of mass of H₂ bond axis. The calculations have revealed the existence of a conical intersection of the lowest excited (3d)²B₂ potential surface with the ground state one. The obtained global energy minimum of the (3d)²B₂ potential surface lying 0.683 eV below the asymptote indicates a possible stabilization of the Ca⁺-H₂ complex towards formation of an exciplex in the (3d)Ca⁺-H₂(v = 0) collision process. The dependence of the vibrational energy levels of H₂ on the distance from Ca⁺ in the C_2v configuration has also been studied.     

The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

Lysozyme crystallization was conducted in the ionic liquid (IL) 1‐butyl‐3‐methylimidizolium tetrafluoroborate ([BMIm][BF₄]) with different buffer/IL proportions. It was found that the addition of [BMIm][BF₄] could promote the crystallization process, during which more lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. A probable explanation was proposed based on the influence of the ionic polarization and kinetics in the lysozyme crystallization. Moreover, the transform in coordination number and the relative growth rate of different crystal faces were discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Reaction mechanism of tylosin tartrate with lysozyme

Under simulated physiological conditions, the interaction between tylosin tartrate and lysozyme was investigated at pH?=?7.40 by fluorescence spectroscopy. The results indicated that tylosin tartrate could strongly quench the intrinsic fluorescence of lysozyme. By determining the quenching constants of the reaction between tylosin tartrate and lysozyme at different temperatures, the quenching mechanism was proven to be a static quenching process. The thermodynamic parameters (ΔH°, ΔS°) of the reaction between tylosin tartrate and lysozyme were obtained by the Van’t Hoff equation, and were 27.80?kJ mol^?1 and 166.28?J mol^?1 K^?1, respectively. The results showed that hydrophobic interaction between tylosin tartrate and lysozyme was dominant. Synchronous fluorescence spectra revealed that the conformation of lysozyme was changed. This method could be applied to measure the content of tylosin tartrate.     

A thermodynamic study of protein-induced lipid lateral phase separation. Effect of lysozyme on mixed lipid vesicles

In this work we study by differential scanning calorimetry (DSC) the lateral phase separation induced by a globular protein (lysozyme) on vesicles built-up by charged (phosphatidic acid) and neutral (phosphatidylcholine) lipids.The adsorption of the positively charged protein onto the negative vesicle surface induces the formation of micro-domains richer in the charged lipid component. This phenomenon is revealed as a splitting of the excess heat capacity peak associated to the melting of the lipid hydrocarbon chains.Also, the peak associated to the protein denaturation is shifted, suggesting the presence of adsorbed proteins onto the vesicle surface. The surface electrostatic potentials, both of proteins and vesicles, have been modulated by pH and ionic strength variations, showing a deep influence of the electric charges in modifying protein adsorption, rate of denaturation (related to unfolding enthalpy variation), and lipid micro-domain formation.Some of the present results have been rationalized on the basis of a theoretical model recently developed by the authors.     

鸡蛋清溶菌酶提取工艺及其应用初探

以鸡蛋清为原料，进行溶菌酶提取条件的探索，制定溶菌酶生产工艺，提供一种酶活力测定方法，并对其应用进行了初步研究。