疏水表面上吸附态溶菌酶的DSC和FTIR研究

联合差示扫描量热 (DSC)和傅里叶变换红外吸收光谱(FTIR)分别研究了鸡蛋白溶菌酶(Lyz)在适度疏水吸附剂(PEG-600)表面上吸附和折叠时,不同盐(硫酸铵)浓度、表面覆盖度和变性剂(盐酸胍)浓度对无水环境的吸附态天然和变性溶菌酶构象变化及热稳定性的影响。研究发现：随着硫酸铵浓度和溶菌酶表面覆盖度的增加,吸附态天然和变性溶菌酶的吸热峰温度都逐渐降低,同时在较高温度下的微扰也增多。吸附发生后,α-螺旋结构减少,β-折叠和β-转角结构增多。在FTIR图谱中,吸附态变性溶菌酶在1 400～1 425 cm-1处的C—C拉伸振动峰和1 650～1 670 cm-1处的酰胺Ⅰ带特征峰都能明显观测到。但是,与之相比,相同条件下吸附态天然溶菌酶在1 650～1 670 cm-1处特征峰却几乎看不到。吸附态天然溶菌酶发生了结构丢失,表现得更加不稳定。     

溶菌酶在裸金电极和疏基乙酸或正十二疏烷基醇修饰电极上的吸附

电化学石英晶体阻抗系统;疏基乙酸;溶菌酶在裸金电极和疏基乙酸或正十二疏烷基醇修饰电极上的吸附     

Development of Amino Acids Functionalized SBA-15 for the Improvement of Protein Adsorption

Ordered mesoporous materials and their modification with multiple functional groups are of wide scientific interest for many applications involving interaction with biological systems and biomolecules (e.g., catalysis, separation, sensor design, nano-science or drug delivery). In particular, the immobilization of enzymes onto solid supports is highly attractive for industry and synthetic chemistry, as it allows the development of stable and cheap biocatalysts. In this context, we developed novel silylated amino acid derivatives (Si-AA-NH₂) that have been immobilized onto SBA-15 materials in biocompatible conditions avoiding the use of toxic catalyst, solvents or reagents. The resulting amino acid-functionalized materials (SBA-15@AA) were characterized by XRD, TGA, EA, Zeta potential, nitrogen sorption and FT-IR. Differences of the physical properties (e.g., charges) were observed while the structural ones remained unchanged. The adsorption of the enzyme lysozyme (Lyz) onto the resulting functionalized SBA-15@AA materials was evaluated at different pHs. The presence of different functional groups compared with bare SBA-15 showed better adsorption results, for example, 79.6 nmol of Lyz adsorbed per m² of SBA-15@Tyr compared with the 44.9 nmol/m² of the bare SBA-15.     

Preparation of a novel lysozyme molecularly imprinted polymer using uniformly sized functionalized poly(glycidyl methacrylate) microspheres as the matrix and its application to lysozyme purification

A novel lysozyme imprinted polymer based on uniformly sized functionalized poly(glycidyl methacrylate) microspheres has been synthesized in aqueous solution using the surface imprinting technique. The microspheres were modified with hydroxyl ethyl methacrylate to allow for the introduction of polymerizable double bonds, with β‐cyclodextrin and acrylamide being grafted onto the surface as functional monomers. The selective recognition properties of the resulting molecularly imprinted polymers (MIPs) were investigated by HPLC. Various factors were also investigated in terms of their influence on the retention behaviors of the imprinted polymers, including the pH and salt concentration of the mobile phase. The binding capability properties of the MIPs were evaluated, and the P_GMA/EDMA‐MIPs showed a high adsorption capacity for lysozyme. Furthermore, this MIP was used to separate and enrich lysozyme from egg whites. The results revealed that the lysozyme surface‐modified MIP could be used to efficiently separate and purify lysozyme from egg whites. Copyright © 2013 John Wiley & Sons, Ltd.     

蛋清溶菌酶基因的密码子优化及其在毕赤酵母中的分泌表达

作为一种天然的抑菌物质,溶菌酶对抗生素耐药菌有较强的杀伤作用，具有无耐药性、无残留等特点，被认为在诸如动物饲料、药品等领域可有效代替抗生素，改善滥用抗生素所引起的一系列问题，市场前景巨大。通过微生物发酵大规模生产重组溶菌酶符合市场发展趋势，由于目前生产的菌株表达量偏低，限制了溶菌酶的应用和产业化发展。为提高蛋清溶菌酶的发酵表达量，根据毕赤酵母密码子的偏爱性,对其编码基因进行密码子优化，并构建了真核分泌表达载体pPIC9K-coEWL，经遗传霉素G418抗性筛选后，获得了一株酵母转化子(G-p-coEWL)，其对G418的抗性浓度高达15 mg·mL^-1。在28℃、pH 6.0、转速240 r·min^-1和甲醇浓度1%的诱导条件下,酵母重组子G-p-coEWL经摇瓶培养72 h，实现了蛋清溶菌酶的分泌表达。在发酵上清液中，总蛋白浓度为 607 mg·L^-1，酶活达到677 U·mL^-1。此外，本实验还比较了葡聚糖凝胶柱Sephadex G-50和强酸性阳离子交换柱SP-Sepharose FF两种方法对目的蛋白的分离效果,得到 Sephadex G-50的分离效果更好，并在14.4 kDa处得到单一的溶菌酶条带。     

美洛昔康与溶菌酶作用机制的光谱分析及理论模建研究

为了探究美洛昔康与溶菌酶的作用机制,在pH=7.40的实验条件下,采用荧光光谱、同步荧光光谱和理论模建分析技术研究了类风湿性关节炎药物美洛昔康与溶菌酶分子之间的相互作用。结果表明,美洛昔康能够以静态猝灭形式有效地猝灭溶菌酶的内源荧光,形成1∶1的复合物,并使溶菌酶的构象发生改变。热力学结果表明,美洛昔康-溶菌酶体系的主要作用力类型为疏水作用力。理论模建结果表明,该体系除疏水作用外还存在氢键作用,且美洛昔康被溶菌酶的活性氨基酸残基Glu35和Asp52包围,结合作用改变了溶菌酶催化活性中心处氨基酸残基的微环境。当患者服用15 mg美洛昔康时,美洛昔康与溶菌酶的蛋白结合率W(B)为3.71%~8.79%,说明美洛昔康与溶菌酶的结合对溶菌酶自身抗炎、抗菌功能的影响不大,体系药物结合率W(Q)为1.08%~1.14%,说明溶菌酶与美洛昔康结合不会影响美洛昔康的药效。该研究从理论上证明了溶菌酶在血浆环境中与药物美洛昔康结合后,对溶菌酶本身功能和美洛昔康的药效不会产生严重影响。     

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological‐based specificity of the biomolecule–ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye‐affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α‐chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N‐hexyl pendant chain, with a ligand density of 1.8 × 10^?2 mmol of dye/g of chromatographic support, to isolate lysozyme, α‐chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α‐chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.     

溶液中金属盐对溶菌酶高级结构的影响

探索蛋白质结构稳定性及其淀粉样纤维化的环境条件具有重要意义.本文采用了荧光光谱法研究溶液中的金属盐对鸡卵清溶菌酶内源荧光和淀粉样纤维化的影响.结果表明,金属盐能够增加溶菌酶的热稳定性,减小淬灭剂对内源荧光的作用,对溶菌酶的高级结构具有一定的稳定作用.另一方面,在长时间热胁迫的情况下,金属盐可促进溶菌酶分子的聚集而纤维化.金属盐的这种双重作用分别与其阳离子和阴离子的性质有关.     

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.     

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.