流加操作与稀释添加耦合辅助高浓度变性蛋白质复性动力学

研究了流加操作与稀释添加耦合辅助高浓度变性还原溶菌酶的复性,建立了相关的过程动力学模型.利用三态复性的过程模型对实验数据进行了拟合,获得了较好的拟合效果.利用模型分析了复性过程的动力学特性,结果表明,两者耦合可在较低的盐酸胍浓度(1 mol•L^-1)存在下使较高浓度的（5 mg•ml^-1）变性溶菌酶获得80%以上的复性收率;并且发现添加剂乙酰胺的辅助复性作用与盐酸胍相同,降低盐酸胍浓度,适当提高乙酰胺浓度即可使聚集体生成速率常数最小,酶的复性收率最大;但甘油与盐酸胍具有协同作用,只有在适当的盐酸胍浓度下添加甘油才可获得理想的复性效果.     

分子动力学模拟HEWL晶体在不同环境中的动力学行为

简要阐述分子动力学模拟的原理及步骤,介绍研究溶菌酶的一般方法和优缺点。在Ubuntu操作系统环境下,利用Gromacs软件和其自带的Gromos96力场,通过分子动力学模拟(MD)鸡蛋清溶菌酶晶体(chicken egg-whitelysozyme,HEWL)溶液,考察真空、水溶液和加入NaCl 3种不同环境条件对溶菌酶晶体构象动力学行为的影响,发现无论从均方根位移(rmsd)、回旋半径、还是从B因子值的轨迹图分析,HEWL在水溶液特别是加入抗衡离子(Na~+,Cl~-)的水溶液的环境下的结构更稳定、合理,与(protein data bank)数据库的真实情况相符。原因是Cl~-与溶菌酶晶体在界面处发生了吸附现象,局部形成溶菌酶-Cl~-复合物,抑制了蛋白-水合物中水分子在相邻水合位置间的跳跃,从而使单晶体在离子液态中更加稳定。模拟结果表明,在pH值6.5,等电位点13.1,总电荷7.999 6的体系下,影响HEWL的吸附位点为123号残基(色氨酸),对从分子水平上解释HEWL晶体的动力学吸附行为具有重要指导意义。     

Isolation of Lysozyme from Hen Egg Albumen by Alcohol-Insoluble Cross-Linked Pea Pod Solid Ion-Exchange Chromatography

ABSTRACT: Albumen from hen eggs was diluted 3-fold with 0.05 M NaCl solution at pH 4.0 and was further treated with 30% ethanol for 8 h. The supernatant (77900 U/mg protein) thus obtained was further diluted (2.5-fold) with distilled water and its pH value was adjusted to 8.0 before being subjected to alcohol-insoluble cross-linked pea pod solid (AICLPPS) ion-exchange chromatography for lysozyme isolation. Results showed that AI-CLPPS ion-exchange chromatography increased the purification to 68-fold with a 72% lysozyme recovery from the starting albumen.     

溶菌酶在水产品防腐保鲜中的应用

溶菌酶作为天然安全的防腐剂广泛应用于水产品加工和贮运过程中。文章概述了溶菌酶的理化性质、作用机理、抑菌特性等,阐述了溶菌酶在水产品冷藏加工中的应用现状,探讨了被修饰的溶菌酶的安全性、溶菌酶发挥作用的最适条件以及溶菌酶纯化程度对其抑菌活性的影响等问题,对溶菌酶复合生物保鲜剂在国内外的应用以及与超高压处理技术、气调包装(MAP)技术协同保鲜进行了概述,并对溶菌酶与环境管理系统(EMS)、辐照处理、高压超声(MS)处理等保鲜技术结合的应用研究做出展望。      

黄原胶/溶菌酶体系相转变及其DSC分析

为探索黄原胶(XG)/溶菌酶(Ly)复合体系在自动酸化条件下相行为及在制备纳米凝胶过程中分子结构变化,采用透光率及热力学分别分析XG/Ly复合体系连续相转变规律及模拟碱和碱热偶合处理过程中XG和Ly的热力学特性。实验表明在连续酸化过程中,XG/Ly复合体系发生共溶体系、可溶性复合物、三维网络结构连续相转变过程,不同XG/Ly质量条件(3∶1、1∶1和1∶3)下发生相互作用的pH点分别为10.46、10.85和10.64,说明Ly可加快XG/Ly体系相转变进程;热力学分析表明制备纳米凝胶过程中XG及Ly原有结构均发生改变。      

DNA Penetration into a Lysozyme Layer at the Surface of Aqueous Solutions

The interactions of DNA with lysozyme in the surface layer were studied by performing infrared reflection–absorption spectroscopy (IRRAS), ellipsometry, surface tensiometry, surface dilational rheology, and atomic force microscopy (AFM). A concentrated DNA solution was injected into an aqueous subphase underneath a spread lysozyme layer. While the optical properties of the surface layer changed fast after DNA injection, the dynamic dilational surface elasticity almost did not change, thereby indicating no continuous network formation of DNA/lysozyme complexes, unlike the case of DNA interactions with a monolayer of a cationic synthetic polyelectrolyte. A relatively fast increase in optical signals after a DNA injection under a lysozyme layer indicates that DNA penetration is controlled by diffusion. At low surface pressures, the AFM images show the formation of long strands in the surface layer. Increased surface compression does not lead to the formation of a network of DNA/lysozyme aggregates as in the case of a mixed layer of DNA and synthetic polyelectrolytes, but to the appearance of some folds and ridges in the layer. The formation of more disordered aggregates is presumably a consequence of weaker interactions of lysozyme with duplex DNA and the stabilization, at the same time, of loops of unpaired nucleotides at high local lysozyme concentrations in the surface layer.     

紫外分光光度法测定蜂蜜中溶菌酶的含量

建立测定蜂蜜中溶菌酶的含量方法。根据溶菌酶的化学性质,采用透析的方法提取蜂蜜中溶菌酶,0.01mol/L pH6.5的PBS透析5d,聚乙二醇浓缩6h后离心,在紫外分光光度计上290nm处检测溶菌酶含量,结果溶菌酶在50～250μg/mL范围内呈良好的线性关系,相关系数R2=0.9999,平均回收率为96.0%,RSD值为2.10%。方法操作简便,结果准确,可作为蜂蜜的质量评价方法之一。     

Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein

Because sea cucumbers lack a well-developed immune system and can ingest pathogenic bacteria together with food, some form of active antibacterial substances must be present in the body for defense. In this study, the cDNA of an i-type lysozyme from the sea cucumber Stichopus japonicus (designated SjLys) was cloned by RT-PCR and RACE PCR techniques. The full length cDNA of SjLys was 713 bp with an open reading frame of 438 bp coding for 145 amino acids. Two catalytic residues (Glu34 and Asp47), conserved in i-type lysozymes, and a highly conserved region near the active site, MDVGSLSCG(P Y)(Y F)QIK, were detected in SjLys. In addition, the domain structure analysis of SjLys showed that it is highly similar to the medicinal leech destabilase, which belongs to a new phylogenetic family of invertebrate lysozymes possessing both glycosidase and isopeptidase activities. To gain insight into the in vitro antimicrobial activities of SjLys, the mature peptide coding region was heterologously expressed in Escherichia coli. The recombinant SjLys protein displayed an inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. A remarkable finding is that the recombinant SjLys exhibited more potent activities against all tested bacterial strains after heat-treating at 100 °C for 50 min. These results indicated that the S. japonicus lysozyme is an enzyme with combined enzymatic (glycosidase) and nonenzymatic antibacterial action.     

Evaluation of the effects of sodium chloride,potassium sorbate,nisin and lysozyme on the probability of growth of Clostridium sporogenes

The aim of this study was to evaluate the combined effect of salt (sodium chloride, 0–8% w/v), sorbate (potassium sorbate, 0–4.5% w/v), nisin (0–500 ppm) and lysozyme (0–500 ppm) on the survival of Clostridium sporogenes as a non‐toxigenic surrogate of Clostridium botulinum in terms of the probability of growth by using a central composite rotatable design. The results indicated that salt and sorbate were the most effective factors in preventing the growth of Cl. sporogenes in high‐moisture (>95%) and low‐acid conditions. The probability of growth of Cl. sporogenes in broth was reduced by combinations of salt and sorbate. Nisin and lysozyme had insignificant effects on the probability of growth of Cl. sporogenes (P > 0.05). Lysozyme individually and in combination with nisin had no inhibitory effect on Cl. sporogenes. Overall, the addition of sorbate and lysozyme may allow the salt concentration to be reduced while preventing growth.     

鸡蛋壳溶菌酶分离纯化工艺研究

以鲜鸡蛋壳为原料,1.0％NaCl(pH3.0,40℃)提取溶菌酶, 724大孔阳离子树脂色谱纯化,10％(NH_4)_2SO_4洗脱,盐析 结晶。用羧甲基纤维素CM52柱色谱分析,显示产品纯度 较高。