珍禽蛋溶菌酶制备工艺优化及稳定性研究

溶菌酶广泛存在于动、植物及微生物中,以鸡蛋蛋清中含量最高.珍禽蛋蛋清中溶菌酶酶活力远高于普通鸡蛋中溶菌酶酶活力,更适宜作为优质溶菌酶生产来源.文中以珍禽蛋与阳离子交换树脂为原料,建立一条适合工业化生产的珍禽蛋溶菌酶的制备工艺,研究主要分为以下几个方面：溶菌酶提取原料及树脂型号的选用,高原乌鸡鸡蛋溶菌酶制备工艺的建立,高原乌鸡鸡蛋溶菌酶质量评价.     

矽肺肺泡巨噬细胞溶菌酶的促成纤维细胞增殖作用

溶菌酶(MW～14KD)是一种水解细菌细胞壁多糖的水解酶.危害严重的职业病矽肺患者血清溶菌酶显著升高.本文报道,采用高效液相色谱技术,包括凝胶过滤柱层析和反相C-4高效液相色谱柱层析,从实验性矽肺大鼠的肺泡灌洗液(BALF),肺泡巨噬细胞裂解液(AME)及其条件培养液(AMCM)中,分离纯化了一种可诱导的巨噬细胞来源的细胞因子(iSPMF-P 15),其分子量约为15.4KD,它具有较明显的刺激成纤维细胞增殖的作用.因此,矽肺肺泡巨噬细胞来源的溶菌酶可能是矽肺纤维化病变中的一种致纤维化的细胞因子.     

Adaptive evolution in the stomach lysozymes of foregut fermenters

The convergent evolution of a fermentative foregut in two groups of mammals offers an opportunity to study adaptive evolution at the protein level. The appearance of this mode of digestion has been accompanied by the recruitment of lysozyme as a bacteriolytic enzyme in the stomach both in the ruminants (for example the cow) and later in the colobine monkeys (for example the langur). The stomach lysozymes of these two groups share some physicochemical and catalytic properties that appear to adapt them for functioning in the stomach fluid. To examine the basis for these shared properties, we sequenced langur stomach lysozyme and compared it to other lysozymes of known sequence. Tree analysis suggest that, after foregut fermentation arose in monkeys, the langur lysozyme gained sequence similarity to cow stomach lysozyme and evolved two times faster than the other primate lysozymes. This rapid evolution, coupled with functional and sequence convergence upon cow stomach lysozyme, could imply that positive darwinian selection has driven about 50% of the evolution of langur stomach lysozyme.     

盐酸溶液中溶菌酶对Q235钢的缓蚀作用

运用失重法、动电位极化曲线法、交流阻抗法研究了溶菌酶在盐酸溶液中对Q235钢的缓蚀作用和吸附行为,并用扫描电子显微镜(SEM)观察腐蚀表面的形貌.结果表明,溶菌酶是一种缓蚀效果显著的混合型缓蚀剂,缓蚀效率受温度影响较小,吸附行为属于自发的单分子层化学吸附,符合Langmuir等温吸附式.     

离子交换色谱流动相组成对溶菌酶复性的影响

目的 研究了复性缓冲液组成对还原变性溶茵酶复性的影响。方法用离子交换色谱法对还原变性溶菌酶进行复性。结果低离子强度的Tris缓冲液比高离子强度的PBS缓冲液能明显提高复性收率；当复性缓冲液中不合其他种类的盐时，脲浓度为2．0mol／L时复性产率最高；当脲浓度低时，复性缓冲液中含有氯化钠和硫酸铵的复性效果均不如不合这些盐的复性效果；当脲浓度高时，硫酸铵能很好地提高溶菌酶的复性回收率。结论认为Hofmeister效应是造成这些现象的主要原因。     

铜(Ⅱ)促进溶菌酶的选择性水解切割反应

采用聚丙烯酰胺凝胶电泳技术,研究了中性条件下CuC l2与鸡蛋清溶菌酶的选择性水解切割反应。研究结果表明,在近中性条件下,CuC l2可以高效且有选择地切割溶菌酶。提高CuC l2对溶菌酶的摩尔比、温度和增加恒温时间均可显著增加切割效率,但过高的温度和配比反而会降低切割的选择性。Cu2+对溶菌酶的切割没有缓冲效应,而且Cu2+对溶菌酶的选择性切割位点与溶菌酶的二级结构有关。     

Synergistic actions of complement and lysozyme in clearance of Escherichia coli from amphioxus Branchiostoma belcheri

Amphioxus appears lacking free circulating blood cells. How it clears invading pathogens from its body remains unknown to date. We demonstrate here that amphioxus Branchiostoma belcheri is capable of efficiently eliminating the invading bacterium Escherichia coli from its humoral fluid, and the complement and lysozyme are both involved in the elimination of the invading pathogen. Both the complement and lysozyme act in concert against the invading bacterium, but the complement appears playing a more dominant role than the lysozyme.     

Expression of recombinant human lysozyme in the milk of transgenic mice

Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal of improving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic mice was 0.2mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.     

溶菌酶标准酶活曲线测定方法的改进

天然溶菌酶活性（标准酶活）的测定是蛋白质复性研究过程中的一个关键步骤。为了增加测定数据的准确性和可比性,改进了测定标准酶活的方法。在固定其他条件的前提下,对于影响标准酶活的重要因素-温度,采用空调控制室温的方法,使其规律性由低到高变化。同时将测活液放置在与所控制室温相同的水浴环境中保持温度,测出各种温度下曲线的斜率,用直线进行拟合。对于其他没有测定的温度点,可以从拟合曲线中求出。这样,通过一组实验便可以得到任意温度下的标准酶活。     

溶菌酶标准酶活曲线测定方法的改进

天然溶菌酶活性(标准酶活)的测定是蛋白质复性研究过程中的一个关键步骤.为了增加测定数据的准确性和可比性,改进了测定标准酶活的方法.在固定其他条件的前提下,对于影响标准酶活的重要因素-温度,采用空调控制室温的方法,使其规律性由低到高变化.同时将测活液放置在与所控制室温相同的水浴环境中保持温度,测出各种温度下曲线的斜率,用直线进行拟合.对于其他没有测定的温度点,可以从拟合曲线中求出.这样,通过一组实验便可以得到任意温度下的标准酶活.     

螺旋藻基因组DNA制备方法的摸索与比较

为了得到高质量的螺旋藻基因组DNA,采用超声波法和溶菌酶法两种方法提取基因组DNA,并对实验方法进行了摸索与比较。结果表明:影响螺旋藻基因组DNA制备质量和得率的重要因素之一是螺旋藻细胞壁的破壁程度。本实验中采用两种破壁方法均可得到高质量高得率的螺旋藻基因组DNA,但超声波法破壁不易掌控,而适当浓度的溶菌酶法可获得较理想的结果。     

偶氮类食品着色剂诱惑红与蛋溶菌酶的相互作用研究

采用荧光光谱、紫外吸收光谱和计算机模拟分子对接研究了偶氮类食品着色剂诱惑红与蛋溶菌酶的相互作用.讨论了诱惑红对蛋溶菌酶荧光的猝灭机理,属于静态猝灭.测定了诱惑红与蛋溶菌酶反应体系的结合常数和结合位点数,根据热力学参数焓变(ΔH>0)和熵变(ΔS>0)可推断诱惑红和蛋溶菌酶的相互作用力为典型的疏水作用力.在298、304、310 K温度下,两者间的结合常数分别为17897、19865、23267.     

Meso-四(对-羟基苯基)卟啉和溶菌酶的相互作用机理

在模拟生理条件下, 利用荧光光谱和同步荧光光谱法研究Meso-四(对-羟基苯基)卟啉(THPP)与溶菌酶的相互作用. 结果表明: THPP对溶菌酶的猝灭过程为静态猝灭; 291 K时, THPP与溶菌酶的结合常数为5.97×10⁴ L/mol; 疏水作用力为THPP与溶菌酶的主要作用力; 金属离子Fe²⁺,Fe³⁺,Cu²⁺,Mg²⁺,Ca²⁺和Zn²⁺的存在不影响THPP与溶菌酶的结合常数; THPP与溶菌酶的结合距离为3.35 nm; THPP可使色氨酸残基周围的极性减弱, 疏水性增强.     

Monoclonal suppressor T-cell factor displaying V H restriction and fine antigenic specificity

The production of stable T-cell clones is essential for the study of T-cell-derived, specific immunoregulatory products and of specific T-cell receptors. T-cell clones have been established by radiation leukaemia virus (RadLV)-induced transformation of suppressor T lymphocytes specific for hen egg white lysozyme (HEL). We report here that culture supernatant obtained from these T-cell clones can, when injected into mice, specifically suppress the anti-HEL antibody response. This monoclonal T-cell product suppresses the antibody response induced by HEL and human lysozyme, but not that induced by ring-necked pheasant egg white lysozyme (REL), thus displaying fine antigenic specificity probably restricted to an epitope involving phenylalanine at amino acid residue 3, present in the N-terminal region of HEL and shared by human lysozyme but absent in REL. The suppression induced by this monoclonal T-cell product is restricted by both H-2 and Igh-1 genes whereas anti-HEL antibodies bearing a predominant idiotype are induced in all mice strains tested, irrespective of their H-2 haplotype or Igh-1 allotype.     

反胶团法提取溶菌酶的研究

本文对AOT/异辛烷反胶团用于从鸡蛋清中萃取溶菌酶进行了研究.对商品AOT、精制AOT和重复使用的AOT加以比较后发现未经提纯处理的商品AOT对萃取虽有影响,但仍具实用性.AOT/异辛烷反胶团系统能有效地保护溶菌酶的活性.此外,对AOT浓度、水相pH值、离子强度等对萃取过程的影响也作了讨论.     

氯化镁促进溶菌酶淀粉样聚集的分子机制研究

高浓度氯化镁能够在一定的条件下促进溶菌酶发生聚集,但其具体的作用机制尚不明确.采用分子生物学实验和分子动力学模拟相结合的方法研究了鸡溶菌酶在不同浓度的MgCl2溶液中的聚集特性和结构稳定性.分子生物学实验结果表明,200 mmol/L MgCl2能够显著促进鸡溶菌酶形成二聚体和三聚体,但效果没有NaCl显著.分子动力学模拟分析显示,200 mmol/L MgCl2能够引起螺旋3和螺旋4的显著偏移,同时能够减弱溶菌酶聚集关键区域（40-110位残基）内二硫键Cys76-Cys94的结合强度;另一方面,MgCl2也能减少疏水核心内部氢键的数量,促使疏水核心的稳定性显著降低,从而促进了溶菌酶分子间的相互作用.     

氯化钠促进溶菌酶聚集机制的分子动力学研究

氯化钠能够在一定的条件下促进溶菌酶发生聚集,但其具体的作用机制目前还尚不明确.采用分子动力学模拟的方法研究了鸡溶菌酶单体在含500 mmol/L NaCl和不合NaCl两种体系中的结构变化.模拟的结果表明:NaCl能够使溶菌酶聚集关键区域(40～110位残基)内二硫键cys76-cys94的结合强度减弱,同时破坏了该区...     

Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme

Measurements of changes in structure and stability caused by 13 different substitutions for threonine 157 in phage T4 lysozyme show that the most stable lysozyme variants contain hydrogen bonds analogous to those in the wild-type enzyme and that structural adjustments allow the protein to be surprisingly tolerant of amino-acid substitutions.     

Substantial increase of protein stability by multiple disulphide bonds

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.