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.     

人工伴侣和盐酸胍促进溶菌酶复性的协同效应

研究了表面活性剂十六烷基三甲基溴化铵 (CTAB)加 β -环糊精 (β -CD) (人工伴侣系统 )和盐酸胍对高浓度变性 -还原溶菌酶氧化复性的影响 ,利用基于二态复性模型的表观动力学模型分析了溶菌酶的复性过程 .结果表明 ,存在最佳盐酸胍浓度 ,使酶的复性率最大 ;当盐酸胍浓度一定时 ,提高人工伴侣系统的浓度使复性反应速率常数增大、逆反应速率常数降低 ,从而提高复性收率 ,证明人工伴侣和盐酸胍对促进变性溶菌酶复性具有协同效应 .在最佳盐酸胍浓度和较高浓度的人工伴侣存在下 ,6 0min内可使浓度为 1.0 5mg·ml^-1的变性酶复性收率达到81% ,而相同条件下的自发复性收率仅为 49% .     

高浓度变性-还原溶菌酶的流加复性动力学特性

研究了高浓度变性-还原溶菌酶的流加复性过程动力学特性,重点考察了影响复性速率和复性收率的主要因素,包括盐酸胍浓度、酶浓度和氧化还原剂浓度.结果表明,与直接稀释复性相比,流加操作可有效降低肽链分子间聚集,提高蛋白质的复性收率;在流加复性条件下,随着酶浓度的提高,复性速率和复性收率均有所下降,但适当提高复性液中盐酸胍浓度仍可获得高浓度蛋白质的高复性收率.另外,随着变性酶浓度的提高,需要适当提高复性液中氧化型谷胱甘肽浓度,以加快溶菌酶分子内二硫键的形成,提高复性反应速度.     

海藻糖对变性溶菌酶的促进复性作用及动力学

研究了海藻糖促进变性还原溶菌酶的复性作用,考察了不同盐酸胍浓度下海藻糖浓度对复性收率的影响,并利用表观竞争反应动力学模型分析了海藻糖辅助溶菌酶的复性动力学特性.结果表明,在海藻糖存在下,聚集体生成速率常数k_A明显减小,但复性速率常数k_N变化不大;随海藻糖浓度的增大,k_A先降低后升高,在合适浓度时k_A出现最小值,由于此时k_N/k_A出现最大值,因此复性收率最大.说明海藻糖的主要作用是有效抑制蛋白质分子间聚集,因此在一定范围内提高其浓度可促进蛋白质复性,从而提高复性收率.     

分子伴侣GroEL促进溶菌酶复性动力学

建立了溶菌酶复性的表观动力学模型 ,研究了分子伴侣GroEL促进变性溶菌酶复性的动力学行为 ,包括酶浓度、三磷酸腺苷 (ATP)浓度、GroEL与酶的摩尔比对复性率和复性速率常数的影响 .酶浓度对复性速率常数的影响比对复性率的影响显著 ;酶的复性率和复性速率常数随ATP浓度的增大而提高 ;在蛋白质浓度一定的情况下 ,存在适宜的GroEL和ATP浓度 ,使复性率和复性速率常数最大     

渗透剂对苹果酸脱氢酶包含体的复性作用

将甜菜碱和海藻糖两种渗透剂作为添加剂,考察了其促进苹果酸脱氢酶包含体的复性作用。结果显示,甜菜碱和海藻糖均可有效促进大肠杆菌苹果酸脱氢酶包含体的复性。在本文实验条件下,随添加甜菜碱浓度的升高,复性后苹果酸脱氢酶的比活增大;而海藻糖则出现最佳添加浓度使复性后苹果酸脱氢酶的比活最大;同时在盐酸胍浓度较高、不添加渗透剂时复性效果较差的复性体系中,若添加合适浓度的甜菜碱或海藻糖即可有效促进苹果酸脱氢酶的复性,显示出渗透剂作为添加剂在实际包含体蛋白质复性过程中的应用前景。另外,通过外源荧光分析发现,添加甜菜碱和海藻糖均可降低苹果酸脱氢酶表面疏水性区域的暴露程度;圆二色光谱分析则表明,甜菜碱和海藻糖可以促进苹果酸脱氢酶α-螺旋结构的生成。     

人工伴侣与凝胶过滤色谱耦合作用促进高浓度溶菌酶复性研究

研究了人工伴侣系统,即十六烷基三甲基溴化铵(CTAB)和β 环糊精(β CD)促进高浓度变性 还原溶菌酶的氧化复性。确定CTAB与β CD的浓度比为1∶4,CTAB与变性溶菌酶的结合时间在10—50min内复性收率较高。同时发现,利用人工伴侣和凝胶过滤色谱的耦合作用可有效提高溶菌酶的复性收率,在连续复性操作条件下可使进料质量浓度为1mg/mL的变性酶获得87%的总复性收率。     

β-环糊精促进变性-还原溶菌酶复性

研究了 β -环糊精 (β -CD)和表面活性剂十六烷基三甲基溴化铵 (CTAB)作为复性液添加剂对促进变性 -还原溶菌酶的氧化复性 .β -CD自身具有抑制肽链分子间聚集 ,提高复性率的作用 ,但其相对于变性酶的浓度需达到 30 0 0～ 5 0 0 0倍以上 ,否则作用效果不明显 .与其相比 ,利用CTAB和 β -CD联合作用系统具有更佳的促进复性效果 ,在适宜的盐酸胍浓度存在下可使较高浓度的变性 -还原酶实现完全复性 .确定了CTAB +β -CD系统促进变性酶复性的最佳操作 pH值和温度     

人工伴侣辅助溶菌酶复性的研究

研究了去污剂十门烷基三甲基溴化铵（ＣＴＡＢ）和β－环糊精（β－ＣＤ）联合作用（人工伴侣系统），对变性－还原溶菌酶的复性作用。考察了操作条件对溶菌酶复性收率的影响。结果表明，在人工伴侣系统辅助下，６０ｍｉｎ可使变性溶菌酶的复性收率达８９％，自发复性仅为２７％；并且其最佳复性温度为３７℃，最佳ｐＨ值为７￣９；同时发现，单独使用环糊精也可在一定程度上辅助溶菌酶复性，复性收率为４６％。     

CTAB辅助溶菌酶复性过程动力学

研究了溶菌酶在十六烷基三甲基溴化铵（CTAB）溶液中的复性过程,通过测定溶液表面张力与酶活力的变化证实变性溶菌酶首先与CTAB形成复合物,进而在氧化-还原剂作用下开始复性并与CTAB发生解离.非还原型SDS-PAGE分析结果表明,复性反应的主要产物有三类：具有天然结构的溶菌酶单体、溶菌酶多聚体及溶菌酶单体与CTAB形成的无活性复合物.不同产物的含量取决于溶液中CTAB与溶菌酶的摩尔比.当变性溶菌酶浓度为0.1～0.4mg•ml^-1,CTAB与溶菌酶摩尔比为5～20时,采用“变性-复性”二态复性模型分析了CTAB辅助溶菌酶复性过程的宏观动力学.结果表明,随CTAB与溶菌酶摩尔比的增大,变性反应的速率常数显著增大,而折叠反应的速率常数先增大后减小,CTAB与溶菌酶的摩尔比为10时,复性率最高.而蛋白质浓度提高则导致折叠反应速率常数减小,变性反应速率常数增大,复性率缓慢下降.     

Refolding of proteins in a CSTR

A model to predict refolding of proteins in a continuous stirred tank reactor (CSTR) was developed and compared to a batch refolding process with simple dilution of the protein in a stirred tank reactor. For experimental verification of the model a continuous refolding of a model protein (α-lactalbumin) was performed in a CSTR. The refolding process of denatured and fully reduced α-lactalbumin could be accurately predicted by a set of differential equations assuming a first order reaction rate for folding and a second order reaction rate for aggregation. The system composed of a CSTR with an additional diafiltration circuit for removal of denaturing agents from the feed solution and to maintain constant refolding conditions. Based on the folding kinetic the dynamic behavior of such a continuous refolding reactor was simulated under different operating conditions. It was shown that the refolding efficiency was higher compared to batch dilution under certain conditions, namely high residence times. The yield of refolded protein could further increased by recycling the outlet stream containing unfolded protein to the reactor entrance.     

酰胺类物质辅助变性碳酸脱水酶复性及动力学

研究了酰胺类物质对促进变性牛碳酸脱水酶（CAB）的复性作用,考察了在较低盐酸胍浓度下添加剂浓度对复性收率的影响,并利用三级反应动力学方程对酰胺类化合物辅助CAB复性的动力学过程进行了深入分析.结果表明,含有酰胺基团的多种添加剂均能有效促进CAB复性;酰胺化合物与盐酸胍的作用相似,即复性速率常数k_N值均大于无添加剂时的自发复性,且随添加剂浓度增加而降低;但聚集体生成速率常数k_A值则在最佳添加剂浓度下出现最小值,从而使k_N/k_A值最大,复性收率最大.     

水溶性醇类对溶菌酶空间结构和折叠过程的影响

利用多角度激光光散射仪和圆二色谱仪研究小分子水溶性一元醇和多元醇对溶菌酶分子的稳定性以及对变性溶菌酶的空间构象和折叠复性的影响.发现在一元醇和二元醇溶液中溶菌酶分子的稳定性均随着醇浓度的增大而提高,而变性酶分子在醇溶液中形成过多的α螺旋和β折叠等二级结构不利于酶的折叠复性;只有较低浓度的多元醇和甲醇有辅助变性溶菌酶复性的作用,其中10％甲醇、20％甘油和30％乙二醇有利于促进酶的复性.     

The role of net charge on the renaturation of reduced lysozyme by the sulfhydryl-disulfide interchange reaction

Reduced and acetylated lysozymes are basic proteins. When theiramino groups were variously acetylated and then renatured bysulfhydryl-disulfide (SH-SS) interchange reaction at pH 8.0,the final folding yield decreased as the number of positivecharges decreased. The final folding yield of native and Ac1lysozyme, with one positive charge eliminated, was less sensitiveto increasing protein concentration than that of Ac2 lysozyme,where two positive charges had been eliminated. The final foldingyield of reduced Ac2 lysozyme increased in the presence of 1M urea, which reduced the aggregation of unfolded lysozyme.Thus, the aggregation of unfolded lysozymes, which leads toa decrease in the final folding yield, was found to be heavilydependent on their net charges. Moreover, the final foldingyield of reduced lysozyme was shown to be increased by use ofcystamine as an oxidizing reagent in comparison with 2-hydroxyethyldisulfide or dithiodiglycolic acid. This may support the ideathat the final folding yield is influenced by electrostaticinteraction between unfolded lysozymes in the early stage ofrenaturation. In contrast, the concentration dependency of thefinal folding yield of Ac1 lysozyme was different from thoseof carboxymethylated His15 and Asp106 lysozymes whose positivenet charges were similar to that of Ac1 lysozyme. On the basisof the observations, it is suggested that the formation of theaggregates in the renaturation process might also be affectedby the structure of the unfolded state of lysozyme in solution.     

Mechanistic studies of protein refolding facilitated by like-charged polymers

The mechanism of lysozyme refolding facilitated by like-charged polymers was studied using cationic polyelectrolytes of different molecular weights and structures. Lysozyme refolding yield increased with increasing the total charge ratio (R) of the charged polymer to lysozyme in the refolding solution till reaching a plateau at a critical total charge ratio (R_c). The same R_c was observed for different polymers. Similarly, there was a critical minimum polymer molecular weight (M_c) for the facilitated protein refolding, below which the refolding yield decreased. The refolding yield was independent of the charge group structures in polymer. Fluorescence spectroscopy revealed that the polymers had no effect on the protein folding kinetics. Two physical models were proposed to explain the mechanism of the facilitated refolding and the meanings of R_c and M_c. The facilitating effect was attributed to the electrostatic interaction-induced oriented alignment of multiple protein molecules near the polymer chains, which maximizes the electrostatic repulsion between neighboring protein molecules, leading to the inhibition of protein aggregation. The studies provided insight into the mechanism of like-charged polymer-facilitated protein refolding, which would help develop more efficient polymers/particles for facilitated protein refolding applications.     

胆固醇配基色谱在蛋白质复性中的应用

以三聚氯氰为连接臂,采用两步法将疏水配基胆固醇修饰到交联琼脂糖（Sepharose）介质上,构建了胆固醇配基色谱。以溶菌酶（Lys）为模型蛋白,考察了胆固醇配基色谱的复性效果,并研究了流速、上样质量浓度和上样量对色谱复性效果的影响,确定了溶菌酶复性的最佳方法。研究结果表明,当溶菌酶上样量为1.00 mL,介质含0.25 mg溶菌酶时,在0.02 mL/min操作流速下进行吸附-洗脱复性,其最终蛋白收率和活性收率分别为96.3%和91.4%,有效促进了溶菌酶的复性。