多壁碳纳米管在线提取蛋清溶菌酶的研究
摘要
溶菌酶是一种专门作用于微生物细胞壁的水解酶,广泛应用于食品工业、包装工业、医学临床及生物工程中。随着各种新的生物分离技术的出现,用于溶菌酶分离纯化的方法也愈来愈多,从传统的结晶法、沉淀法、色谱法、离子交换法,到现在采用的新技术如亲和膜色谱、反胶团萃取、双水相萃取等。其中固相萃取法具有回收率高、有机溶剂消耗少、富集倍率高、操作简单、省时、省力、易于自动化等优点,是最常使用的提取蛋清溶菌酶的方法。
溶菌酶是蛋清主要蛋白质中唯一一种碱性蛋白质,在水溶液中带正电荷；碳纳米管经氧化处理后,在水溶液中其表面带负电荷；基于物质之间电荷的相互作用,本文建立了多壁碳纳米管在线提取蛋清中溶菌酶的新方法。将经适当纯化处理后的碳纳米管装入自制的固相萃取柱,对蛋清中溶菌酶进行选择性吸附后,用碳酸盐缓冲溶液进行定量洗脱。本文在顺序注射系统中,对洗脱剂、洗脱剂的pH以及富集流速、洗脱流速、洗脱剂体积等实验参数进行了优化选择,并考察了洗脱剂的离子强度对洗脱结果的影响。实验结果表明,多壁碳纳米管微柱可以有效的定量萃取溶菌酶,富集倍率为12,试样处理量为10个h-1。
对实际样品鸡蛋清进行提取,得到电泳纯度为100%的溶菌酶,100 mL鸡蛋清可提取溶菌酶0.40 g。若蛋清中所含蛋白质按13%计算,该方法所提取的溶菌酶占蛋清中蛋白质总量的3.6%,与标准值相符。与其它提取蛋清溶菌酶的方法相比较,本法具有试剂用量少、提取速度快等优点。此外,由于引用了顺序注射这种在非平衡状态下快速进样的分析技术,使得该法还具有在线、微量、密闭、自动等优点。
Lysozyme is a hydrolytic enzyme widely applied in food industry, packaging industry, medicine and bioengineering. Quite a few isolation and purification approaches are currently available for lysozyme, including crystallization, precipitation, chromatography, ion-exchange, reverse micellar extraction, aqueous two-phase systems. Solid phase extraction (SPE) is a highly efficient simple sample pretreatment technique with advantages of high enrichment factor, low running cost and ease of automation. It is also widely used in the isolation of lysozyme from egg white.
Lysozyme is the only alkali protein in egg white and is positively charged in aqueous solution. Carbon nanotubes's isoelectric point is 5.0 and after oxidation it is negatively charged. A novel method for the extraction of lysozyme from egg white using carbon nanotubes was developed based on the electrostatic interactions. Carbon nanotubes were packed into a SPE microcolumn after appropriate pretreatment, and the microcolumn was incorporated into a sequential injection system which facilitates online selective sorption of lysozyme in egg white. The retained protein was afterwards quantitatively eluted by using carbonate buffer. Various parameters affecting the adsorption and elution process were investigated to achieve optimized purification conditions. The results showed that lysozyme could be efficiently extracted by the microcolume packed with multi-walled carbon nanotubes. An enrichment factor of 12 and a sampling throughput of 10 h-1 were obtained.
The purity of the lysozyme isolated from egg white was 100% and ca.0.4 g lysozyme could be extracted from 100 mL egg white. When considering that 13% of the egg-white is protein species, the isolated lysozyme counts to 3.6% of the total protein mass, which matched well the standard value.
溶菌酶是蛋清主要蛋白质中唯一一种碱性蛋白质,在水溶液中带正电荷；碳纳米管经氧化处理后,在水溶液中其表面带负电荷；基于物质之间电荷的相互作用,本文建立了多壁碳纳米管在线提取蛋清中溶菌酶的新方法。将经适当纯化处理后的碳纳米管装入自制的固相萃取柱,对蛋清中溶菌酶进行选择性吸附后,用碳酸盐缓冲溶液进行定量洗脱。本文在顺序注射系统中,对洗脱剂、洗脱剂的pH以及富集流速、洗脱流速、洗脱剂体积等实验参数进行了优化选择,并考察了洗脱剂的离子强度对洗脱结果的影响。实验结果表明,多壁碳纳米管微柱可以有效的定量萃取溶菌酶,富集倍率为12,试样处理量为10个h-1。
对实际样品鸡蛋清进行提取,得到电泳纯度为100%的溶菌酶,100 mL鸡蛋清可提取溶菌酶0.40 g。若蛋清中所含蛋白质按13%计算,该方法所提取的溶菌酶占蛋清中蛋白质总量的3.6%,与标准值相符。与其它提取蛋清溶菌酶的方法相比较,本法具有试剂用量少、提取速度快等优点。此外,由于引用了顺序注射这种在非平衡状态下快速进样的分析技术,使得该法还具有在线、微量、密闭、自动等优点。
Lysozyme is a hydrolytic enzyme widely applied in food industry, packaging industry, medicine and bioengineering. Quite a few isolation and purification approaches are currently available for lysozyme, including crystallization, precipitation, chromatography, ion-exchange, reverse micellar extraction, aqueous two-phase systems. Solid phase extraction (SPE) is a highly efficient simple sample pretreatment technique with advantages of high enrichment factor, low running cost and ease of automation. It is also widely used in the isolation of lysozyme from egg white.
Lysozyme is the only alkali protein in egg white and is positively charged in aqueous solution. Carbon nanotubes's isoelectric point is 5.0 and after oxidation it is negatively charged. A novel method for the extraction of lysozyme from egg white using carbon nanotubes was developed based on the electrostatic interactions. Carbon nanotubes were packed into a SPE microcolumn after appropriate pretreatment, and the microcolumn was incorporated into a sequential injection system which facilitates online selective sorption of lysozyme in egg white. The retained protein was afterwards quantitatively eluted by using carbonate buffer. Various parameters affecting the adsorption and elution process were investigated to achieve optimized purification conditions. The results showed that lysozyme could be efficiently extracted by the microcolume packed with multi-walled carbon nanotubes. An enrichment factor of 12 and a sampling throughput of 10 h-1 were obtained.
The purity of the lysozyme isolated from egg white was 100% and ca.0.4 g lysozyme could be extracted from 100 mL egg white. When considering that 13% of the egg-white is protein species, the isolated lysozyme counts to 3.6% of the total protein mass, which matched well the standard value.
引文
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