Nicolás Paula, Lassalle Verónica L, Ferreira María L
Planta Piloto de Ingeniería Química (PLAPIQUI-UNS-CONICET), Universidad Nacional del Sur, Camino La Carrindanga km 7, 8000, Bahía Blanca, Argentina.
Instituto de Química del Sur (INQUISUR-UNS-CONICET), Avda. Alem 1253, 8000, Bahía Blanca, Argentina.
Enzyme Microb Technol. 2017 Feb;97:97-103. doi: 10.1016/j.enzmictec.2016.11.009. Epub 2016 Nov 25.
The aim of this manuscript was to study the application of a new method of protein quantification in Candida antarctica lipase B commercial solutions. Error sources associated to the traditional Bradford technique were demonstrated. Eight biocatalysts based on C. antarctica lipase B (CALB) immobilized onto magnetite nanoparticles were used. Magnetite nanoparticles were coated with chitosan (CHIT) and modified with glutaraldehyde (GLUT) and aminopropyltriethoxysilane (APTS). Later, CALB was adsorbed on the modified support. The proposed novel protein quantification method included the determination of sulfur (from protein in CALB solution) by means of Atomic Emission by Inductive Coupling Plasma (AE-ICP). Four different protocols were applied combining AE-ICP and classical Bradford assays, besides Carbon, Hydrogen and Nitrogen (CHN) analysis. The calculated error in protein content using the "classic" Bradford method with bovine serum albumin as standard ranged from 400 to 1200% when protein in CALB solution was quantified. These errors were calculated considering as "true protein content values" the results of the amount of immobilized protein obtained with the improved method. The optimum quantification procedure involved the combination of Bradford method, ICP and CHN analysis.
本手稿的目的是研究一种蛋白质定量新方法在南极假丝酵母脂肪酶B商业溶液中的应用。证明了与传统考马斯亮蓝法相关的误差来源。使用了八种基于固定在磁铁矿纳米颗粒上的南极假丝酵母脂肪酶B（CALB）的生物催化剂。磁铁矿纳米颗粒用壳聚糖（CHIT）包覆，并用戊二醛（GLUT）和氨丙基三乙氧基硅烷（APTS）进行改性。随后，CALB吸附在改性载体上。所提出的新型蛋白质定量方法包括通过电感耦合等离子体原子发射（AE-ICP）测定（CALB溶液中蛋白质的）硫含量。除了碳、氢和氮（CHN）分析外，还应用了四种不同的方案将AE-ICP与经典考马斯亮蓝法相结合。当对CALB溶液中的蛋白质进行定量时，以牛血清白蛋白为标准使用“经典”考马斯亮蓝法计算的蛋白质含量误差范围为400%至1200%。这些误差是将改进方法获得的固定化蛋白质含量结果视为“真实蛋白质含量值”计算得出的。最佳定量程序涉及考马斯亮蓝法、ICP和CHN分析的组合。
