Technical University Dortmund, Faculty of Biochemical and Chemical Engineering, Laboratory of Plant and Process Design, Emil-Figge-Str. 70, 44227 Dortmund, Germany.
J Biotechnol. 2012 Oct 15;161(2):138-46. doi: 10.1016/j.jbiotec.2012.01.030. Epub 2012 Feb 3.
Purification and separation of proteins play a major role in biotechnology. Nowadays, alternatives to multistep operations suffering from low product yields and high costs are investigated closely amidst which one of the promising options is foam fractionation. The molecular behavior at the gas-liquid interface plays an important role in the formation and stabilization of enriched foam. This study for the first time correlates the physico-chemical parameters to the molecular structure in view of protein enrichment during foam fractionation of the two relatively different proteins lysozyme and β-casein employing biophysical techniques such as circular dichroism (CD) spectroscopy and infrared reflection absorption spectroscopy (IRRAS). In case of lysozyme, high enrichment was achieved at pH<pI in contrast to current opinion. This is due to partial unfolding and aggregation of the lysozyme molecules under favorable foaming conditions that resulted with high enrichment of foamed protein. Under these favorable conditions, CD spectra and IRRA spectra show that the unfolding of lysozyme is partially irreversible. However, the unfavorable foaming conditions, giving low enrichment, promote only minor structural changes and these changes are fully reversible. In case of β-casein, no pronounced unfolding can be observed using CD spectroscopy and IRRAS. The β-casein molecules adsorb and purely reorient at the gas-liquid interface, depending on favorable or unfavorable conditions.
蛋白质的纯化和分离在生物技术中起着重要作用。如今，人们正在深入研究替代多步操作的方法，这些方法的缺点是产率低、成本高，其中一种很有前途的选择是泡沫分离。在富氧泡沫的形成和稳定过程中，分子在气液界面上的行为起着重要作用。本研究首次采用圆二色性（CD）光谱和红外反射吸收光谱（IRRAS）等生物物理技术，将物理化学参数与分子结构相关联，研究了两种相对不同的蛋白质溶菌酶和β-酪蛋白在泡沫分离过程中的蛋白质浓缩情况。对于溶菌酶，与目前的观点相反，在 pH<pI 的情况下实现了高浓缩。这是由于溶菌酶分子在有利的起泡条件下部分展开和聚集，导致泡沫蛋白的高浓缩。在这些有利条件下，CD 光谱和 IRRA 光谱表明溶菌酶的展开部分是不可逆的。然而，低浓缩的不利起泡条件只促进了较小的结构变化，这些变化是完全可逆的。对于β-酪蛋白，CD 光谱和 IRRAS 都没有观察到明显的展开。β-酪蛋白分子在气液界面上吸附并根据有利或不利的条件重新定向。
