Departamento de Biocatálisis. ICP-CSIC, C/Marie Curie 2, Campus UAM-CSIC Cantoblanco, 28049 Madrid. Spain; Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto 14040-901, SP, Brazil.
Departamento de Biocatálisis. ICP-CSIC, C/Marie Curie 2, Campus UAM-CSIC Cantoblanco, 28049 Madrid. Spain.
Int J Biol Macromol. 2024 Apr;263(Pt 2):130403. doi: 10.1016/j.ijbiomac.2024.130403. Epub 2024 Feb 27.
Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.
固定化酶在氨化载体上使用戊二醛化学可能涉及三种不同的相互作用，阳离子、疏水和共价相互作用。为了尝试了解这种杂功能性的影响，我们研究了黑曲霉β-半乳糖苷酶在氨化载体（MANAE）和带有一个（MANAE-GLU）或两个戊二醛分子的氨化载体（MANAE-GLU-GLU）上的物理吸附。为了消除戊二醛的化学反应性，使用硼氢化钠还原载体。在酶吸附后，使用不同的 NaCl 浓度、Triton X100、离子型洗涤剂（SDS 和 CTAB）或不同的温度（4°C 至 55°C）从载体上释放酶。使用 MANAE 载体，在 0.3 M NaCl 下几乎所有固定化酶都被释放。使用 MANAE-GLU，在 0.3 M 和 0.6 M NaCl 下得到了类似的结果。然而，在 1 M 或 2 M NaCl 下孵育时，许多酶分子没有从载体上释放出来。对于 MANAE-GLU-GLU 载体，没有一种测试的 NaCl 浓度足以释放与载体结合的所有酶。只有使用高温、0.6 M NaCl 和 1% CTAB 或 SDS 才能从载体上释放出全部蛋白质。本文的结果证实了用戊二醛修饰的氨化载体的杂功能性。
