Betancor Lorena, Hidalgo Aurelio, Fernández-Lorente Gloria, Mateo Cesar, Fernández-Lafuente Roberto, Guisan José M
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain.
Biotechnol Prog. 2003 May-Jun;19(3):763-7. doi: 10.1021/bp025785m.
Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.
牛肝过氧化氢酶被固定在不同的载体上。由于亚基解离，发现这种酶的四聚体性质会导致其在稀释条件下迅速失活，这一事实可能排除其工业用途。使用高度活化的乙醛酸琼脂糖进行多亚基固定不足以使所有酶亚基参与其中。事实上，洗涤衍生物会导致酶活性大幅下降。将先前固定化的酶与特制的葡聚糖醛进行进一步交联，使用固定在高度活化的乙醛酸琼脂糖载体上的多亚基制剂或固定在活化程度较低的乙醛酸琼脂糖上的单亚基酶，都能使多聚体结构完全稳定。使用与葡聚糖醛交联的多亚基固定化衍生物时，观察到最终生物催化剂具有最高的稳定性。最佳衍生物保留了约60%的固定化活性，在SDS存在下煮沸后不释放任何酶亚基，洗涤过程中不失活，其稳定性不依赖于稀释。该衍生物用于10 mM过氧化氢的分解反应10个循环，酶活性没有任何下降。
