Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

被引:50
作者
Bolivar, Juan M. [1 ]
Rocha-Martin, Javier [1 ]
Mateo, Cesar [1 ]
Cava, Felipe [2 ]
Berenguer, Jose [2 ]
Vega, Daniel [2 ]
Fernandez-Lafuente, Roberto [1 ]
Guisan, Jose M. [1 ]
机构
[1] CSIC, Inst Catalisis, Dept Biocatalisis, Madrid 28049, Spain
[2] CSIC UAM, Ctr Biol Mol Severo Ochoa, Dept Biol Mol, Madrid 28049, Spain
关键词
Thermophilic enzymes; Multimeric enzymes; Enzyme stabilization; Enzyme immobilization; Protein purification; Oriented immobilization; Multipoint covalent attachment; LARGE PROTEINS; REVERSIBLE IMMOBILIZATION; ADDITIONAL STABILIZATION; ENZYME IMMOBILIZATION; SELECTIVE ADSORPTION; MULTIMERIC ENZYMES; GLYOXYL AGAROSE; HEAVY-METALS; DEHYDROGENASE; STRATEGIES;
D O I
10.1016/j.molcatb.2008.12.010
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme-support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions). This method could be extended to any other multimeric enzyme expressed in mesophilic hosts. (C) 2008 Elsevier B.V. All rights reserved.
引用
收藏
页码:158 / 163
页数:6
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