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Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila
Author(s) -
Guisán José M.,
Alvaro Gregorio,
FernandezLafuente Roberto,
Rosell Cristina M.,
Garcia Jose L.,
Tagliani Auro
Publication year - 1993
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260420408
Subject(s) - covalent bond , chemistry , enzyme , penicillin amidase , immobilized enzyme , agarose , sodium dodecyl sulfate , combinatorial chemistry , biochemistry , organic chemistry
We have developed a strategy for immobilization–stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose–aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme–support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000‐fold more stable than soluble and one‐point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)]. © 1993 John Wiley & Sons, Inc.