STABILIZATION OF HETERODIMERIC ENZYME BY MULTIPOINT COVALENT IMMOBILIZATION - PENICILLIN-G ACYLASE FROM KLUYVERA-CITROPHILA

We have developed a strategy for immobilization-stabilization of penic illin G acylase (PGA) from Kluyvera citrophila by controlled multipoin t covalent attachment to agarose-aldehyde gels. This enzyme is compose d by two dissimilar subunits noncovalently bound. Thus, in this articl e 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 supp ort. We have demonstrated that important thermal stabilizations of der ivatives were only obtained through a very intense enzyme-support mult ipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activ ity and being more than 1000-fold more stable than soluble and one-poi nt attached enzyme. In addition, the involvement of the two subunits i n 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 unfol ding with urea and sodium dodecyl sulfate (SDS)]. (C) 1993 John Wiley & Sons, Inc.