Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila

Abstract

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)].

Immobilization of lipases on glyoxyl–octyl supports: Improved stability and reactivation strategies

“…The previous immobilization of the enzymes on supports via covalent linkages may help the refolding step [25][26][27][28]. In fact, if several enzyme-support linkages are established, the refolding may be facilitated because the relative positions of these groups cannot be altered, and those may act as reference points during refolding [29].…”

Use of immobilized lipases for lipase purification via specific lipase–lipase interactions

“…Moreover, this support has been used to yield a very intense enzyme-support multipoint attachment between the enzyme and the support, and a fully inert surface after immobilization. Thus, many enzymes have been stabilized using this technique, e.g., Penicillin G acylase from Escherichia coli [26] and K. citrophila [27], trypsin [28], chymotrypsin [29], alcalase [30], carboxypeptidase A [31], FNR NADP-reductase [32], esterase [33], thermolysin [34], DAAO [35], catalases [36,37], and lipases from different sources [18,38], urokinase [39], l-aminoacylase [40], chitosinase [41]. The final reduction of the immobilized preparations with sodium borohydride permits to have very stable secondary amino bonds and very inert hydroxy groups in the support surface (Fig.…”

Enhancing the thermal stability of lipases through mutagenesis and immobilization on zeolites

“…Concerns with enzyme stability in the presence of organic solvents [1], surfactants [2,3], extreme pH values [3] or high temperatures [5][6][7] have stimulated much research from both academia and industrial R&D scientists [8,9] to provide substantial improvements for many biocatalysts [10,11]. Enzymatic thermal stability is a very desirable property, since reaction rates typically increase exponentially with temperature, up until enzyme denaturing becomes prevalent [12].…”