Stabilization of multimeric enzymes via immobilization and post-immobilization techniques

Controlled and directed immobilization plus post-immobilization techniques are proposed to get full stabilization of the quaternary structure of most multimeric industrial enzymes. The sequential utilization of two stabilization approaches is proposed: (a) Multi-subunit immobilization: a very intense multi-subunit covalent immobilization has been achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed by large internal surfaces and covered by a very dense layer of reactive groups secluded from the support surface through very short spacer arms. (b) Additional cross-linking with poly-functional macromolecules: additional chemical modification of multi-subunit immobilized derivatives with polyfunctional macromolecules promotes an additional cross-linking of all subunits of most of multimeric enzymes. A number of homo and hetero-dimeric enzymes has been stabilized by the simple application of multi-subunit immobilization but more complex multimeric enzymes (e.g., tetrameric ones) were only fully stabilized after the sequential application of both strategies. After such stabilization of the quaternary structure these three features were observed: no subunits were desorbed from derivatives after boiling them in SDS, thermal inactivation becomes independent from enzyme concentration and derivatives became much more stable than soluble enzymes as well as than non-stabilized derivatives. For example, thermal stability of D-amino acid oxidase from Rhodotorula gracilis was increased 7.000 fold after stabilization of its quaternary structure. (C) 1999 Elsevier Science B.V. All rights reserved.