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 stabilizat ion approaches is proposed: (a) Multi-subunit immobilization: a very intens e multi-subunit covalent immobilization has been achieved by performing ver y long immobilization processes between multimeric enzymes and porous suppo rts composed by large internal surfaces and covered by a very dense layer o f reactive groups secluded from the support surface through very short spac er arms. (b) Additional cross-linking with poly-functional macromolecules: additional chemical modification of multi-subunit immobilized derivatives w ith polyfunctional macromolecules promotes an additional cross-linking of a ll subunits of most of multimeric enzymes. A number of homo and hetero-dime ric 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 strateg ies. After such stabilization of the quaternary structure these three featu res were observed: no subunits were desorbed from derivatives after boiling them in SDS, thermal inactivation becomes independent from enzyme concentr ation and derivatives became much more stable than soluble enzymes as well as than non-stabilized derivatives. For example, thermal stability of D-ami no acid oxidase from Rhodotorula gracilis was increased 7.000 fold after st abilization of its quaternary structure. (C) 1999 Elsevier Science B.V. All rights reserved.