Immobilization of functionally unstable catechol-2,3-dioxygenase greatly improves operational stability

Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearoth ermophilus has been immobilized on highly activated glyoxyl agarose beads, The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a hi gh retention of activity (around 80%). Enzyme immobilized under these condi tions showed little increase in thermostability compared with the soluble e nzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibitin g a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentiall y independent of protein concentration whereas free enzyme was rapidly inac tivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catech ol 2,3-dioxygenases at protein concentrations of 10 mu g/ml. Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully in activated and enhanced the resistance to inactivation during catalysis. The se results suggest that the immobilization of the enzyme under controlled c onditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and inc reased the rigidity of the subunit structures. (C) 2000 Elsevier Science In c. All rights reserved.