Purified endoglucanase (apparently to homogeneity) from Arachniotus citrinus was immobilized in polyacrylamide gel with 53.4% yield of immobilization. Immobilization improved the pH optimum from 4.9 (free) to 5.6 (immobilized) and temperature optimum by 10degreesC. The V-max of immobilized endoglucanase at 70 and 90degreesC were 1.4- and 3.0-fold improved over its free counterpart. Immobilized endoglucanase retained about 30% residual activity at 90degreesC while its soluble counterpart retained only 6% of its original activity. Similarly there was highly significant impact on improvement of k(cat), k(cat)/K-m and DeltaG(E-S) values. The immobilized endoglucanase remained entrapped in the gel and did not leak out even after five cycles of repeated applications and exhibited the same conformation even after 25 days of the sixth application. The immobilized endoglucanase exhibited almost the same V-max, K-m, k(cat), specificity constant values in all six applications. Immobilization improved the quality of endoglucanase by lowering the energy of activation from 27.9 to 11.4 kJ mol(-1), confirming thermal stabilization by immobilization. The results of thermodynamic analysis for CMC hydrolysis indicated that the immobilized enzyme required the same amount (-23.4 kJ mol(-1)) of free energy (DeltaG(E-T)) to form the transition state. Similarly the same amount (3 +/- 0.12 kJ mol(-1)) of substrate binding energy (DeltaG(E-S)) remained constant during all applications. The melting temperature of the immobilized enzyme improved from 60 to 70degreesC and was in good agreement to values for thermostable enzymes. Irreversible thermal denaturation of soluble and immobilized endoglucanases indicated that immobilization significantly decreased entropy and enthalpy of deactivation and made the immobilized endoglucanase thermodynamically more stable. (C) 2004 Elsevier Ltd. All rights reserved.