Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-beta-glucanase (endo-beta-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilization. The enzyme immobilized in alginate beads showed a K-m value (1.02% w/v) lower than that of the free enzyme (1.31%). The apparent V-max of immobilized cellulase preparations (238.3 mu mol glucose/ml x h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60 degrees C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calcium-alginate beads was 4.6 h at 55 degrees C and 5.4 h at 60 degrees C, while that of the free enzyme was 3.0 h at 55 degrees C and 1.2 h at 60 degrees C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and beta-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250-720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized-beta-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced the hydrolytic efficiency to half.