Activity modulation and reusability of Beta-D-Galactosidase confined in a sol-gel derived porous silicate

MARINA C. CRESCIMBENI; VERONICA M. NOLAN; PEDRO D. CLOP; GRACIELA N. MARIN; MARIA A. PERILLO

COLLOIDS AND SURFACES B-BIOINTERFACES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2010 vol. 72 p. 387 - 396

0927-7765

In the present work we applied the sol–gel method to obtain glass lentils entrapping Beta-D-galactosidase (B-Gal) (EB-Gal) within a silicate matrix. The effect of pH, temperature, polarity and salt concentration on the activity of EB-Gal was studied. Apparent kinetic parameters for ortho-nitro-phenyl-d-galactopyranoside hydrolysis catalyzed by EB-Gal (V´max, K´M) were lower compared to the soluble enzyme (SB-Gal), reflecting the solute diffusion restriction imposed by the matrix observed in the time curves, a partial protein inactivation upon encapsulation, and an improvement in the affinity of EB-Gal for the substrate as compared with SB-Gal. At pH < 4, EB-Gal stability was higher than that of SB-Gal. EB-Gal could be reused after storage at 4 °C for up to 90 days, and retained its activity profile within the range of pH = 2–10 and saline concentration 0–400 mM. Pre-incubation at 75 °C for 30 min fully inactivated SB-Gal while BB-Gal retained approximately 90% of its activity, even in the reused samples. Encapsulation did not introduce additional impairments to the reaction rate measured in heterogeneous dispersions, beyond those derived from their own particle-crowded environment. This reusable EB-Gal was resistant to typical technological conditions applied in milk processing that would lead to the unfolding and inactivation of SB-Gal. The results are discussed from the biophysical viewpoint.