Activity modulation and reusability of beta-D-galactosidase confined in sol-gel derived porous silicate glass.
In the present work we applied the sol-gel method to obtain glass lentils entrapping beta-D-galactosidase (beta-Gal) (Ebeta-Gal) within a silicate matrix. The effect of pH, temperature, polarity and salt concentration on the activity of Ebeta-Gal was studied. Apparent kinetic parameters for ortho-nitro-phenyl-beta-D-galactopyranoside hydrolysis catalyzed by Ebeta-Gal (V'max, K'M) were lower compared to the soluble enzyme (Sbeta-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 Ebeta-Gal for the substrate as compared with Sbeta-Gal. At pH<4, Ebeta-Gal stability was higher than that of Sbeta-Gal. Ebeta-Gal could be reused after storage at 4 degrees 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 degrees C for 30 min fully inactivated Sbeta-Gal while Ebeta-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 Ebeta-Gal was resistant to typical technological conditions applied in milk processing that would lead to the unfolding and inactivation of Sbeta-Gal. The results are discussed from the biophysical viewpoint.
Crescimbeni
Marina C
MC
Biofísica Química, Cátedra de Química Biológica, Department Química/ ICTA, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, X5016CAG, Córdoba, Argentina.
Nolan
Verónica
V
Clop
Pedro D
PD
Marín
Graciela N
GN
Perillo
María A
MA
D055162:Biocatalysis; D004795:Enzyme Stability; D005139:Eyeglasses; D005782:Gels / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009598:Nitrophenylgalactosides / Q000737:chemistry; D016062:Porosity; D017510:Protein Folding; D012492:Salts / Q000737:chemistry; D017640:Silicates / Q000737:chemistry*; D013499:Surface Properties; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism*
D005782:Gels; D012492:Salts; D017640:Silicates; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase