DOI: 10.1002/cctc.201100174 Application of Hierarchical Porous Silica with a Stable Large Porosity for b-Galactosidase Immobilization Claudia Bernal, Ligia Sierra, and Monica Mesa* [a] Introduction The b-galactosidase (E.C. 3.2.1.23), also called lactase, catalyzes the hydrolysis of b-galactosidic linkages present in molecules such as lactose, generating high power reduction sugars such as glucose and galactose. b-Galactosidase (b-gal) has been used for lactose hydrolysis in milk and whey and for the syn- thesis of polysaccharides. However, this soluble enzyme can be used only for a single run in batch reactors, increasing the processing costs. Immobilization on solid supports has allowed its implementation in continuous and batch processes, offering enzymatic stability and the possibility of reuse. [1] The immobili- zation of b-gal by using the physical adsorption method in al- ginate [2] and by forming chemical bonds on agarose modified with different functional groups has been performed. [3–5] The main disadvantage of these organic supports is the low me- chanical resistance, especially under high pressure, at which they lose their characteristics and, therefore, their catalytic effi- ciency in continuous reactors. After the discovery of organized mesoporous silicas in 1992, [6] the synthesis of inorganic materials with controlled porous structures was widely studied. These materials offer some special properties for use as enzyme supports because of their high surface area, thermal and mechanical stability, non-toxicity, and high resistance against microbial attacks and organic solvents. Also, they have adjustable pore sizes, which enable the immobilization of a large variety of biomolecules through hydrogen bonding and electrostatic interactions owing to the existence of surface silanol groups. Some relative- ly small enzymes have been immobilized in these types of silica, [7–12] but an enlargement of their mesopore size is desired for the immobilization of bigger enzymes, such as b-gal from Kluyveromyces lactis (K. lactis), with a diameter of approximate- ly 14 nm and a molecular weight (MW) of approximately 220– 240 kDa, [13] to allow the normal conformational changes during the catalytic process and to avoid lixiviation at the same time. The support pore size, with regard to the enzyme molecule diameter, as well as the pore structure, affects the enzymatic activity and stability. [12, 14–16] The synthesis parameters, such as the type of silica precursor and surfactant, composition of the reaction mixture, silica polycondensation conditions (pH, tem- perature, stirring), and the presence of swelling agents, play an important role in the final pore structure and surface proper- ties of a mesoporous material template using surfactants. [17] For mesoporous silica synthesis using cetyltrimethylammonium bromide (CTAB) as the surfactant, the maximum porosity ob- tained, even in the presence of swelling agents, is below 12 nm. [18–20] Larger pores can be obtained as textural porosity formed by particle aggregation and controlled by the hydroly- sis and condensation rates of silica, [21–22] stirring time, [23] and the surfactant removal procedure. [24] For instance, a modulated polycondensation rate of silicate species, controlled by the hy- drolysis of ethyl acetate (EtAc), leads to silicas with large meso- pores of different sizes. [25, 26] Schulz-Ekloff et al. [25] prepared bi- modal micrometric silica particles with approximately 3 nm and 10–30 nm pore sizes by polycondensation of sodium met- asilicate in the presence of the CTAB surfactant and EtAc. The larger porosity was ascribable to the high CTAB/Na 2 SiO 3 molar ratio, in which the concentration of silicate anions was insuffi- cient to form stable silica walls. Hierarchical porous silica, particles or monoliths, were synthe- sized by polycondensation of sodium silicate in the presence of cetyltrimethylammonium bromide and ethyl acetate at dif- ferent concentrations under hydrothermal conditions. They were used as the support for the immobilization of b-galactosi- dase from Kluyveromyces lactis by adsorption. The enzyme loading capacity (higher than 50 mg g 1 support) and the re- tention ability (lixiviation less than 20 % after 72 h of catalysis) of these supports were explained as a function of the hierarch- ical porosity, mesopore sizes of 10–40 nm, and macropore sizes of 0.07–20 mm and the presence of ionized silanol groups on the surface. The optimum pH value and temperature for the maximum activity of the obtained hybrid biocatalyst were evaluated, indicating that the three-dimensional structure of the lactase was not significantly affected during the immobili- zation process. The stability under extreme conditions was im- proved in comparison with soluble lactase. The porous sup- ports exhibited morphological and porous stability under the immobilization and catalytic processes. These results show that the obtained materials are good candidates for the immobiliza- tion of large enzymes, such as b-galactosidase. [a] C. Bernal, Prof. L. Sierra, Prof. M. Mesa Grupo Ciencia de los Materiales Instituto de Química, Universidad de Antioquia. Cra 53 N. o 61-30, SIU, laboratory 301, Medellin (Colombia), AA 1226 Fax: (+ 574) 2191040 E-mail : mmesacad@gmail.com ChemCatChem 0000, 00, 1 – 8  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1& These are not the final page numbers! ÞÞ