Highly Efficient “Tight Fit” Immobilization of α‐Chymotrypsin in Mesoporous MCM‐41: A Novel Approach Using Precursor Immobilization and Activation

Abstract

The zymogen alpha-chymotrypsinogen A is bound to mesoporous silica MCM-41 with a protein loading of 170 mg/g solid (MCM-Z) by a simple stirring in aqueous tris-HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain alpha-chymotrypsin immobilized on MCM-41 (MCM-E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available alpha-chymotrypsin (MCM-E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM-E.I) has been used for resolution of N-acetyl-dl-amino acid esters and racemic trans-4-methoxy-3-phenylglycidic acid (PGA) methyl ester.

Nanostructures for enzyme stabilization

“…First, the pore size of mesoporous materials affects the adsorption behavior and leaching of enzymes (Diaz and Balkus, 1996;Takahashi et al, 2000Takahashi et al, , 2001Yiu et al, 2001;Fadnavis et al, 2003;Fan et al, 2003b;Jie et al, 2004, Vinu et al, 2004b. As explained above, larger enzymes than the pore size of mesopores cannot be adsorbed into mesoporous materials.…”

Effect of pore diffusional resistance on biocatalytic activity of Burkholderia cepacia lipase immobilized on SBA-15 hosts

“…Immobilization processes employing adsorption on porous glass beads (Marlot et al, 1985), ion exchange resins, alumina (Brady et al, 1986), silica (Brady et al, 1988), (bio) polymers (Gitlesen et al, 1997;Yasuda et al, 2002) have been applied to lipases. More recently, novel ordered mesoporous silicas have been investigated as potential hosts for lipase immobilization (Fadnavis et al, 2003;Takahashi et al, 2001;Thomas, 1999). Mesoporous materials are characterized by high specific surface areas (up to about 1500 m 2 /g) and pore volumes (up to about 1.5 cm 3 /g), making them ideal candidates as immobilization hosts.…”

Synthesis and Functionalization of Ordered Large‐Pore Mesoporous Silica Nanoparticles for Biomedical Applications

“…The pore dimension (7 nm) of the as‐synthesized SBA‐15 MSNs is sufficient for adsorbing a large variety of important biomolecules , . The narrow pore size distribution of the nanoparticles can also be favorable for adsorbing enzymes possessing a comparable size, as there appears to be evidence indicating that mesopores matching the size of encapsulated enzymes can enhance enzyme activity compared to hosts with pore sizes larger than the enzyme . In addition, 2D hexagonal pore systems confined in small particles result in short channel lengths, thus, facilitating mass transfer in the mesostructure that can be favorable for immobilizing and releasing proteins , .…”