One-step preparation of bioactive enzyme/inorganic materials

Simultaneous exfoliation of crystalline alpha-zirconium phosphate (alpha-ZrP) nanosheets and enzyme binding, induced by shearing, without the addition of any toxic additives is reported here for the first time. These materials were thoroughly characterized and used for applications. The bulk alpha-ZrP material (20 mg mL(-1)) was exfoliated with low concentrations of a protein such as bovine serum albumin (BSA, 3 mg mL(-1)) in a shear reactor at 10k rpm for <80 minutes. Exfoliation was monitored by powder X-ray diffraction with samples displaying a gradual but complete loss of the 7.6 angstrom (002) peak, which is characteristic of bulk alpha-ZrP. The fully exfoliated sample loaded with the protein was characterized by transmission and scanning electron microscopy in addition to other biophysical methods. Lysozyme, glucose oxidase, met-hemoglobin, and ovalbumin also induced exfoliation and directly produced enzyme/ZrP biocatalysts. Thus, exfoliation, biophilization and enzyme binding are accomplished in a single step. Several factors contributed to the exfoliation kinetics, and the rate increased with alpha-ZrP and BSA concentrations and decreased with pH. However, the exfoliation efficiency inversely depended on the isoelectric point of the protein with ovalbumin (pI = 4.5) being the best and lysozyme (pI = 11.1) being the worst. A strong correlation between the protein size and exfoliation efficiency was noted, and the latter suggests the role of hydrodynamic factors in the process. Exfoliation was also achieved by simple stirring using a magnetic stirrer, under low volumes, and model enzymes, indicating 60-90% retention of bound enzymatic activities. The addition of BSA to enzymes as the diluent and stabilizing agent also prevents enzymes from the denaturing effect caused by stirring. This new method requires no pre-treatment of alpha-ZrP with toxic exfoliating agents such as tetrabutyl ammonium hydroxide and provides bioactive enzyme/inorganic materials in a single step. These protein-loaded biocompatible nanosheets may be useful for biocatalysis and biomedical applications.