Precursor concentration effects on crystallite size and enzyme immobilization efficiency of Enzyme@ZIF-8 composite

• ZIF-8 forms via an amorphous phase composed of precursors and enzyme. • ZIF-8 cannot form without enzymes at low molar ratio of precursors (Hmim/Zn). • Enzymes reversed correlation between the ZIF-8 size and the precursor molar ratio. • pH-dependent coordination state of Zn ion affects ZIF-8 formation via an amorphous phase. • Low molar ratio precursors efficiently immobilize enzymes in ZIF-8. Composites of zeolitic imidazolate framework-8 (ZIF-8) and biomolecules have garnered much attention because the environmental tolerance of biomolecules is enhanced in the composites with ZIF-8. Those composites are formed easily by dissolving biomolecules with precursors of ZIF-8 in an aqueous solution. However, correlation between the composites’ properties and forming conditions remains unclear. In this study, we specifically examined composites of ZIF-8 and an enzyme (glucose oxidase): a much-researched composite for application purposes. For such composites, the crystallite size dependence of ZIF-8 on prepared molar ratio of precursors (2-methyimidazole / Zn ion: Hmim/Zn) was assessed and compared to that found in an earlier study for pure ZIF-8 without seeding. Results show that the ZIF-8 crystallite size decreased along with the decrease of the molar ratio of precursors. This correlation is opposite to that in cases of pure ZIF-8. Thus, the formation process of ZIF-8 in presence of enzyme is supposed to be much different from that of pure ZIF-8, and such process was discussed focusing on the pH-dependent coordination state of Zn ion in preformed amorphous phase of composite. Furthermore, the molar ratio of precursors affected the enzyme immobilization efficiency. Along with molar ratio of precursors, variations of counter anion of Zn were also investigated and discussed.