Preparations Of Papain-inorganic Hybrid Nanomaterials By Assembly

Enzyme immobilization is a well-known essential process that improves enzyme stability and storage and that enables the industrial reuse of enzymes for more reaction cycles; thus, enzyme immobilization has extensive applications in sustainable chemical processes. Most of the immobilization methods lead to enhanced stability compared with free enzymes in solution; however, several drawbacks exist, including mass-transfer limitation, activity loss caused by harsh synthesis conditions, and conformational change in enzyme structure. Over the past decade, researchers focus in finding some breakthrough in nano/microsized hybrid materials that can synchronously improve enzyme stability, activity, and lifespan. Materials, such as nanoparticles, nanotubes, nanofibers, nanocomposite, mesoporous material, have already been proposed as substrates in enzymatic immobilization because of their large surface-to-volume ration.In this paper, the novel of enzyme-inorganic nanomaterials with a hierarchical flower-like structure was synthesized. And the enzyme-inorganic hybrid nanoflowers’ structure was confirmed by Fourier transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy. The hierarchical flower-like structure formed through self-assembly using papain as organic component and Cu3(PO4)2?3H2O as inorganic component.By changing the incubation conditions of nanoflowers, including incubation time, temperature, pH and enzyme concentration, each condition that might affect the morphology of nanoflower. A new method was applied to calculate the amount of papain that was actually embedded in nanoflowers to increase the accuracy of enzyme-activity. The dried hybrid nanoflowers were heated to 700 oC in order to remove the organic enzyme in hybrid nanoflowers. The enzyme activity of papain embedded in the hybrid nanoflowers was further evaluated using BAEE as substrate. Combined with the morphology of nanoflowers, the research also studied each condition that might affect the nanoflowers enzyme activity.The results showed that nanoflowers grow gradually over time, the papainCu3(PO4)2?3H2O scaffold were formed initially, and the complexes further agglomerated, after 72 h the hierarchical flower-like nanostructure was formed. When we changed the pH value of the buffer solution, the structure of nanoflowers has a significant change. When the enzyme concentration was 0.25 mg/m L, with pH value increasing from 6.0 to 9.0, the structure of nanoflowers changed from compact sphere to loose flower-like structure. When the pH value was 9.0 and the enzyme concentration was 0.25 mg / mL, the hybrid nanoflowers exhibited extremely high enzyme activity(13273 U/mg) compare with the free enzyme(183 U/mg). In addition, the study of pseudo-first-order kinetics, indicating the high catalytic efficiency of the nanoflowers. In cycle analysis, although the enzyme activity decreased significantly after the sixth reaction cycles, activity was still much higher than that of free papain. The SEM figures showed that the structure of nanoflowers petals was damaged after recycling, which fully demonstrated the integrity of nanoflowers structure has a great influence on its enzyme activity.