| The ability of nature to synthesize minerals has motivated researchers to look for biotemplates to create functional materials.Biologists,chemists,and materials scientists are doing their best to find "green" methods for the synthesis and assembly of nanocatalytic materials.Diatoms can synthesize advanced nanostructures that live in porous silica shells called diatom shells.Through the sol-gel process,mimicking this model,silica-based bio-hybrids have been produced.Biomolecules such as proteins,enzymes or antibodies can be captured in the silica matrix to obtain corresponding biosensors and bioreactors.The main issues in this paper are as follows.1.Inspired by diatoms,we introduced lactase during the sol-gel formation of silicates,using lactase to mediate the appearance of silica carrier cages,and then covalently linked lysozyme to the surface of the silica carrier.A "Lactase-silica-lysozyme" complex was constructed,and the catalytic conversion efficiency of the complex to low-lactose milk and the characteristics of inhibiting bacterial growth during the conversion process were studied.In the process of constructing the "Lactase-silica-lysozyme" complex,we optimised the entrapment of lactase and the covalent attachment of lysozyme.The results showed that when the addition amount of enzyme was 0.6 g and the aging time of silica carrier was 72 hours,the immobilization efficiency of lactase reached 96.2%;when the concentration of lysozyme was 2.0 mg/ml,the concentration of glutaraldehyde was 2.0%,the reaction time of the covalent linkage was 6 hours and the p H of the reaction medium was 8.0,the immobilized efficiency of lysozyme reached 93.5%.The experimental results further confirmed that the "Lactase-Silicon-Carrier-Lysozyme" complex showed 96.3% of the antimicrobial activity compared to that of the free lysozyme,and the enzymatic hydrolysis activity of the embedded ?-galactosidase did not change before and after the covalent binding of lysozyme.Small-angle X-ray diffraction(XRD)patterns showed that the "lactose-silica-lysozyme" complex had three diffraction peaks(100),(110),and(200)of a two-dimensional hexagonal P6 mm structure.It was confirmed that the presence of lactase did not disturb the mesoporous structure of the "Lactase-silica-lysozyme" complex.The N2 adsorption and desorption curves indicated that the silica-based support had a pore size of 2.7 nm,which facilitated the diffusion of substrates and products.Fourier infrared spectroscopy confirmed that the "lactase-silica-lysozyme" complex had characteristic bands of amide I,amide II and Si-O-Si,confirming the presence of ?-galactosidase in the silica matrix.The "Lactase-silica-Lysozyme" complex showed perfect hydrolysis activity and antibacterial activity.After 10 consecutive runs,the covalently bound lysozyme still retained 96.6% of initial antibacterial activity;the encapsulated ?-galactosidase still had 99.8% of initial hydrolysis activity.The S.aureus ATCC653 and E.coli ATCC 1122 treated with the "Lactase-silica-Lysozyme" complex showed 95.5% and 89.6% of growth inhibition rates,respectively.The hydrolysis activity and antibacterial activity of the "Lactase-silica-lysozyme" complex remained almost unchanged within three months.Finally,we confirmed that after the lactose solution was treated in a packed column reactor loaded with the "Lactase-silica-lysozyme" complex for 60 days,the reactor could still hydrolyze 90.26% and 90.02% of lactose when the flow rates were 6 m L h-1 and 7.5 m L h-1,respectively.The packed column reactor could provide 87.66% of the lactose hydrolysis rate and 15.75% of the bacteria residual rate when the residence time of skim milk in the “Lactase-Silica Carrier-Lysozyme” complex packed column was 160 min.2.With an in-depth understanding of biomineralization,we have noticed that bacteria can synthesize crystalline magnetic nanoparticles for navigation and positioning.Algae,plants,and bacteria produce metallic nanoparticles through detoxification pathways.These phenomena inspired us to try to prepare protein-mediated copper phosphate nanocrystals in vitro.We had the privilege of discovering that laccases could construct enzymes-nanoflowers,and used the constructed laccase-nanoflowers to synthesize resveratrol dimers that are more antioxidant than resveratrol in vitro.Scanning electron microscopy showed that the sample consisted of a large number of flower-like nanoparticles with diameters ranging from 7 to 10 microns.The morphology of nanoflowers was very similar to that of marigold in nature.The characteristic absorption bands of PO43-,amide I and II in laccase-nanoflowers were observed by Fourier transform infrared spectroscopy,confirming the presence of laccase in the nanoflowers.Energy dispersive X-ray spectroscopy showed that the sample consisted mainly of C,N,Cu,P,and oxygen,revealing the successful incorporation of laccase into nanoflowers.When ABTS was used as a substrate,laccase in nanoflowers showed a specific activity of 350% of free laccase.After optimizing the reaction conditions,the specific activity of laccase in nanoflowers was 2.2 times higher than that in the buffer solution without copper(II)ions at p H 5.0,at 35°C and 0.1M Na Cl.The specific activity of laccase in nanoflowers was 1.8 times than that of laccase in copper(II)ion buffer solution.The laccase nanoflowers had good reusability and could maintain 93.2% of an initial specific activity even after ten cycles.The enhanced activity of laccase in nanoflowers might be attributed to the synergistic interaction between matrix and laccase during nanoscale encapsulation.In the p H range of 3.0-7.0,laccases in nanoflowers were less sensitive to p H changes.After being incubated at 45°C,55°C or 65°C for different time,the specific activity of laccase in nanoflowers was lower than that of free enzyme.3.In view of the fact that amino acids are the basic structural units of proteins,we used copper(II)ions as the inorganic component and the basic unit of protein L-amino acids as organic components to synthesize amino acid hybrid nanoflowers.The results showed that amino acids successfully mediated the appearance of copper phosphate crystals.Energy dispersive X-ray spectroscopy results showed that the amino acid hybrid nanoflowers were mainly composed of carbon,nitrogen,copper,phosphorus and oxygen,and the Cu/P atomic ratio was about 3:2.Raman spectra showed that the amino acid hybrid nanoflowers exhibited characteristic Cu-O bond peaks at 297 cm-1(Ag),352 cm-1(Bg1),and 642 cm-1(Bg2).The amino acid-copper phosphate hybrid crystals depending on the catalytic principle of Fenton-like reagents showed a peroxidase-like catalytic activity during the degradation of Rhodamine B.In this paper,we assembled and obtained a series of "enzyme-nanocarrier" complexes.These "enzyme-nanocarrier" complexes were used to prepare low-lactose milk,obtain resveratrol dimer and degrate the dye in wastewater.The "enzyme-nanocarrier" complexes showed the excellent catalytic activity and could be stable in multiple cycles of the reaction.The research of the dissertation focused on the topic of biotemplates-mediated synthesis and assembly of nanocatalytic materials.It is expected that our research results will lay a foundation for the new “green bio-manufacturing” approach. |
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