Effect of substrate (ZnO) morphology on enzyme immobilization and its catalytic activity
© Zhang et al; licensee Springer. 2011
Received: 24 March 2011
Accepted: 13 July 2011
Published: 13 July 2011
Skip to main content
© Zhang et al; licensee Springer. 2011
Received: 24 March 2011
Accepted: 13 July 2011
Published: 13 July 2011
In this study, zinc oxide (ZnO) nanocrystals with different morphologies were synthesized and used as substrates for enzyme immobilization. The effects of morphology of ZnO nanocrystals on enzyme immobilization and their catalytic activities were investigated. The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent. The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system. The surface of as-prepared ZnO nanoparticles was functionalized with amino groups using 3-aminopropyltriethoxysilane and tetraethyl orthosilicate, and the amino groups on the surface were identified and calculated by FT-IR and the Kaiser assay. Horseradish peroxidase was immobilized on as-modified ZnO nanostructures with glutaraldehyde as a crosslinker. The results showed that three-dimensional nanomultipod is more appropriate for the immobilization of enzyme used further in catalytic reaction.
The composition, size, and surface characteristics of substrates have been considered as the important factors that affect the physical/chemical properties of the immobilized enzymes [1–3]. However, for conventional bulk solid substrates, such as glass slides , polymer monoliths [5, 6], and silica beads , the control on their morphologies and functionalizing their surfaces are usually laborious. Nanoscaled materials, such as metal  and metal oxide nanospheres , carbon nanotubes [9, 10], graphene oxide nanosheets , have also been utilized as the substrates for enzyme immobilization. It has been demonstrated that the size of nanostructured materials might play significant roles to regulate the catalytic activity of the immobilized enzymes . The preparation of nanostructured materials with controlled chemical composition, size, morphology, and the surface functionalization have witnessed great progressive achievements during the last two decades [12, 13]. However, the systematic study of the effects of the morphology of the nanoscale substrates on the enzyme immobilization remains to be expanded. ZnO nanocrystals have unique physical/chemical properties and pronounced biocompatibility, which are beneficial for many practical applications. In addition, a variety of nanostructures of ZnO, such as nanospheres , nanowires , nanorods , nanonails , nanotubes , nanotetrapods , nanotablets , and nanoflowers , have been prepared successfully. Therefore, ZnO nanocrystals are ideal materials to study the effect of the morphology of the substrate on the catalytic efficiency of the immobilized enzymes. Horseradish peroxidase (HRP) was used as a model enzyme because it has been wildly studied and used in many fields, such as organic syntheses , phenol removal , biosensor, and drug delivery .
In this study, we report the effects of the morphology of the ZnO nanocrystals on the enzyme immobilization. ZnO nanocrystals with different morphologies, including nanosphere, nanodisk, and nanomultipod, were fabricated simply through a hydrothermal procedure. The surface of ZnO nanocrystal was functionalized with the amino groups using 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) . Glutaraldehyde was used as a crosslinker to immobilize the HRP enzyme molecules on the surface of as-modified ZnO nanocrystals. Then the enzyme loading and catalytic activity were evaluated.
The loadings and kinetic properties of the HRP immobilized on ZnO nanocrystals
Enzyme loading (mg/m2)
K m (mM)
K cat/K m (mM-1s-1)
HRP on ZnO nanospheres
HRP on ZnO nanodisks
HRP ZnO nanomultipods
The catalytic property of the immobilized HRP was further characterized by the catalytic efficiency (K cat/K m ). The catalytic efficiency of immobilized HRP was much lower than that of free HRP, which coincide with the report of the literature . As shown in Table 1, the catalytic efficiency values of immobilized HRP on the nanospheres, nanodisks and nanomultipods were 0.78, 1.09, 1.28 mM-1 s-1, respectively. Through comparing the K cat/K m value of the HRP immobilized on three ZnO nanocrystals with different morphologies, the nanomultipod ZnO nanocrystals are apparently favorable for enzyme immobilization.
Those all may due to the three dimensional structural feature of nanomultipods, which could affect the enzyme interaction with the immobilized substrate and conformation of the enzyme, and result in increasing the enzyme loading on the solid substrate and catalytic efficiency. Because the nanomultipods have several pods, and the spaces among the pods are limited, the glutaraldehyde is difficult to self-polymerize, and the amino groups on the multipods are more efficiency to immobilize HRP. While, nanospheres and nanodisks have opened spaces, glutaraldehyde tends to polymerize. Thus, HRP loadings on nanospheres and nanodisks need more glutaraldehyde to reach the maximum loadings. Due to the opened structure and lower surface density of amino groups, the loading of HRP on nanospheres is lowest. Thus, HRP loadings on nanospheres and nanodisks need more glutaraldehyde to reach the maximum loadings. Compared with nanodisks and nanospheres, the HRP loading on ZnO nanomultipods reached the highest when its glutaraldehyde to amino groups ratio was lower, which indicates that every HRP molecule needs less glutaraldehyde to immobilize HRP on the surface of ZnO nanocrystal and less conformation happened. Therefore, the 3D structure of nanomultipods and the lowest ratio of N glutaraldehyde/N-NH2 may result in the stabilization of HRP immobilized on nanomultipods, which indicates that the 3D nanomultipod is more appropriate for the immobilization of enzyme and its catalytic efficiency.
ZnO nanocrystals with different morphologies, including nanosphere, nanodisk, and nanomultipod, were prepared via hydrothermal reactions using the mixtures of methanol/water with different volume ratios. The surfaces of ZnO nanocrystals were modified with amino groups using TEOS and APTES to study the effect of the morphology of the materials to the enzyme immobilization. The surface density of the amino groups and the thickness of coating were controlled by tuning the ratio of TEOS to APTES. It was demonstrated when the ratio of TEOS to APTES was 1:4, a layer of uniform thickness of approximately 2 nm can be generated on surface of the ZnO nanocrystals.
HRP molecules were immobilized on the modified ZnO nanocrystal surfaces using glutaraldehyde as a crosslinker. It was illustrated that the enzyme loading on the ZnO nanostructure was in the order of nanospheres < nanomultipod < nanodisks, while the nanomultipod reached the highest loading when its glutaraldehyde to amino groups ratio was lower than the other two, causing less conformation change of HRP on the ZnO surface, leading to a higher catalytic efficiency. In brief, the 3D nanomultipod is more appropriate for the immobilization of enzyme and for being used in catalytic reaction than the other two, which has great implications for the many ongoing studies of enzyme immobilization and applications of the immobilized enzymes.
Zn(Ac)2·2H2O, (CH3)4NOH (25%), Glutaraldehyde (25%), phenol (99%), 4-aminoantipyrine (4-AAP), methanol, and H2O2 (30%) were purchased from Sinopharm Chemical Reagent Company, Shanghai, China. APTES (≥98.0%) was bought from Sigma-Aldrich, USA. TEOS was obtained from Linfeng Chemical Reagent Company, Shanghai, China. HRP was purchased from Majorbio Biotech Company, USA. All reagents were used as-received.
The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent. The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system. In a typical experiment, Zn(Ac)2·2H2O (5 mmol) was dissolved in 15 mL of the mixture of methanol and water with different volume ratios in a flask under vigorous stirring. Then, 15 mL of (CH3)4NOH was dropped into the flask as mineralizing agent. The as-obtained turbid suspension was transferred into a Teflon inner reactor of stainless-steel autoclave. The autoclave was heated at 200°C for 24 h for the hydrothermal reaction. The reaction solution was then cooled down to room temperature and the solid product was separated from the reaction mixture by centrifugation (4000 rpm), and was washed with ethanol and water alternately, each for three times.
The size, morphology, BET surface area, and crystallinity of the ZnO nanocrystals before and after the surface modification were characterized using scanning electron microscope (Zeiss ultra 55, Germany), transmission electron microscopy (TEM) (JEM-2100, Japan), accelerated surface area, porosimetry system (Micromeritics ASAP 2010 M+C, USA), and X-ray powder diffraction (XRD) (BRUKER-AXS, Germany). The surface functionalities of the ZnO nanocrystals after the modification were studied using FT-IR with a Nicolet 5700 Fourier transform infrared spectrometer (Thermo Electron, USA), and Zeta potentials obtained on Nicomp 380/ZLS (America).
A typical surface functionalization process was as follows.
In general, 100 mg of ZnO nanocrystals was suspended into 20 mL of ethanol (pH = 10.8) under sonication. Then, 15 μL of TEOS and 60 μL of APTES were added into the ethanol solution, and the mixture was stirred for 5 h. The solid product was filtered and washed with ethanol, and then dried at room temperature. The amounts of amino groups on the surface of ZnO nanocrystals were measured by Kaiser Assay. At first, the standard curve of glycine obtained by Kaiser Assay. Glycine were mixed with 2 mL of acetate buffer (0.6 M, pH = 4.5) and 2 mL of 10 mg/mL ninhydrin ethanol solution. The mixture was treated at 90°C for 20 min. After centrifugation at 10,000 rpm for 3 min, the amino groups' concentration in the supernatant was monitored spectrophotometrically 565 nm, and then, 10 mg of modified ZnO was utilized to monitor spectrophotometrically as glycine. Then, the amount of amino groups was calculated based on the standard curve of glycine.
100 mg modified ZnO was suspended into 20 mL pH 7.0 0.1 M phosphate buffers, and then 750 μL of 25% glutaraldehyde solution was added to the mixture. The mixture was incubated at room temperature, 200 rpm for 2 h. After filtration and washing with the phosphate buffer, the samples were utilized to immobilize HRP.
For HRP immobilization, 100 mg of functionalized ZnO nanocrystals was added into 1 mL, 0.1 M, and pH 7.0 of potassium phosphate buffer containing 100 μg of HRP. The mixture was incubated at 4°C for 2 h with 240 rpm shaking, and then centrifuged at 10,000 rpm for 3 min. The supernatant was collected. The sediments were centrifuged and rinsed alternately three times with 0.1 M, pH 7.0 phosphate buffer solution to remove non-specifically adsorbed enzymes. The solid was stored at 4°C for further measurements. The supernatants were employed to determine the enzyme loading on modified ZnO.
The enzyme loading is obtained by subtracting the amount of the left HRP in the supernatant from the total HRP added. Free HRP and the immobilized HRP activity was assayed by colorimetric method using 4-AAP as previously described . In brief, the immobilized enzyme was added into 1 mL of 0.1 M, pH 7.0, phosphate buffer which contained 60 mM of phenol, 14.38 mM of 4-AAP, and 1.21 mM of hydrogen peroxide, and then reacted at 30°C for 3 min. The initial catalytic reaction rates of the enzyme in the supernatant and the immobilized enzyme were determined by measuring the UV absorbance of the reaction mixture at 510 nm. The double reciprocal plots of the rates and substrate concentrations were plotted to obtain K m and K cat according to the Lineweaver-Burk equation.
transmission electron microscopy
X-ray powder diffraction
This study was financially supported by the National "973" Program (Nos. 2007CB936000 and 2010CB933900), the NSFC (No. 20774029 and No. 20906055) of China, the State key laboratory of bioreactor engineering (No. 2060204), and China postdoctoral science foundation (No. 20100470131).
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.