1. Introduction
In recent years, the enzyme has been employed in numerous applications in the form of immobilized enzyme preparation [1–3]. By nature, the immobilization of the enzyme is divided into two groups. (I) When the enzyme is immobilized on the outer surface of a support and hence directly exposed to the solution, the enzyme is easily in contact with the substrate, which may result in high catalytic efficiency in a catalytic reaction. On the other hand, the enzyme molecular is sensitive to adverse effects of some reagents in the solution, which may cause the loss of the activity of the enzyme. (II) In contrast, for the enzyme which is embedded in the support and finitely contacts with the solution, the catalytic activity of the enzyme in the support is only observed if the diffusion of the product and the substrate in the support is similar to the case in the solution [4].
In order to prevent the enzyme from direct exposure to the solution, some methods such as entrapment in semi-permeable membrane [5], sol-gel method [6] and physical adsorption [7] have been adopted. The encapsulation of the enzyme which possesses relatively large spatial size is often achieved by the semi-permeable membrane and sol-gel method. In the semi-permeable membrane approach, low mechanical stability of the membrane results in poor reusability of immobilized enzyme. In the sol-gel method, the support presents poor mechanical stability and the pore size distribution cannot easily be controlled. These disadvantages block practical application of immobilized enzyme in industry. In 2006, the encapsulation of fumarase, trypsin, lipase and porcine liver esterase was fulfilled through the “fish-in-net” approach in our experiment [8]. Taking into account the problem that three-dimensional size of the enzyme might be bigger than the pore size of the silica matrix, the enzyme and performed precursors were placed in a non-denaturing environment simultaneously to complete the encapsulation of the enzyme. The encapsulated enzyme presented perfect enzymatic activity and excellent reusability. The silica-based matrix, acting as the support of the enzyme, simultaneously possessed an ordered mesoporous structure and regular macroporous structure. Such technology has been used in practical applications such as the hydrolysis of lactose in the preparation of low lactose milk [9] and the removal of aniline in industrial wastewater [10].
When the spatial size of the enzyme molecule is smaller than the pore size, many kinds of mesoporous molecular sieves are used as support to achieve immobilization through physical adsorption. Due to their high mechanical stability, good chemical stability, large special surface area and pore volume, a silica-based matrix such as MCM-41 [11], SBA-15 [12] is widely applied to the adsorption of enzyme. Adsorbed enzyme preparations are easily separated from the reaction system and possess perfect reusability. Attributed to the merits mentioned above, adsorbed enzyme in mesoporous silica materials is suitable for industrial production.
However, the change of temperature, pH value and ionic strength can alter the surface charge property of the absorbed enzyme and mesoporous silica, leading to the disappearance or the decrease of translational and rotational restriction of the enzyme molecule after absorption [13]. Thus, the leakage of the adsorbed enzyme molecules occurs.
In order to overcome the leakage problem, two-step immobilization was carried out to achieve the immobilization of the enzyme in this experiment. Herein, myoglobin and lysozyme were selected as a model protein owing to its well known physicochemical property and commercial availability. First, amino-functionalized mesoporous molecular sieve was used to absorb myoglobin. Second, lysozyme was linked to the amino group of the amino-functionalized mesoporous molecular sieve to avoid the leaching of the absorbed myoglobin. Finally, the bifunctional bioreactor was obtained (Figure 1).
2. Results and Discussion
2.1. Characterization of Amino-Functionalized Mesoporous Silica
The aminopropyl-functional SBA-15 mesoporous material was first synthesized by one-pot co-condensation synthesis in 1996 [14]. The amino-functionalized mesoporous silica produced by co-condensation synthesis possessed higher loading of the functional group [15]. The co-condensation synthesis provided more homogenous distribution of organosilane functionalities without collapse of the mesoporous framework [16]. In 2005, the conventional co-condensation method was improved by a prehydrolysis-step of the silica resource before the adding of APTES [17]. In this experiment, the improved co-condensation synthesis was adopted to simplify the synthesis procedure and prepare the desired amino-functionalized mesopcorous silica.
As suggested by Soofin et al., the mesoporous ordering of amine-silane decreased with the increasing molar composition of APTES in the synthesis system [17]. When the molar composition was close to 0.2, X-ray diffraction peaks of amine-silane began to weaken. In order to simultaneously acquire the maximum loading of amino group and ordered mesostructure, 0.2 was chosen as the molar composition of the APTES in this experiment. The sample showed the H1 type hysteresis loop (Figure 2a). The pore size distribution of the sample was very sharp (Figure 2b). The BET surface area, pore volume and BJH pore diameter were 250 m2/g, 0.41 cm3/g and 5.7 nm, respectively. It is necessary that the pore size should be larger than the molecular size of the enzyme to ensure successful adsorption [18]. Furthermore, when the size of the mesoporous material matches the dimension of the enzyme molecule, the enzyme is deeply absorbed into the internal surface of mesoporous material rather than on the external surface [7,19]. Myoglobin (Mr 16700) contains a single polypeptide of 153 amino acid residues with one molecule of heme as a prosthetic group. The myoglobin possesses a molecular dimension of 4.5 nm × 3.5 nm × 2.5 nm [20]. Obviously, amino-functionalized mesoporous silica synthesized in this experiment was suitable for the adsorption of myoglobin.
The DTG profile of amino-functionalized mesoporous silica showed two peaks at 50 °C and 320 °C, respectively (Figure 3). The evaporation of physically adsorbed water gave rise to the weight loss at temperature lower than 100 °C. The decomposition of aminopropyl group of amino-functionalized mesoporous silica resulted in weight loss at 250 °C–500 °C [13]. Moreover, either the dehydroxylation of the Si-OH group or the elimination of the residual ethoxy group originated from incomplete hydrolysis of silicon ethoxide might lead to the slight weight loss above 500 °C [21–22].
In FTIR spectra, the broad peak at around 3500 cm−1 was assigned to the stretch vibration of N–H (Figure 4). Three absorbance peaks in the range of 2800–3000 cm−1 were associated with stretching and bending vibration of C–H. Two negative peaks at 2200–2400 cm−1 were attributed to the contribution of CO2 in the environment. The absorption peak at 1630 cm−1 might be due to bending vibration of the adsorbed water molecules. The formation of polysiloxane was confirmed by the Si-O-Si band at 804 cm−1. The incorporation of the amino group was verified by symmetric –NH2 bending vibration at 1515 cm−1 [17].
2.2. Assignment of UV/VIS and FTIR Spectra of Amino-Functionalized Mesoporous Silica after Adsorption Myoglobin
The isoelectric point of myoglobin is 7.0 [20]. The isoelectric point of amino-functionalized mesoporous silica synthesized by the co-condensation synthesis increases in the range of 7.9~8.7 with the increasing molar composition of APTES in the range of 0~0.2 [23]. Because the maximum molar composition APTES (0.2) was used in the synthesis, the isoelectric point of amino-functionalized mesoporous silica was 8.7. The adsorption process was carried out at pH 7.5 in this experiment. At pH 7.5, myoglobin carried net negative charge while amino-functionalized mesoporous silica presented net positive charge. The electrostatic attraction between the amino-functionalized mesoporous silica and myoglobin could facilitate the adsorption of myoglobin.
After adsorption, three absorbance peaks in the range of 2800–3000 cm−1 presented band broadening, which was associated with the contribution of the methylene of myoglobin (Figure 4). Amide I at 1640 cm−1 and amide II at 1548 cm−1 band were assigned to α-helix-rich protein, which verified the presence of myoglobin in amino-functionalized mesoporous silica.
In UV/VIS spectroscopy, the UV absorbance of amino-functionalized mesoporous silica acted as the baseline (Figure 5). The intensive peak at around 400 nm was assigned to the soret-band of porphyrin [24]. The two weak peaks at around 500 nm and one weak peak at 600 nm were assigned to the Q-band of porphyrin, respectively [25]. These peaks exhibited characteristic absorption of heme of myoglobin. The UV/VIS spectroscopy verified that myoglobin was successfully adsorbed into amino-functionalized mesoporous silica.
2.3. Evaluation of Peroxidative Activity of Myoglobin
After adsorption, lysozyme was covalently tethered to the surface of glutaraldehyde activated amino-functionalized mesoporous silica. The aldehyde group of the glutaraldehyde molecule connected with the amino group of amino-functionalized mesoporous silica and lysozyme through Schiff’s base reaction [26]. As can be seen from Figure 6, the absorbance peak of the produced bisazobiphenyl appeared at 472 nm, which was in accordance with the result of Claiborne and coworkers [27], suggesting that the adsorbed myoglobin still exhibited peroxidative activity after covalent attachment of lysozyme.
2.4. Antibacterial Property of Lysozyme
The antibacterial activity of immobilized lysozyme was determined. As shown in Figure 7, there was no antibacterial circle for sample No. 1, suggesting that amino-functionalized mesoporous silica adsorbed myoglobin had no antibacterial activity. After the lysozyme was covalently linked to the amino-functionalized mesoporous silica, the immobilized enzyme was washed with buffer and the antibacterial activity of the washing supernatant was detected. No activity of lysozyme could be found in the supernatant after washing over four times, suggesting that the free or adsorbed lysozyme was totally removed. The bacteriostatic circle of the final immobilized enzyme (sample No. 8) clearly verified that covalently tethered lysozyme still possessed antibacterial activity (Figure 7).
The final immobilized enzyme was suspended in the buffer (pH 9.0 Gly-NaOH) for two hours and centrifuged. No protein could be detected in the supernatant, which demonstrated that the leaching of myoglobin did not occur. The final immobilized enzyme exhibited the activity of both myoglobin and lysozyme (Table 1) and the myoglobin/lysozyme ratio in the final immobilized enzyme was 0.624. According to the Betancor’s report, the monomer form of glutaraldehyde was grafted on the surface of amino-functionalized mesoporous silica when the concentration of glutaraldehyde applied was 5% (v/v) [28]. In this experiment, the concentration of glutaraldehyde used was 2.5% (v/v) so that the smaller spacer arm (monomer) was presented on the surface of amino-functionalized mesoporous silica. The shorter spacer arm (monomer) should offer a higher rigidity which was propitious to avoiding the leaching of the adsorbed myoglobin.