Dopamine-functionalized mesoporous onion-like silica as a new matrix for immobilization of lipase Candida sp. 99-125

Dopmine functionalized mesoporous onion-like silica (DPMS) was synthesized via a biomimetic coating, and lipase Candida sp. 99-125 (LCS) was immobilized in DPMS (LCS@DPMS) by physical adsorption in this study. The DPMS was characterized by SEM, TEM, BET and FT-IR, and it was shown that the DPMS had clear multishell structures with large surface area of 419 m2/g. The activity, pH stability, thermal stability, storage stability, and reusability of the LCS@DPMS were investigated in detail. The stabilities of LCS@DPMS were improved significantly compared to the free lipase and LCS@MS (LCS immobilized in unfunctionalized mesoporous onion-like silica by physical adsorption). All the results indicated that the DPMS had high efficiency and improved stability for lipase immobilization.

Enzyme-catalyzed processes are widely used in the pharmaceutical or food industry due to their ease of production, green chemistry and substrate specificity 1 . The use of free enzymes is often hampered by several drawbacks such as low stability, high cost and poor reusability 2 . Immobilization of enzymes on solid supports is one of the best methods for biocatalysis to increase the stability and reusability of enzymes, simplify purification and recycling processes 3,4 . In the last few decades, numerous immobilization approaches and supports materials have been investigated 5 . In particular, mesoporous silica supports have attracted much attention due to their well-defined pore sizes and geometry, large specific surface area, and mechanical stability 6,7 . These advantages of mesoporous silica supports enable the improvement of activity and stability of enzymes. Recently, a novel meso-structured onion-like silica (Meso-Onion-S), which had highly curved mesopores and highly ordered onion-like multilayer, was synthesized and used as a host for the immobilization of enzyme, and the stability of the enzyme was effectively improved 8,9 . However, there are only two reports regarding the study of Meso-Onion-S, and therefore further studies for the immobilization of enzyme in this novel porous material are indeed required.
Typical strategies for immobilizing enzymes onto mesoporous silicas rely on surface grafting via polymers containing functional groups or low molecular weight linkers to which enzymes are reacted by covalent conjugation [10][11][12] . But such immobilization methods are usually accompanied by enzymes deactivation 13 . Thus, despite numerous reported approaches for immobilizing enzymes on mesoporous silicas, there is still the need for a simple, facile and cost-effective surface modification approach. Recently, inspired by the adhesive protein secreted by mussels, a mild approach of surface modification utilizing dopamine as functionalization reagent which can be simply attached onto almost all types of material surfaces, has been reported by Messersmith and co-workers 14 . Furthermore, the dopamine molecules contain both catechol and primary amine, which are capable of immobilizing biomolecules 15,16 . Therefore, immobilizing enzymes onto different substrates modified with dopamine can be easily achieved by facile conjugation of proteins 12,[15][16][17] . However, to the best of our knowledge, Meso-Onion-S modified with dopamine has never been used as enzyme immobilization support. Ongoing efforts are necessary for the study on immobilization of enzyme in this new matrix.
Lipase is widely used in the food, fine chemical and pharmaceutical industries because of the versatile reactions it catalyses, such as hydrolysis, transesterification and esterification [18][19][20][21] . Lipase from Candida sp. 99-125 is an important enzyme with high activity and stability, and it is also cost-effective compared to other lipases 22 . Therefore, lipase Candida sp. 99-125 has great potential for applications in industry. However, to the best of our knowledge, there is no report for the immobilization of lipase Candida sp. 99-125 onto supports modified with
Preparation of dopamine-functionalized Meso-Onion-S (DPMS). The Meso-Onion-S was synthesized according to the method reported by Jun et al. 8 . Meso-Onion-S was functionalized with dopamine using the post-grafting method. Typically, 0.5 g of Meso-Onion-S was added into 100 mL of 1.25 g/L dopamine solution, which was freshly prepared in Tris-HCl buffer (pH 8.5), and the suspension was stirred for 2 h. Then, the solid was filtered and washed with phosphate buffer (pH 8.0) for three times. The as-prepared solid product was denoted as DPMS. Thermal and storage stability. The thermal stability was determined by immersing the free and immobilized lipase in isooctane at 60 °C and incubated for a certain period of time, and the residual activities were measured at each time point. The relative activities were calculated as described above.

Immobilization of lipase in DPMS.
To determine the storage stability, the free and immobilized lipase were immersed in 0.1 M phosphate buffer solutions with pH 8.0, and stored in a fridge under 4 °C. The residual activities were measured at each time point, and the relative activities were calculated.
Stability of free and immobilized lipase in shaking conditions. The free lipase, LCS@MS and LCS@ DPMS were immersed in 0.1 M phosphate buffer solutions with pH 8.0 at room temperature and shaken at a speed of 200 rev/min. The residual activities were measured at each time point, and the relative activities were calculated.
Determination of kinetic parameters. The Michaelis-Menten kinetic parameters of free and immobilized lipase were determined by measuring the lipase activity using p-NPP as substrate in the initial concentration range from 1.32 to 13.2 μ mol/L at 298 K and pH 8.0. The values of kinetic parameters K m and V m were obtained by fitting the experimental data to the Michaelis-Menten model using non-linear regression code.
Reusability. The reusability of immobilized lipase was determined by conducting the enzymatic activity assay method described in activity assay section. At the end of each batch reaction, the immobilized lipase was recovered with centrifugation, washed excessively with buffer solution in order to remove the product or substrate and dried at room temperature. Then, the lipase was applied again in the next reaction cycle with fresh substrates.
Characterizations. SEM images of DPMS were obtained by a scanning electron microscopy (S-4800, Hitachi, Japan). TEM images were realized by a transmission electron microscopy (JEM-2100F, JEOL, Japan) operating at 200 kV. The surface-area measurements of Meso-Onion-S and DPMS were based on the Brunauer-Emmett-Teller method (BET, BELSORP-max, BEL, Japan), and the pore size distributions were obtained from adsorption isotherm branches by the Barrett-Joyner-Halenda (BJH) method. The Fourier transform infrared (FT-IR, VECTOR22, Bruker, Germany) spectra of Meso-Onion-S, DPMS and LCS@DPMS were recorded using the KBr pellet method.

Results and Discussion
Characterization of the immobilized lipase. The SEM photograph revealed that the DPMS was made up of hundreds of nanometer sized onions which aggregated into secondary micrometer sized particles (Fig. 1), and this result was in accordance with the previous reports 8,9 . The TEM image showed that the DPMS had clear multishell structures with mesopore size of about 13 nm between the layers (Fig. 2). Figure 3 shows the nitrogen adsorption/desorption isotherms of Meso-Onion-S and DPMS. The specific surface area of DPMS was estimated as 419 m 2 /g, and the BJH adsorption cumulative volume and pore size were 1.14 cm 3 /g and 12.3 nm, respectively. Such pore size and pore volume were large enough to ensure the accommodation of enzymes. Compared with unfunctionalized Meso-Onion-S, the reduction of specific surface area, cumulative volume and pore size of DPMS indicated the success of modification. Figure 4 exhibits the FTIR spectra of Meso-Onion-S, DPMS and LCS@DPMS. Typical peaks at 467 cm −1 , 796 cm −1 and 1086 cm −1 corresponded to the bending vibration, symmetric stretching and asymmetric stretching of Si-O-Si bonds from all of the samples, suggesting that the structure of Meso-Onion-S was well preserved in DPMS and LCS@DPMS 23 . Characteristic band at 962 cm −1 represented the symmetric stretching vibration of Si-OH groups 24 . The adsorption peak at 1495 cm −1 of DPMS was belong to the stretching of benzene ring in the polydopamine 17 , which indicated the successful immobilization of dopamine. For the LCS@DPMS, the peaks at 1545 cm −1 and 1465 cm −1 could be assigned to the deformation vibration of -NH 2 groups and bending vibration of the -C-H groups 2 . This observation confirmed the successful immobilization of the lipase in DPMS. Figures 5 and 6 shows the effect of lipase concentration on the lipase loading amount and activities of LCS@ DPMS, respectively. According to the results, the lipase concentration was set at 10 mg/mL in the subsequent enzyme immobilization experiments. The results indicated that the lipase loading amount of LCS@DPMS was 81.6 mg/g carrier, and the immobilized yield was 52.4%. In the control experiment, the lipase was also immobilized in Meso-Onion-S by physical adsorption (named LCS@MS) under the same preparation conditions. However, the enzyme loading amount of LCS@MS was 62.2 mg/g carrier, and the immobilized yield was 45.3%, which was lower than that of LCS@DPMS. The improved lipase loading capacity and specific activity of LCS@ DPMS could be attributed to the strong conjugation of covalent bond between the lipase molecules and the dopamine functionalized Meso-Onion-S, which might promote the lipase loading amount of LCS@DPMS.
Effect of pH on the activities of free and immobilized lipase. The effect of pH on the activities of free and immobilized lipase is shown in Fig. 7. The optimum pH for free lipase and LCS@MS was observed at 8.0, while LCS@DPMS reached its maximal activity at 7.0. Compared to the free lipase and LCS@MS, the optimum pH of LCS@DPMS exhibited a shift toward the acidic region. The polydopamine on the surface of DPMS contains quinones, which are reactive toward nucleophilic groups and can react with the amino or thiol groups of the lipase, and this reaction can lead to the immobilization of lipase in DPMS via covalent bond formation between the polydopamine and the lipase molecules 16,25 . The more acidic optimal pH of LCS@DPMS might be caused by the following two reasons: (i) the covalent attachment between the lipase and polydopamine on the surface of the support that limited the transition of lipase conformation against the change of pH 12,26 , and (ii) the amine or imines groups on the surface of polydopamine near the active sites of lipase molecules could diminish the concentration of protons 15,17 . Additionally, the pH profile of LCS@DPMS was wider range than free lipase and LCS@MS, which enhanced stability of LCS@DPMS under acidic conditions. Therefore, the LCS@DPMS was more suitable for widespread practical applications.
The thermal stability of free and immobilized lipase. Thermal stability is an important property of enzyme for industrial applications. The results of thermal stabilities of free lipase, LCS@MS and LCS@DPMS are shown in Fig. 8. Obviously, after 20 min of incubation at 60 °C, the free lipase and LCS@MS retained 54% and 65% of their original activities, respectively, while the remaining activity of LCS@DPMS was 73%. After 60 min the remaining activities of free lipase and LCS@MS decreased to 17% and 28%, respectively, and that of LCS@DPMS  sites [27][28][29][30] . Moreover, the covalent bonds between the lipase molecules and polydopamine could enhance the rigidity of lipase, and protected them from unfolding and then prevented their denaturation at high temperature [31][32][33] . For these reasons, the thermal stability of LCS@DPMS was meliorated comparing with the free lipase and LCS@MS.  The storage stability of free and immobilized lipase. The results of storage stability of the free and immobilized lipase are shown in Fig. 9. Evidently, the activity of the immobilized lipase decreased slowly than that of the free lipase. After 30 days, the relative activity of the LCS@DPMS could maintain 64%, whereas the relative activities of the free lipase and LCS@MS remained 16% and 42%, respectively. Thus, the LCS@DPMS exhibited higher storage stability than the free lipase and LCS@MS. The lipase was adsorbed in Meso-Onion-S via relatively weak physical force, and in contrast, the polydopamine on the surface of DPMS could react with the lipase molecules to form covalent conjugation, which could increase the conformational stability of lipase 34,35 . Therefore, the reason for the improved storage stability of LCS@DPMS might be that the LCS@DPMS could protect the active sites from deactivating more effectively under the same conditions compared with the free lipase and LCS@MS.
The stability of free and immobilized lipase in shaking conditions. The stability of the immobilized enzyme in shaking conditions is one of the important issues for most enzymatic reactions that are conducted in vigorous shaking for reducing mass transfer limitations 2 . Figure 10. shows the stability of free and immobilized lipase in shaking conditions (200 rev/min). It could be seen that the activity of the free lipase decreased fast and only 9.7% of the initial activity was retained the next day, and the reason for the decrease of activity was that the vigorous shaking could result in the autolysis and denaturation of the lipase 9,36 . Adsorption of the enzyme in Meso-Onion-S and DPMS improved the stability of lipase, and the LCS@MS retained 24% of its initial activity after shaking for 7 days, and in contrast, the LCS@DPMS retained 53% of its initial activity under the same conditions. The activity reduction was ascribed to the leaching of enzyme from the immobilized lipase 37,38 . Compared to the LCS@MS, the improved storage stability of LCS@DPMS might be ascribed to the strong conjugation between the lipase molecules and the polydopamine on the surface of Meso-Onion-S, which could prevent the lipase leaching effectively.
Kinetics of free and immobilized lipase. The    than that of the free lipase, respectively, indicating that the substrate's affinity capacity for the immobilized lipase was lower compared to the free lipase. The change in the substrate's affinity for the lipase was probably caused by conformational change in the adsorption process of lipase onto supports, and resulting in a lower accessibility of the substrate toward the active sites of the immobilized lipase 9,39 . Furthermore, the K m value of the LCS@ DPMS was lower than that of the LCS@MS, which might be ascribed to that the polydopamine on the surface of Meso-Onion-S could keep the conformation of the immobilized lipase more integrated and then enhance the affinity capacity between the lipase and the substrate 7 .

Reusability of the immobilised lipases.
For practical applications, the reuse of immobilised lipase is necessary. The reusability of lipase immobilized in Meso-Onion-S and DPMS is shown in Fig. 11. It could be found that the LCS@MS and LCS@DPMS retained 63 and 71% of their initial activities after 6 consecutive operations, respectively. The decrease in activity was mainly caused by the denaturation and leaching of enzyme from the immobilised lipases 35,40 . Contrasting to LCS@MS, the activity of LCS@DPMS decreased slowly. The low operating stability of LCS@MS might result from that the lipase was adsorbed on Meso-Onion-S through physical force which was relatively weak and the lipase was easy to leach during the catalytic process 34 . The improved operational stability of LCS@DPMS was caused by the strong conjugation of covalent bond between the lipase molecules and the dopamine functionalized Meso-Onion-S, and thus the leakage of lipase from the DPMS was   prevented. Moreover, the covalent bond could prevent the denaturation of lipase molecules upon use [31][32][33] . The more stable performance of LCS@DPMS in repeated use will make it desirable for practical applications.

Conclusions
In summary, a simple and cost-effective method was used to immobilize lipase Candida sp. 99-125 in mesoporous onion-like silica based on grafting polydopamine onto the silica, and the pH, thermal and storage stability of the immobilized lipase LCS@DPMS were improved significantly. Additionally, the pH profile of LCS@DPMS was wider range than free lipase and LCS@MS, and the performance of its reusability was more stable than LCS@MS. Taken together, the DPMS had great potential as a matrix for practical applications in the immobilization of lipase.