Immobilization of Penicillin G Acylase on Mesostructured Cellular

Article pubs.acs.org/IECR

Immobilization of Penicillin G Acylase on Mesostructured Cellular Foams through a Cross-Linking Network Method Hang Shi, Yujun Wang,* and Guangsheng Luo State Key Laboratory of Chemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China ABSTRACT: A novel immobilization method that combines the advantages of physical adsorption and covalent binding was developed for penicillin G acylase (PGA) immobilization. In this method, a polymer network was formed by linear polymer molecules and enzymes were connected to the network through multipoint immobilization. This method is different from the conventional covalent binding method that creates chemical bonds between PGA molecules and support. Initially, PGA was physically adsorbed into the support because of the interaction between Si−OH and enzyme molecules. Afterward, chitosan and glutaraldehyde were added to generate a network connected with PGA, thereby successfully preventing enzyme leaching. Using the novel method, the PGA loading amount reached 555.0 mg/g (dry support), the specific activity was up to 27.7 U/mg (enzyme), and 85% activity was retained after five reuses. In addition, the immobilized PGA showed relatively high operation stability in pH and thermal stability.

1. INTRODUCTION Penicillin G acylase (PGA; penicillin amidohydrolase, E.C. 3.5.1.11) is a crucial hydrolytic enzyme used in β-lactum antibiotic production.1 However, industrial applications are limited by their low stability, low tolerance to organic solvents, as well as difficult recovery and reuse of the free enzyme. These problems can be surmounted by enzyme immobilization which is influenced by the design of support materials and the immobilization methods.2−10 Physical adsorption is one of the most widely used methods in PGA immobilization because of its convenient experimental procedure and nontoxic solvents. In addition, in the condition of physical adsorption, the activity site is often unaffected; thus the immobilized enzyme can retain nearly full activity.11 PGA molecules have been physically adsorbed on MCM-48,12 hollow silica nanotubes,13 mesoporous silica,14 and epoxyactivated magnetic cellulose microspheres.15 However, the weak connection between PGA molecules and support materials leads to a critical problem, namely, the adsorbed enzymes are continuously leached out from the support, resulting in poor operational stability.16 Numerous efforts have been exerted to solve this leaching problem. For instance, Zhao et al.17 successfully grafted the amine group onto the mesocellular siliceous foams (MCFs) surface with 3-aminopropyltriethoxysilane (APTS), PGA molecules can then be fixed more firmly through this covalent method on aminefunctionalized MCFs materials than physical adsorption on blank MCFs. PGA molecules were also immobilized on oxirane-modified mesoporous silicas,18 self-prepared immobilized metal affinity membrane,19 and amine-functionalized PVC membranes.20 These covalent binding methods can prevent enzyme leaching from the surface. However, such methods can also lead to more complicated procedures in support preparation and contamination from toxic solvents. Moreover, the surface area and pore volume can be wasted as a result of the surplus modifier. Therefore, a novel method that combines the advantages of physical adsorption and covalent binding possesses an important meaning. © 2014 American Chemical Society

In the present work, a novel immobilization method that combines the advantage of physical adsorption and covalent binding was proposed for PGA immobilization (Figure 1). Through this cross-linking network method, PGA can be firmly immobilized on MCFs compared with physical adsorption method. Moreover, unlike surface modification in the covalent binding method, this novel method can provide a simpler procedure. Mesoporous silica materials were chosen as the support because of their large surface area, special pore structure, high thermal stability, biocompatibility, and environmental friendliness.21 First, PGA was immobilized onto MCFs through physical adsorption. The immobilization was not adequately stable because the interaction between PGA molecules and MCFs was only a weak hydrogen bond. Afterward, chitosan and glutaraldehyde (GA) were added to the immobilized enzyme. Chitosan is a natural cationic polysaccharide that shows good biocompatibility and biodegradability. Moreover, chitosan has numerous reactive amino and hydroxyl groups, which means it can be adsorbed into MCFs and is able to connect with the amino group on PGA molecules in the presence of GA. In addition, PGA molecules will be cross-linked with one another in the chitosan network. This multipoint immobilization provides higher stability compared with conventional single point immobilization method. Interestingly, in this method, no new chemical bond was created between PGA molecules and the support, which can simplify the experimental procedure and get rid of the toxic solvents. The physical properties of the immobilized PGA were characterized, the influence of chitosan types and enzyme concentration on immobilized PGA were studied. In addition, the stability of immobilized PGA in pH and temperature was investigated. Received: Revised: Accepted: Published: 1947

November 10, 2013 January 8, 2014 January 14, 2014 January 14, 2014 dx.doi.org/10.1021/ie403806d | Ind. Eng. Chem. Res. 2014, 53, 1947−1953

Industrial & Engineering Chemistry Research

Article

Figure 1. Procedure of cross-linking network immobilization method.

shaken at 160 rpm at 30 °C for another 20 min. In the following step, the mixture was centrifuged to separate the PGA solution and the MCFs with the immobilized PGA. The protein concentration of the supernatant was measured to calculate the immobilization yield [IMY (%)]. The resulting MCFs were washed by 15 mL phosphate buffer (pH = 7.9, 0.05 M) three times in order to measure the amount of leaching enzyme. The PGA loading amount [qE (mg/g)] and the IMY (%) were calculated according to the following formulas: q − q2 qE(mg/g) = 1 m q − q2 IMY(%) = 1 × 100% q1

2. MATERIALS AND METHODS 2.1. Materials. Tetraethyl orthosilicate (TEOS), hydrochloric acid (HCl, 36.5 wt %), and sodium hydroxide (NaOH) were produced by Beijing Chemical Company (Beijing, China). Ammonium fluoride (NH4F) was purchased from Xilong Chemical (Shantou, China). P123 triblock copolymer (poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide), EO20-PO70-EO20, (M = 5800) was purchased from Sigma-Aldrich (Dorset, UK). Sodium dihydrogen phosphate (NaH 2 PO 4 ), dibasic sodium phosphate (Na2HPO4), glutaraldehyde (GA), and 1,3,5-trimethylbenzene (TMB) were obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). PGA (E.C. 3.5.1.11) was purchased from Shunfeng Haideer Company, Ltd. (Zhejiang, China). Penicillin G potassium (Pen G) was provided by the North China Pharmacy Factory (Hebei, China). Chitosan A (DD 95.7%) was produced by Acros Organics (New Jersey, USA); chitosan B (DD 85.1%) was purchased from Sinopharm Chemical Reagent Company (Beijing, China); and chitosan C (DD 70.6%) was purchased from Yuhuan Chemical Company (Zhejiang, China). 2.2. Synthesis. MCFs were synthesized according to ref 22. In this method, 2 g P123 was dissolved in 75 g HCl solution with 1.6 mol/L concentration at room temperature. Afterward, 4.0 g TMB was added, and the resulting solution was magnetically stirred at 39 °C for 3 h. Subsequently, 4.6 mL TEOS was dropped into the solution, and the mixture was stirred for another 24 h. Afterward, 2.5 mL NH4F aqueous solution (1 wt %) was added, and the resulting solution was transferred into an autoclave and kept at 100 °C for 24 h. The obtained white precipitate was filtered, washed with plenty of water and ethanol, dried, and calcined at 550 °C for 6 h. 2.3. PGA Immobilization. Up to 0.1 g MCFs was incubated with 15 mL PGA solution, which was prepared by diluting the original PGA solution with phosphate buffer (pH = 7.9, 0.05 M). The mixture was then shaken in the SHA-B Incubator Shaker at 160 rpm at 30 °C for 24 h. Afterward, 1 mL chitosan solution, which was prepared by dissolving 0.5 g chitosan in 100 mL of 0.5% v/v HCl, was added into the mixture with 1 mL of 1.0 wt.% GA. The resulting mixture was

where q1 (mg) is the total amount of protein in the solution before immobilization, q2 (mg) is the unimmobilized enzyme in the residual solution, and m (g) is the weight of the support. 2.4. Enzyme Activity Assays. The activity of immobilized PGA was measured by titrating phenylacetic acid (PAA) produced in the hydrolysis reaction with NaOH. A certain amount of immobilized PGA was added to 100 mL of 5% (w/ v) Pen G buffer solution, which was previously heated to 37 °C. Then, 0.25 mol/L NaOH aqueous solution was used to titrate PAA produced in the hydrolysis reaction. The pH of the buffer solution was maintained at 7.9 for 10 min; whereas, the temperature was maintained at 37 °C by a circulating water bath. The apparent activity (A, U/g) and the specific activity (SA, U/mg-enzyme) were calculated according to the following formula: A(U/g) =

C NaOHVNaOH × 1000 mt

SA(U/mg) =

A qE

where CNaoH (mol/L) is the concentration of NaOH aqueous solution, VNaoH (mL) is the NaOH volume used in titration, and t (min) is the reaction time. 1948

dx.doi.org/10.1021/ie403806d | Ind. Eng. Chem. Res. 2014, 53, 1947−1953


Article

Figure 2. (a) TEM images of MCFs before immobilization. (b) TEM images of MCFs after immobilization. (c) SEM images of MCFs before immobilization. (d) SEM images of MCFs after immobilization.

2.5. Characterization of MCFs before and after Immobilization. A scanning electron microscopy (SEM) system (JEOL JSM7401F) operated at 1.0 kV was used to characterize the samples. The morphologies of the samples were characterized using transmission electron microscopy (TEM) on a JEOL JEM2010 microscope operating. Nitrogen adsorption−desorption isotherms were carried out at 77 K using a Quantachrome Autosorb-1-C chemisorption−physisorption analyzer. Thermogravimetry (TG) measurement was performed on a TG 2050 analyzer, infrared spectroscopic analysis was acquired from a Shimadzu FTIR-8100 Fourier infrared spectroscopy. The enzyme concentration was analyzed on an Agilent 8453 Ultraviolet−visible (UV−vis) spectrophotometer.

difference of blank MCFs, physical adsorption, and chitosan cross-linking (Figure 3). The structural parameters including pore volume, pore diameter, and specific surface are shown in Table 1. From the figure and the structural parameters, both the pore volume and the specific surface decreased noticeably after physical adsorption, while after chitosan cross-linking, the pore volume and specific surface were reduced further. The result indicated that PGA molecules entered the MCFs channels and occupied most of the space, the chitosan added can also enter the pores of MCFs and results in the change of the structural parameters. Furthermore, in the image of BJH pore size distribution, only the pores larger than 7 nm showed a decrease. The pore distributions were very similar to each other before and after immobilization. Considering that the molecule size of PGA is 7.0 nm × 5.0 nm × 5.5 nm,1 the result indicated that during the immobilization process, the PGA molecule can enter and distribute evenly in all the pores larger than its own size. This finding may also explain the slight reduction in the average pore diameter. Further experiments were performed to prove the successful immobilization of PGA molecules on MCFs. The TG measurement of blank MCFs, physical adsorption, and chitosan cross-linking are shown in Figure 4. The different slopes of the three curves indicated that along with the increase of

3. RESULTS AND DISCUSSION 3.1. Characterization of the Immobilized PGA. TEM and SEM images of MCFs (Figure 2) were obtained to observe the structure of the supports. The image before immobilization showed a uniform array of silica struts. In the image after immobilization, this framework structure was still retained, which provided channels for the mass transfer of the substrates. The isotherms of nitrogen adsorption and desorption and BJH pore size distributions were also investigated to show the 1949



Article

Figure 4. TG curves of blank MCFs, physical adsorption, and chitosan cross-linking.

Figure 3. (a) Isotherms of nitrogen adsorption and desorption. (b) BJH pore size distributions of blank MCFs, physical adsorption, and chitosan cross-linking.

Table 1. Structural Parameters of Blank MCFs, Physical Adsorption, and Chitosan Cross-Linking sample

pore volume (mL·g )

pore diameter (nm)

blank MCFs physical adsorption chitosan crosslinking network

2.06 1.07 0.75

16.83 15.05 14.55

−1

specific surface (m2·g−1) 510.90 303.40 206.50

Figure 5. FTIR spectra of (a) blank MCFs, (b) physical adsorption, and (c) chitosan cross-linking.

band in the IR spectrum of physical adsorption, indicating that more free Si−OH were released because of the connection between the chitosan. 3.2. Comparison of Different Immobilization Methods. The performance of chitosan cross-linking network method was compared with different immobilization methods, including free enzyme, physical adsorption, and methods reported in other works. The conditions and results are listed in Table 2. Under the same PGA solution concentration, chitosan cross-linking method showed a larger enzyme loading amount than physical adsorption. One possible reason for this finding was that the amide groups on chitosan provide much more link points to PGA enzyme in the presence of GA; thus, the PGA molecule can be attached to the chitosan network firmly. The cross-linking between PGA molecules and the chitosan network also shortened the average distance between these molecules, which released part of the pore volume, leading to the increase of loading amount. Moreover, the

temperature, the chitosan and PGA in the support decomposed slowly. The weight change also revealed that the weight of enzyme that was physically adsorbed onto MCFs was about 18% of the whole support. By contrast, after chitosan crosslinking, the weight of enzyme together with chitosan was about 23% of the whole support. The result corresponded to the enzyme loading amount, which will be presented in the succeeding section. FTIR spectroscopic analyses of blank MCFs, physical adsorption, and chitosan cross-linking were conducted from 400 to 4000 cm−1 (Figure 5). The characteristic absorptions were displayed at 1654 and 1548 cm−1, which were attributable to amide bands I and II, respectively. These values corresponded to the amide group, indicating the presence of the enzyme in the support so the peak at 3435 cm−1 of the chitosan cross-linking MCFs was much wider than the hydroxyl 1950



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Table 2. Comparison of Different Immobilization Methods

a

samples

CEa (mg/mL)

qE (mg/g-dry support)

IMY (%)

A (U/g)

SA (U/mg-enzyme)

activity retention (%)

free enzyme physical adsorption chitosan cross-linking network-1 chitosan cross-linking network-2 amine−functionalized MCFs17

2.4 2.4 5.4 5.5

243.0 313.4 555.0 443

67.5 87.1 68.5

7184.0 7130.4 8483.9 4138

39.7 29.6 22.8 15.3 9.3

100 74.6 57.4 38.5

WLb (mg) 0.9 0 0 0

Concentration of the PGA solution. bEnzyme leaching amount.

Table 3. Influence of Chitosan on Immobilized PGA samples

chitosan types

DDa (%)

chitosan amounts (g/g-dry support)

qE (mg/g-dry support)

IMY (%)

A (U/g)

SA (U/mgenzyme)


S1 S2 S3 S4 S5

A B C B B

95.7 85.1 70.6 85.1 85.1

0.050 0.050 0.050 0.025 0

324.1 313.4 247.0 307.3 243.0

90.1 87.1 68.6 85.4 67.5

7343.7 7130.4 6853.3 7356.0 7184.0

22.7 22.8 27.7 23.9 29.6

57.1 57.4 69.8 60.2 74.6

a

Deacetylating degree.

enzyme leaching amount of physical adsorption was 0.9 mg; whereas, the chitosan cross-linking method was zero. These findings indicate that the interaction between enzyme and support was sufficiently strong to prevent enzyme leaching. As shown in Table 2, the SA of the immobilized PGA inevitably decreased because the chitosan network may influence the contact between the substrate and the active site. However, compared with other immobilization methods reported in the literature, the SA still maintained a relatively high level. Under the same concentration of PGA solution, the enzyme loading amount and the SA of the immobilized PGA in this work were much higher than that of PGA immobilized on aminopropylfunctionalized MCFs through Schiff base reaction.17 3.3. Influence of Chitosan on Immobilized PGA. Three samples (S1, S2, S3) cross-linked with same amounts of chitosan with different deacetylating degrees (DD) were investigated under the same PGA solution concentration (CE = 2.4 mg/mL). The experimental results are shown in Table 3. Chitosan with higher DD resulted in a larger enzyme loading amount and higher IMY. The amide groups on chitosan were believed to depend on the DD of the chitosan. Higher DD can provide more amide groups (link points) for the PGA molecules, which leads to the increase of the enzyme loading amount. However, higher DD can also lead to SA decrease of the immobilized PGA. Therefore, the strong connection between PGA molecules and chitosan, together with the cross-linking between PGA molecules, may limit the contact between the substrate and the active site, which ended in the decrease of SA. In addition, three samples (S2, S4, S5) crosslinked with different amounts of chitosan with the same DD were also investigated under the same CE (2.4 mg/mL). On the basis of the data, with the increase of chitosan amount, the enzyme loading amount was shown to increase remarkably. One possible reason for this finding was that the chitosan amount can affect the cross-linking network, which is a thicker network with higher capacity. More amide groups can easily fix more PGA molecules. SA also changed with the change of chitosan amount because of the same reason stated before in the DD influence. 3.4. Influence of the Enzyme Concentration on Immobilized PGA. Table 4 shows the effect of varying enzyme concentration from 0.80 to 5.40 mg/mL on

Table 4. Influence of Concentration of Enzyme on Immobilized PGA samples

CE (mg/mL)

qE (mg/gdry support)

IMY (%)

S6 S2 S7 S8

5.4 2.4 1.6 0.8

555.0 313.4 225.7 114.4

68.5 87.1 94.0 95.3

A (U/g)

SA (U/ mgenzyme)


8483.9 7130.4 5634.2 3167.9

15.3 22.8 24.9 27.7

38.5 57.3 62.8 69.8

immobilized PGA cross-linked with chitosan B. From these results, we can clearly see that with the decrease in the original concentration of enzyme solution, the enzyme loading amount decreased while IMY increased. Interestingly, the enzyme activity decreased when SA was increased. Therefore, crosslinking immobilization was a process of adsorption equilibrium. A higher concentration of enzyme naturally leads to a higher enzyme loading amount. With increased SA, less PGA molecules immobilized in the support will create larger space for the contact between the active site and the substrate. In addition, the hydrolysis reaction became easier; thus, the immobilized PGA in low concentration of enzyme presented a particularly high IMY and activity retention. 3.5. Stability of the Immobilized PGA. Evaluating the stability of immobilized PGA is important because obtaining a stable enzyme is one of the main purposes of enzyme immobilization. As shown in Figure 6a, the percentage enzyme activity of chitosan cross-linking method after normalization was determined in substrate solutions with varied pH values. The optimum pH did not change after immobilization. However, according to the results, the pH profile of chitosan cross-linking method was broader than that of free enzyme and physical adsorption. Particularly in the alkaline region, nearly no activity loss was observed for the immobilized enzyme in the pH range 7.9−10. Therefore, PGA after immobilization became more stable against the pH change. Figure 6b shows the thermal stability of chitosan crosslinking method compared with the free enzyme and physical adsorption. The free enzyme is quite sensitive to temperature change, especially in the region of high temperature. The percentage activity was reduced to 27.2% at 57 °C. The method of physical adsorption could improve the thermal stability at a 1951



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support prevented the conformation transition of the PGA molecules at high temperature. Reusability is also a crucial factor in economics. To evaluate reusability, the immobilized enzyme was reused five times. The enzyme activities are shown in Figure 6c. The immobilized PGA retained 85% activity after five times of reuse. This stability can be further improved if we reduce the loss during the process in centrifugation.

4. CONCLUSION PGA was successfully immobilized on MCFs through the crosslinking network method. The PGA molecules were connected to chitosan through multipoint immobilization, which prevented enzyme leaching. The PGA loading amount can reach 555.0 mg/g (dry support), and the SA was up to 27.7 U/ mg-enzyme. Nearly no activity loss was observed for the immobilized enzyme in the pH range 7.9−10, and in the temperature range 37 to 57 °C. Eighty-five-percent activity was retained after five reuses. These performances indicate the possible application of immobilized PGA in biotechnology.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 86-10-62783870. Fax: 86-10-62770304. E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation (Grant Nos. 21276140, 91334201, 20976096, and 21036002), the National Basic Research Foundation of China (Grant No. 2013CB733600), and the Innovative Science and Technology Foundation of PetroChina (Grant No. 2011D-5006-0407).

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REFERENCES

(1) Duggleby, H. J.; Tolley, S. P.; Hill, C. P.; Dodson, E. J.; Dodson, G.; Moody, P. C. E. Penicillin acylase has a single-amino-acid catalytic centre. Nature 1995, 373, 264. (2) Bajpai, A. K.; Bhanu, S. Immobilization of α-amylase in vinylpolymer-based interpenetrating polymer networks. Colloid Polym. Sci. 2003, 282, 76. (3) Petri, A.; Gambicorti, T.; Salvadori, P. Covalent immobilization of chloroperoxidase on silica gel and properties of the immobilized biocatalyst. J. Mol. Catal. B: Enzym. 2004, 27, 103. (4) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451. (5) Huang, X.-J.; Yu, A.-G.; Xu, Z.-K. Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application. Bioresour. Technol. 2008, 99, 5459. (6) Hung, Y.-J.; Peng, C.-C.; Tzen, J. T.; Chen, M.-J.; Liu, J.-R. Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity. Bioresour. Technol. 2008, 99, 8662. (7) Vaidya, B. K.; Ingavle, G. C.; Ponrathnam, S.; Kulkarni, B.; Nene, S. N. Immobilization of Candida rugosa lipase on poly (allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous polymer particles. Bioresour. Technol. 2008, 99, 3623. (8) Hu, X.; Wang, P.; Hwang, H.-M. Oxidation of anthracene by immobilized laccase from Trametes versicolor. Bioresour. Technol. 2009, 100, 4963.

Figure 6. (a) PH stability. (b)Thermal stability. (c)Reusability of immobilized PGA.

certain extent because of the protection of MCFs. Using the cross-linking network immobilization, the optimum temperature was not changed, but the thermal stability was improved significantly, the percentage activity maintained more than 96% at the temperature of 57 °C. The good performance in thermal stability was caused by the fact that the chitosan network and 1952



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(9) Zhou, H.; Yang, L.; Li, W.; Shou, Q.; Xu, P.; Li, W.; Wang, F.; Yu, P.; Liu, H. Improving the stability of immobilized penicillin g acylase via the modification of supports with ionic liquids. Ind. Eng. Chem. Res. 2012, 51, 4582. (10) Zhou, H.; Yang, L.; Li, W.; Wang, F.; Li, W.; Zhao, J.; Liang, X.; Liu, H. Immobilizing penicillin g acylase using silica-supported ionic liquids: The effects of ionic liquid loadings. Ind. Eng. Chem. Res. 2012, 51, 13173. (11) Chong, A.; Zhao, X. Design of large-pore mesoporous materials for immobilization of penicillin g acylase biocatalyst. Catal. Today 2004, 93, 293. (12) Xue, P.; Lu, G.; Guo, Y.; Wang, Y.; Guo, Y. A novel support of mcm-48 molecular sieve for immobilization of penicillin g acylase. J. Mol. Catal. B: Enzym. 2004, 30, 75. (13) Xiao, Q.-G.; Tao, X.; Zhang, J.-P.; Chen, J.-F. Hollow silica nanotubes for immobilization of penicillin g acylase enzyme. J. Mol. Catal. B: Enzym. 2006, 42, 14. (14) Lü, Y.; Guo, Y.; Wang, Y.; Liu, X.; Wang, Y.; Guo, Y.; Zhang, Z.; Lu, G. Immobilized penicillin g acylase on mesoporous silica: The influence of pore size, pore volume and mesophases. Microporous Mesoporous Mater. 2008, 114, 507. (15) Luo, X.; Zhang, L. Immobilization of penicillin g acylase in epoxy-activated magnetic cellulose microspheres for improvement of biocatalytic stability and activities. Biomacromolecules 2010, 11, 2896. (16) Hwang, E. T.; Gu, M. B. Enzyme stabilization by nano/ microsized hybrid materials. Eng. Life Sci. 2013, 13, 49. (17) Zhao, J.; Wang, Y.; Luo, G.; Zhu, S. Covalent immobilization of penicillin g acylase on aminopropyl-functionalized mesostructured cellular foams. Bioresour. Technol. 2010, 101, 7211. (18) Sun, H.; Bao, X. Y.; Zhao, X. Immobilization of penicillin g acylase on oxirane-modified mesoporous silicas. Langmuir 2009, 25, 1807. (19) Chen, C.-I.; Ko, Y.-M.; Shieh, C.-J.; Liu, Y.-C. Direct penicillin g acylase immobilization by using the self-prepared immobilized metal affinity membrane. J. Membr. Sci. 2011, 380, 34. (20) Eldin, M.; El Enshasy, H.; Hassan, M.; Haroun, B.; Hassan, E. Covalent immobilization of penicillin g acylase onto amine-functionalized pvc membranes for 6-apa production from penicillin hydrolysis process. Ii. Enzyme immobilization and characterization. J. Appl. Polym. Sci. 2012, 125, 3820. (21) Na, W.; Wei, Q.; Sun, H.; Nie, Z.-R. Catalase immobilized on siliceous mesocellular foam with controlled window size. J. Porous. Mater. 2013, 20, 75. (22) Schmidt-Winkel, P.; Lukens, W. W.; Zhao, D.; Yang, P.; Chmelka, B. F.; Stucky, G. D. Mesocellular siliceous foams with uniformly sized cells and windows. J. Am. Chem. Soc. 1999, 121, 254.

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Immobilization of Penicillin G Acylase on Mesostructured Cellular

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