Immobilization of Penicillin G Acylase in Epoxy-Activated Magnetic

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Biomacromolecules 2010, 11, 2896–2903

Immobilization of Penicillin G Acylase in Epoxy-Activated Magnetic Cellulose Microspheres for Improvement of Biocatalytic Stability and Activities Xiaogang Luo and Lina Zhang* Department of Chemistry, Wuhan University, Wuhan 430072, China Received June 11, 2010; Revised Manuscript Received September 10, 2010

We prepared magnetic cellulose porous microspheres (MCM) with mean diameter of ∼200 µm by employing the sol-gel transition (SGT) method from a mixture of magnemite ferrofluid and cellulose dissolved in 7 wt % NaOH/12% urea aqueous solvent precooled to -12 °C. Subsequently, the cellulose microspheres were activated with epoxy chloropropane to enhance loading efficiency of biomacromolecules. Their morphology, structure, and properties were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction, and vibrating-sample magnetometer. The results indicated that the spherical magnetic γ-Fe2O3 nanoparticles with mean size of 10 nm were uniformly dispersed and embedded in the cellulose substrate of MCM, and the structure and nature of γ-Fe2O3 were conserved perfectly. Penicillin G acylase (PGA) as a biocatalyst was immobilized successfully in the porous microspheres, as a result of the existence of the cavity and affinity forces in the activated cellulose matrix. The immobilized PGA exhibited highly effective catalytic activity, thermal stability, and enhanced tolerance to pH variations. Furthermore, the cellulose microspheres loaded with the enzymes could be removed and recovered easily by introducing a magnetic field, leading to an acceptable reusability. Therefore, we have provided a simple and biocompatible support for the enzyme immobilization, which will be promising for the applications in the biomaterial fields.

Introduction Enzymes are versatile biocatalysts having high specificity, and their applications have been found in areas of biomaterials and organic syntheses.1 However, the major drawbacks for their industrial applications are low thermal stability and solvent stability as well as the difficulty in recycling. Immobilized enzymes (IMEs) usually can enhance the stability and achieve ease recycling and have higher activity than native enzymes, whereas the support material is a very important factor.2 Immobilization of proteins onto solid supports is a promising biotechnological application because it is easy to separate proteins from the reaction media for repeated use. Therefore, IMEs have already been extensively investigated.3 For solid support, agarose gel beads have been chemically modified with aˆ-casein to display reactive glutamine (Gln) residues on the support surface.4 Novel calcium titanium phosphate-alginate (CTPalginate) and hydroxyapatite-alginate (HAp-alginate) microspheres have been used as enzyme delivery matrices and bone regeneration templates.5 Alginate-chitosan core-shell microcapsules as a biocompatible matrix for enzyme immobilization have been developed, and the enzyme loading efficiency is higher in the barium alginate core (100%) as compared with the calcium alginate core (60%).6 Films prepared from cellulose with polyamidoamine (PAMAM) dendrimers exhibit a better performance for immobilization of laccase than those prepared by simple mixing of the cellulose and the dendrimer.7 Penicillin G acylase (PGA) is a major industrial biocatalyst, and it has been used in the enzymatic production of 20 000 t a-1 of 6-aminopenicillanic acid (6-APA). PGA is initially * To whom correspondence should be addressed. Tel: +86-27-87219274. Fax: +86-27-68762005. E-mail: [email protected]; linazhangwhu@ gmail.com.

employed as an industrial catalyst for the manufacture 6-APA from penicillin G.8 A simple, safe, and biocompatible support for the enzyme immobilization is essential for successful biotechnology applications. Therefore, considerable efforts are still being made to immobilize PGA on solid supports, including gels, resins or inorganic supports, biopolymer supports, and magnetic beads. Various immobilization methods have been used to improve the biocatalytic activity, stability, and recycling.8-12 It is noted that some biopolymers, such as aldehyde-agarose,13 inclusion in glatine,14 gelatin-chitosan,15 and concanavalin A,16 are satisfactory supports. Cellulose is an almost inexhaustible natural polymer with fascinating structure and properties such as biocompatibility, biodegradability, and safety, leading to increasing demands on its products.17,18 Usually, chemically modified cellulose is the primary candidate of cellulose-based materials.19 Therefore, cellulose derivatives have been used in enzyme immobilization, such as CMcellulose, TEAE-cellulose, DEAE-cellulose, and so on.9 However, direct utilization of pure regenerated cellulose as a support for enzyme immobilization has never been reported because many cellulose solvents such as N,N-dimethylacetamide (DMAc)/ LiCl, DMSO/triethylamine/SO2, ammonia/ammonium salt (NH3/ NH4SCN), and N-methylmorpholine-N-oxide (NMMO) are toxic to the organism system. In our laboratory, a new solvent, 7 wt % NaOH/12 wt % urea aqueous solution precooled to -12 °C, has been developed to dissolve cellulose. It is worth noting that the cellulose dissolution at low temperature arises as a result of a fast dynamic self-assembly process among solvent small molecules (NaOH, urea, and H2O) and the cellulose macromolecules.20 Moreover, magnetic cellulose microspheres (MCMs) have been prepared by in situ synthesis of Fe3O4 in the cellulose pores, showing excellent adsorption capacity for BSA and sensitive magneticinduced delivery.21 Therefore, it might be an exciting idea to

10.1021/bm100642y  2010 American Chemical Society Published on Web 10/04/2010

Immobilization of PGA in Epoxy-Activated MCMs

immobilize enzymes in the MCMs to achieve easy recycling and to enhance their stability and catalytic activity. There are porous structure and numerous functional groups in the regenerated cellulose substrate, in which PGA may be encased and bound on the pore wall to immobilize. Moreover, this is a novel, simple and environmentally friendly pathway, and the sulfate content in the regenerated cellulose products has been determined to be essentially zero.22 The “green” preparation process and free-sulfate in cellulose support are very important for the bioapplication. Until this day, PGA supported on the regenerated cellulose matrixes fabricated via a “green” process has been reported scarcely. Epoxy-activated supports are almost ideal systems to develop a good course for enzyme immobilization. On one hand, epoxy groups are highly stable at neutral pH, even under wet conditions, and commercial supports can be prepared quite far from the position where the enzyme has to be immobilized. On the other hand, epoxy supports are able to react with different nucleophilic groups of the protein (e.g., amino, hydroxyl, or thiol moieties).23 It is not hard to imagine that immobilization of PGA in epoxy-activated MCMs can improve the biocatalytic stability and activities. In the present work, we attempted to fabricate MCMs, followed by activation by epoxyl chloropropane to enhance the enzyme loading efficiency of PGA. The morphology, structure, and properties of the immobilized PGA were characterized. We are optimistic to provide a new “green” pathway for immobilization of PGA in the magnetic cellulose porous matrix, improving the biocatalytic activity and the stability of PGA.

Experimental Details Materials. Cellulose (cotton linter pulp) was provided by Hubei Chemical Fiber Group (Xianfan, China). Its viscosity-average molecular weight (Mη) was determined by the use of an Ubbelohde viscometer in LiOH/urea aqueous solution at 25 ( 0.05 °C and calculated according to the Mark-Houwink equation24 to be 8.1 × 104. PGA (E.C.3.5.11, 804 IU/mL) and penicillin G potassium salt were purchased from Sigma-Aldrich, Shandong Lukang Pharmaceutical, China. Epoxyl chloropropane and phenyl acetic acid (PAA) were obtained from Limited Company of Biochemistry, Shanghai, China. Other chemicals were purchased domestically and used without further purifications. Preparation of Magnetic Cellulose Microspheres. Maghemite (γFe2O3 nanoparticles) ferrofluid was synthesized by coprecipitation of a stoichiometric mixture of ferrous and ferric chlorides in an ammonium hydroxide solution. The resultant magnetite (Fe3O4) precipitate was acidified by nitric acid and oxidized to translate into maghemite (γFe2O3) at 90 °C with iron(III) nitrate. A new reactor device, submerged circulative impinging stream reactor (SCISR), was used for this purpose. To obtain a stable magnetic dispersion compatible with a cellulose gel, particles were coated by citrate anions.25 After precipitation with acetone, the coated particles were dispersed in water to obtain a stable ferrofluid when the pH value was adjusted to 7.25.25,26 The magnetic regenerated cellulose magnetic microspheres were fabricated by the sol-gel transition (SGT) as follows. A solution with NaOH/urea/H2O of 7:12:81 by weight was cooled to -12 °C. A desired amount of cellulose (8 g) and quantitative magnetic fluid were immediately dispersed in the precooled solvent (200 mL) with vigorous stirring to obtain a mixture of magnetic nanoparticles and cellulose solution. A well-mixed suspension containing 300 mL of paraffin oils and 10 g of Span 80 were dispersed in a reactor. The resulting suspension was stirred at 1000 rpm for 30 min. Subsequently, 60 mL of the cellulose solution containing the nanoparticles was dropped into the suspension within 1 h at room temperature. After stirring for 2 h, the suspension in the vessel was kept for an additional 3 h at the same stirring speed. Dilute hydrochloric acid (10%) was added until the pH reached 7, at which time the suspension formed regenerated MCMs containing the γ-Fe2O3 nanoparticles. After the removal of the liquid paraffin, about

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50 mL of the MCM samples in the substratum was obtained. The resulting microspheres were washed with deionized water and then with acetone three times to remove the residual paraffin oils and Span-80.27 Finally, the MCM products were freeze-dried or stored in 20% alcohol at 0-5 °C. Activation of Magnetic Cellulose Microspheres. MCMs (4 g) were added to a solution of 12 mL epoxy chloropropane in 40 mL of DMSO; then, 40 mL of 2 N NaOH was slowly dripped in the reaction mixture system. The mixture was allowed to stand for 6 h at room temperature; then, the resulting solution was removed by magnetic separation. The activated MCMs were obtained after washing with acetone and distilled water until the eluate was neutral.28 The epoxyl groups in MCM were determined by a reaction between the oxirane ring and sodium thiosulfate, followed by titration with 0.01 mol/L hydrochloride acid to neutralize any released OH- in the reaction.29 The amount of epoxy group in the activated MCM was estimated to be 1.83 mmol/g (dry). The epoxide group readily reacts with enzyme. Under relatively mild conditions, these groups in the cellulose support may react with both amino groups and carboxyl group in enzyme. Hence, PGA enzyme can be coupled to supports bearing epoxide groups to improve its stability. This is a type of immobilization, and its advantages have been discussed.30 In our finding, the activated MCM was used as a new support for the immobilization of enzymes. Preparation of the Immobilized PGA. The PGA enzyme immobilization was carried out as follows. About 0.1 g of the activated magnetic carriers was mixed with 2 mL of 100 U/mL PGA enzyme solution in 0.1 mol/L phosphate buffers (pH 8.0). The mixture was shaken at room temperature for 24 h at 250 rpm. The supernatant was removed by magnetic separation, and the immobilized PGA (IMP) was washed with 1 mol/L (NH4)2SO4 and phosphate buffer, respectively, to remove the noncovalently coupled enzymes on the supports. The IME was stored at 4 °C. The total amount of PGA protein was assayed according to the Coomassie Brilliant Blue G-250 method with bovine serum albumin (BSA) as the standard.31 Characterization of the Support Materials. Transmission electron microscopy (TEM) of the Fe2O3 nanoparticles was carried out on a JEOL JEM-2010 (HT) electron microscope using an accelerating voltage of 200 kV. The morphology of the microspheres was observed by scanning electron microscope (SEM) (FESEM, SIRON TMP, FEI). The MCM samples in the wet state were frozen in liquid nitrogen, snapped immediately, and freeze-dried using a lyophilizer (CHRIST Alpha 1-2, Germany). The microspheres were dried at ambient temperature and were coated with Pt for the SEM observation. Their cross-section display was opened with a blade drawn. FT-IR spectra were recorded on a Nicolet FTIR spectrometer (Nicolet NEXUS 670) by KBr pellet method. X-ray diffraction of the magnetic microspheres was carried out on the X-ray diffractometer (Rigaku D/MAX-2400 XRD with Ni-filtered Cu Ka radiation). The XRD patterns were recorded in the region of 2θ from 10 to 60°. Samples were ground into powders and dried in a vacuum oven at 60 °C for 48 h before characterization. The magnetization curves of the dried microspheres were recorded with a vibrating-sample magnetometer (VSM SQUID, MPMS XL-7, Quantum Design) at 25 °C. Measurement of Enzyme Activity. Soluble PGA ActiVity. We determined the enzyme activity of PGA by measuring the penicillin G enzymatic product 6-APA spectroscopically. One unit of PGA (U) is defined as the amount of enzyme that produces 1 mmol 6-APA per min with 2% (w/v) penicillin G as substrate solvated in phosphate buffer (0.1 mol/L, pH 8.0) at 37 °C. The amount of 6-APA was determined with the method of p-dimethylaminobenzaldehyde (PDAB).32 The solution mixture of 20% (v/v) of acetic acid (A), 0.5% (w/v) of NaOH (B), and 0.5% (w/v) of PDAB in methanol (C) was prepared. Subsequently, A, B, and C were added to 0.5 mL of the sample solution by the volume ratio 2:1:0.5. After carrying out the reaction for 20 min at room temperature, we determined the optical density (OD) of the solution by using a UV spectrophotometer at 415 nm.31

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Immobilized PGA ActiVity. The enzyme activity of immobilized PGA was measured in the same manner as in the determination of the soluble PGA activity described in the previous section, except that the reaction mixture was continuously stirred during the reaction. The activity yield remaining after immobilization was defined as

activity yield (%) )

C × 100 A

(1)

and the immobilization yield was calculated by

A-B × 100 A

immobilization yield (%) )

(2)

where A is the total activity of enzyme added in the initial immobilization solution, B is the activity of the residual enzyme in the immobilization and washing solution after the immobilization procedure, and C is the activity of the immobilized PGA. The maximum activity is defined as 100%, and the relative activity refers to the percentage that an enzyme activity accounts for the highest one. Kinetic Constant of PGA. Kinetic study was performed on selected samples in the substrate concentration range of 0.1 to 1.0% (w/v). The Michaelis-Menten equation for a single substrate, uninhibited enzyme reaction is as follows

V)

Vmax[S] Km + [S]

(4)

In this case of the hydrolysis of PenG catalyzed by the IME, the reaction rate was inhibited by PAA. The apparent Michaelis-Menten equation can be expressed as

V)

Vmax[S] [PAA] Km 1 + + [S] Ki,PAA

(

)

Table 1. Physical Properties of MCM and Activated MCM Fe2O3 content Fp w P V S R1 R2 sample (%) (mg/mL) (%) (%) (mL/g) (m2/g) (µm) (nm) MCM activated MCM

15.6 15.8

1.03 1.08

92.4 95.4 91.8 97.6

12.2 11.2

16.6 17.1

200 200

185 180

(3)

where V is the rate of the reaction, [S] is the concentration of the substrate, Km is the apparent constant, and Vmax is the maximum of reaction velocity. The Lineweaver-Burk plot for the Michaelis-Menten equation can be expressed as

Km 1 1 1 ) + V Vmax [S] Vmax

Figure 1. TEM images of γ-Fe2O3 nanoparticles in water.

(5)

where Ki,PAA is the constant of PAA inhibition and [PAA] is the concentration of PAA.

Results and Discussion Structure and Properties of Solid Supports. TEM images of the Fe2O3 nanoparticles in water are shown in Figure 1. The Fe2O3 nanoparticles existed as spheres with mean size of ∼10 nm, showing a good dispersion in water. A two-parameter fit of the magnetization curves allowed the determination of the mean diameter (d0 ) 10 nm) and the distribution width (σ ) 0.4). These were good indications that the Fe2O3 nanoparticles may be dispersed homogeneously in the aqueous solution containing cellulose, leading to good dispersion of Fe2O3 in the cellulose matrix. The physical properties of MCM and activated MCM are summarized in Table 1. Similar to the MCM, the activated MCM microspheres containing iron oxide exhibited relatively

high water content (w), wet density (Fp), porosity (P), pore volume (V), and the specific surface area (S) as well as better average particles size (R1) and mean pore size (R2), providing sufficient cavities to immobilize the PGA enzyme. Both MCM and activated MCM exhibited compatible microporous structures, density in the wet state, and more than 95% porosity. Interestingly, the Fe2O3 content and specific surface areas of activated MCM were higher than those of MCM, whereas its water content was lower than that of MCM. This could be caused by the more compact structure of the microspheres after activation. This suggested that the activated MCM may be an immobilizing enzyme with higher stability than the original MCM. Figure 2 shows the SEM images of the surface morphology (a,b) and the cross-section structure (c,d) of MCM. The microspheres displayed good spherical shape, and the both the surface and the interior of MCM exhibited porous structure. The pore formation was a result of the H2O-induced phase separation during the sol-gel process, where the solvent-rich regions contributed to the pore formation.33 As shown in Figure 2b,d, the large pores in the MCM microspheres containing Fe2O3 nanoparticles almost disappeared, suggesting that the spherical magnetic γ-Fe2O3 nanoparticles were embedded in the porous cellulose matrix. It further confirmed that iron oxide nanoparticles could be readily impregnated into the cellulose matrix and that they were bound to the cellulose macromolecules via electrostatic interactions34 to protect the structure and the nature of γ-Fe2O3 nanoparticles. Figure 3 shows the FT-IR spectra of (a) γ-Fe2O3 nanoparticles, (b) native cellulose, (c) magnetic composite microspheres (MCM), and (d) activated MCM. There was a strong peak at ∼576 cm-1 in Figure 3a, assigned to the characteristic absorbance of Fe2O3.35 In addition, the peaks at 1622 and 1362 cm-1 were assigned to absorbance of the asymmetric and symmetric stretching vibration of COO- of the citric acid modifier, respectively, indicating the presence of γ-Fe2O3 nanoparticles coated by COO- group. Native cellulose in Figure 3b exhibited



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Figure 4. Powder X-ray diffraction pattern of (a) γ-Fe2O3 nanoparticles, (b) native cellulose, (c) magnetic composite microspheres MCM, and (d) and activated MCM. Figure 2. SEM images of the (a) surface morphology and (b) surface structure of MCM and (c) cross-section display and (d) cross-section display structure of MCM.

Figure 3. FT-IR spectra of (a) γ-Fe2O3 nanoparticles, (b) native cellulose, (c) magnetic composite microspheres (MCM), and (d) activated MCM.

characteristic absorbance of cellulose I,36 whereas MCM (b,c) exhibited absorbance regenerated from cellulose I to cellulose II,37 suggesting that the structure of the cellulose has been changed to cellulose II during the preparation process. The peaks at 3300-3450 cm-1, corresponding to stretching vibrations of hydroxyl groups of cellulose, shifted to higher wavenumbers and became broader, indicating that strong interaction between cellulose and Fe2O3 nanoparticles through hydrogen bonding occurred in MCM and activated MCM. This suggested that the stability of Fe2O3 nanoparticles in cellulose microspheres was enhanced. The higher stability is very important for the applications of the composite microspheres. In Figure 3d, the additional absorbance at 560 and 1120 cm-1 appeared in the activated MCM, as compared with MCM, indicating the existence of the epoxy groups. It was clearly demonstrated that the activated MCM contained epoxy groups.23 X-ray diffraction has been used to study the structure and interaction between the inorganic particles and cellulose matrix. The powder X-ray diffraction patterns of (a) γ-Fe2O3 nanoparticles, (b) native cellulose, (c) MCM, and (d) activated MCM are presented in Figure 4. The XRD peaks of the γ-Fe2O3

nanoparticles (Figure 4a) agreed well with those of γ-Fe2O3, and no other crystalline phases were observed. The mean size of γ-Fe2O3 crystalline was also estimated by Scherer’s equation to be 10.2 nm, which is in good agreement with that measured by TEM.26 The diffraction peaks at 2θ ) 14.8, 16.3, and 22.6° for (11j0), (110), and (200) planes, respectively, are characteristic peaks for cellulose I crystal, and those at 2θ ) 12.1, 19.8, and 22.6° for (11j0), (110), and (200) planes are peaks for cellulose II crystal.38 The initial cellulose (Figure 4b) has typical crystalline peaks of cellulose I, whereas the MCM and the activated MCM microsphere (Figure 4c,d) samples exhibited characteristic peaks of the cellulose II. Moreover, they displayed some distinct peaks at 36.02, 43.56, and 57.70° assigned to the (220), (311), and (400) planes of γ-Fe2O3, respectively. The mean size of the Fe2O3 crystallites was estimated using Scherer’s equation to be the same as that of the prepared Fe2O3 nanoparticles (Figure 4a). The results indicated that the γ-Fe2O3 nanoparticles were embedded in the cellulose matrix, and the Fe2O3 structure hardly changed. This clearly indicated that the Fe2O3 nanoparticles in the cavity of the porous cellulose matrix were protected because of the existence of a shell to prevent removal. Figure 5 shows the magnetic hysteresis loops of (a) γ-Fe2O3 nanoparticles, (b) MCM, and (c) activated MCM at 25 °C (left) and a photograph of the immobilized PGA magnetic microspheres attracted rapidly by a magnet (right). A magnified view of the -200 to 200 Oe regions is shown in the insert. All of these samples exhibited an extremely small hysteresis loop, which was characteristic of superparamagnetic nanoparticles. The saturation magnetization intensity of Fe2O3 nanoparticles (Figure 5a) was 42.2 emu g-1, and that of MCM (b) and activated MCM (c) obtained from the hysteresis loop was 7.6 and 7.9 emu g-1, respectively, depending on the Fe2O3 content. This revealed that MCM and activated MCM possessed good magnetic properties. As shown in Figure 5 (right), the immobilized PGA magnetic microsphere could be attracted rapidly by a magnet within 1 min. Therefore, the MCM microsphere had a sensitive magnetic response. It was not hard to imagine that their recovery could be achieved by applying a magnetic field from the reaction system. In view of the results mentioned above, a model describing the process of the epoxy activation of the carrier and enzyme immobilization is proposed in Scheme 1. The microspheres as solid supports had porous structure, and there was an abundance of hydroxyl groups in the cellulose matrix. Therefore, the

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Figure 5. Magnetic hysteresis loops of (a) γ-Fe2O3 nanoparticles, (b) magnetic composite microspheres MCM, and (c) activated MCM at 25 °C (insert: a magnified view of the -200 to 200 Oe regions) (left) and photograph of the immobilized PGA magnetic microspheres attracted rapidly by a magnet (right). Scheme 1. Schematic Depiction of the Activation of Epoxy and Immobilization of Enzyme in the Magnetic Cellulose Microspheres (MCMs)

enzyme could easily bond on the activated cellulose microspheres through hydrogen bonds as a result of the presence of porous structure in the cellulose matrix and the affinity forces from -OH groups and the Fe2O3 nanoparticles. On the basis of the two-step mechanism of enzyme immobilization,39-41 the immobilization of PGA in the activated MCM with epoxy groups proceeded via a physical adsorption of the proteins on the surface of cellulose support; then, the high concentration of reactive groups in the PGA and of epoxy groups in the support could make possible a rapid “intramolecular” covalent reaction among them. It has been reported that the probability of the formation of multiple covalent linkage between PGA and epoxy could be enhanced, leading to the increase in the stability PGA immobilized microspheres.42,43 The immobilization of enzymes inside porous MCM may increase the stability by preventing any intermolecular process and by preventing the enzyme from interactions with external interfaces.44 Therefore, the cavities in the cellulose microspheres could provide not only a space for storage of the enzyme but also a wall shell to protect its structure and nature. Kinetics of Immobilized Penicillin G Acylase. The relative activities of the immobilized PGA were determined in the solutions at different pH, ranging from 5 to 10 at 37 °C. Figure 6 shows the effect of pH (a) and temperature (b) on the enzyme activities. The optimum pH value for the catalysis of native PGA was found to be pH 8. The immobilized PGA displayed the highest activity at pH 8.5, which is in good agreement with the reported value by Wang et al.45 Figure 6b shows the effect of temperature on enzyme activity. After reacting for 3 min,

the activity of PGA enzyme was measured. The immobilized PGA achieved a remarkable increase in the optimum reaction temperature and maintained 100% activity at 45 °C, whereas the optimum reaction temperature of its soluble enzyme was 40 °C. Namely, the optimum reaction temperature of the immobilized PGA was higher than that of its soluble counterpart. The changes of the activity of PGA were investigated in phosphate buffers containing different concentrations of penicillin G. Usually, when the substrate concentration is at a lower level, the enzyme exhibits an improved activity with an increase in the concentration. The IME carriers would be broken before the kinetic constants of the IME were measured, and as a result, the influence of the diffusion problem in the inner carriers can be impaired. The values of kinetic constants, Km and Vmax, were calculated according to the equation of Lineweaver-Burk (eq 4). The values of Km and Vmax were determined to be 1.192 × 10-5 mol/L, 42.95 µmol/min for native enzyme, and 8.92 × 10-5 mol/L and 28.64 µmol/min for IME, respectively. This trend was reasonable, similar to other magnetic hydroxyl enzyme carrier.45 The major kinetic constant, Km, for the IME was much higher than that for native enzyme, indicating better affinity of immobilized PGA to the cellulose substrate. The penicillin G could be hydrolyzed to two products, PAA and 6-APA, which were shown to inhibit PGA. In our findings, 0.5 and 0.25% (w/v) of PAA were added to the phosphate buffer containing 2% (w/v) of penicillin G, respectively, and we observed that PAA inhibited the activity of PGA at a higher concentration compared with that of the control experiment without the PAA.



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Figure 6. Effect of (a) pH and (b) temperature on enzyme activities.

Figure 7. Relative activity of the free and the immobilized PGA incubated in 0.1 M phosphate buffer (pH 8.5) at 45 °C at different time.

Therefore, PAA should be a competitive inhibitor, and the inhibiting constant KPAA was 2.81 × 10-4 mol/L, calculated from the eq 5. Thermal Stability of the Immobilized PGA. The thermal stability of the immobilized PGA is one of the most important criteria dealing with its applications. Figure 7 shows the relative activity of the free and the immobilized PGA incubated in phosphate buffer at 45 °C for different time periods. Both PGA samples exhibited a similar trend; namely, the relative activity decreased with an increase in time. However, as shown in Figure 7b, the relative activity of the immobilized PGA was much higher than that of free PGA at 45 °C for 2 h (normal use time). Clearly, the immobilized PGA was more stable than the free one at relatively high temperature, indicating a better thermal stability. The increase in the thermal stability of immobilized PGA was caused by the stabilization through the weak intermolecular forces and the prevention of the autolysis of the PGA.23 Therefore, the thermal stability of the immobilized PGA was significantly improved through the immobilization in the porous structure of the cellulose matrix. It is worth noting that when the MCMs containing γ-Fe2O3 were applied to immobilize the PGA, it led to further improvement in the yield of immobilization and the biocatalytic stability. Reusability of the Immobilized PGA. The most important and attractive advantage of immobilization is the reusability of PGA. The catalyst reusability was determined by measuring of the stability of the immobilized PGA as a function of number of reusages. Residual activity of the immobilized PGA during the reuse is shown in Figure 8. The activity of the first batch was taken to be 100%. The immobilized PGA exhibited a better reusability, retaining 95.9% residual activity after being used 10 times. Moreover, there was no further loss in activity after these cycles. The enzyme immobilized in the cellulose matrix

Figure 8. Operation stability of immobilized PGA.

has many advantages, such as the prevention of protein contamination in the carrier, leading to high effective activity and good stability of enzyme catalyst, repeated use, easy separation from the reaction mixture, recycling, and so on. Therefore, this work is expected to provide a new support that rendered excellent immobilization of the enzyme catalysts. Furthermore, this new support was fabricated via a “green” process and will be promising for the applications in the biomaterial fields.

Conclusions We successfully activated MCMs by using epoxy chloropropane to promote the covalent immobilization of enzyme. PGA was immobilized successfully in the porous structure of the

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MCMs. The existence of the cavity in the cellulose substrate and affinity forces from -OH groups and the Fe2O3 nanoparticles played an important role in the improvement of the enzyme immobilization, leading to the protection of the structure and nature of biocatalyst. The immobilized PGA exhibited high effective activity, thermal stability, and enhanced tolerance to pH variations as well as good reusability. The immobilization of PGA in the activated MCM with epoxy groups proceeded via a physical adsorption between the proteins and the cellulose support; then, the increased concentration of reactive groups in the PGA and of epoxy groups in the support could make possible a rapid “intramolecular” covalent reaction among them. Moreover, the cellulose magnetic microspheres loaded with PGA could be conveniently and easily separated from reaction solution, leading to recovery of the catalysts. The new immobilization carriers prepared from cellulose solution in aqueous solvent via a simple, easily prepared, and environmental friendly process will have wide applications in the biocatalyst fields. Acknowledgment. This work was supported by National Basic Research Program of China (973 Program, 2010CB732203), National Supporting Project for Science and Technology (2006BAF02A09), the National High Technology Research and Development Program of China (863 Program, 2003AA333040 and 2006AA02Z102), major grants of the National Natural Science Foundation of China (30530850 and 59933070), and the National Natural Science Foundation of China (20474048 and 20874079).

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Immobilization of Penicillin G Acylase in Epoxy-Activated Magnetic

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