Journal of Microbiology and Biotechnology

  • 제26권5호
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  • Pages.829-836
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  • 2016
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Covalent Immobilization of Penicillin G Acylase onto Fe3O4@Chitosan Magnetic Nanoparticles

  • Ling, Xiao-Min (School of Pharmacy, Jiangsu University) ;
  • Wang, Xiang-Yu (School of Pharmacy, Jiangsu University) ;
  • Ma, Ping (School of Pharmacy, Jiangsu University) ;
  • Yang, Yi (School of Pharmacy, Jiangsu University) ;
  • Qin, Jie-Mei (School of Pharmacy, Jiangsu University) ;
  • Zhang, Xue-Jun (United Pharmaceutical Institute of Jiangsu University and Shandong Tianzhilvye Biotechnology Co. Ltd., Jiangsu University) ;
  • Zhang, Ye-Wang (School of Pharmacy, Jiangsu University)
  • 투고 : 2015.11.19
  • 심사 : 2016.02.11
  • 발행 : 2016.05.28
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초록

Penicillin G acylase (PGA) was immobilized on magnetic Fe3O4@chitosan nanoparticles through the Schiff base reaction. The immobilization conditions were optimized as follows: enzyme/support 8.8 mg/g, pH 6.0, time 40 min, and temperature 25 ℃. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 6.2 mg/g-support were obtained. Broader working pH and higher thermostability were achieved by the immobilization. In addition, the immobilized PGA retained 75% initial activity after ten cycles. Kinetic parameters Vmax and Km of the free and immobilized PGAs were determined as 0.113 mmol/min/mg-protein and 0.059 mmol/min/mg-protein, and 0.68 mM and 1.19 mM, respectively. Synthesis of amoxicillin with the immobilized PGA was carried out in 40% ethylene glycol at 25 ℃ and a conversion of 72% was obtained. These results showed that the immobilization of PGA onto magnetic chitosan nanoparticles is an efficient and simple way for preparation of stable PGA.

키워드

Introduction

Owing to recent advances in catalytic engineering, enzymes have been used to produce a number of useful substances [11]. Penicillin G acylase (PGA; E.C. 3.5.1.11) is one of the most important enzymes in the pharmaceutical industry. It catalyzes the hydrolysis of penicillin G to produce 6-aminopenicillanic acid (6-APA) and 7-aminodeacetoxycephalosporanic acid, two key intermediates for the production of β-lactam antibiotics [9]. PGA can also be employed to synthesize β-lactam antibiotics, including amoxicillin [35], ampicillin [4], cefaclor [39], and ceftriaxone [29]. Enzymatic synthesis of penicillins and cephalosporins has advantages like moderate reaction, less reaction steps, and cleaner production [20], which will be a green pharmaceutical production of β-lactam antibiotics in the future. Nevertheless, employing free PGA in the production processes faces some important drawbacks such as poor stability, difficult product separation, and the enzyme recovery [24]. Therefore, immobilization was applied to overcome these drawbacks, making them more economically and efficiently. Until now, various immobilization methods, including entrapment, physical adsorption, covalent coupling, and affinity interaction, have been developed to immobilize enzymes and cells [22]. For example, tannase was entrapped with calcium alginate for removal of tannins from green tea infusion [40]. Sorangium cellulosum, a myxobacterium for production of epothilone, was immobilized in porous ceramics [5].

The carrier is another important factor for preparation of a stable biocatalyst. Nano-scale materials have received increasing attention because of their advantages [7]. In order to make the immobilized biocatalysts more easily recyclable, magnetic particles were introduced as support for immobilization [17].

Owing to their super paramagnetic behavior, large surface area, and low toxicity [6,18], Fe3O4 nanoparticles have been used as supports for immobilization of enzymes, with the advantages including easy separation and effective recycling under an external magnetic field. However, magnetic Fe3O4 nanoparticles were susceptible to agglomeration in solutions because of the strong magnetic dipole–dipole attractions between particles [34]. Moreover, there are not many active groups except a small amount of hydroxyl groups on the surface of Fe3O4 particles. Thus, effective coating of magnetite particles using some biocompatible and biodegradable polymers with specific functional groups is essential to increase its dispersibility and application.

Amongst the biocompatible materials employed for coating, chitosan, a 2-amino-2-deoxy-(1 → 4)-β-D-glucan, has attracted more and more attention because of its cheap, stable, hydrophilic, and biocompatible properties [16]. Many researchers reported the preparation of magnetic Fe3O4@chitosan nanoparticles and their applications for enzyme immobilization [8, 12, 16,]. Various enzymes, including lipase [41], glucose oxidase [30], peroxidase, and hydroperoxide lyase [15], have been immobilized onto chitosan or its derivatives. It can provide an optimal microenvironment for the immobilized enzyme to maintain relatively high biological activity and stability.

In the present work, Fe3O4@chitosan nanoparticles were prepared and used as the carrier for immobilization of PGA. The effects of enzyme/support ratio, immobilization time, and pH on the enzyme loading and efficiency were investigated. Characterization of the immobilized PGA was also performed. The immobilized PGA was also employed for synthesis of amoxicillin to explore the potential industrial applications.

 

Materials and Methods

Enzyme Assay

The activity of PGA was estimated by the p-dimethyl-aminobenzaldehyde method [19] at 40℃ and pH 8.0. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol 6-APA per minute at 40℃ (pH 8.0) from 8% solution (w/v) of penicillin G salt. Experiments were carried out in triplicates, and the standard error was never more than 5%.

Preparation of Magnetic Chitosan Nanospheres

Magnetic chitosan particles were prepared by chemical coprecipitation of Fe2+ and Fe3+ ions by NaOH in the presence of chitosan, followed by hydrothermal treatment [31,36]. Briefly, 2 g of chitosan was dissolved in 100 ml of 2% acetate solution, and then FeSO4 and FeCl3 with a molar ratio of 1:2 were dissolved in the solution. The resulted solution was chemically precipitated at 40°C by adding 30% NaOH dropwise with vigorous stirring under the protection of nitrogen. The suspension was heated to 90℃ and the temperature maintained for 1 h under continuous stirring. The resultant Fe3O4@chitosan nanoparticles were separated with an external magnet, rinsed several times with water, and dried at 50℃ under vacuum.

Immobilization of Penicillin G Acylase onto Fe3O4@Chitosan Nanoparticles

PGA was immobilized on the surface of the Fe3O4@chitosan nanoparticles by cross-linking of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, St. Louis, MO, USA). First, 20 mg of Fe3O4@chitosan particles was dispersed in 0.1 M EDC solution. Then, the suspension was mixed with PGA solution and incubated for the immobilization at 25℃ for 6 h. The protein concentration of the reaction mixture was measured with the Bradford method [2], employing bovine serum albumin as the standard. The immobilization yield (Y) and efficiency (E) were calculated as follows:

where R0 is the total protein content of the crude enzyme preparation; R1 and R2 are the protein concentration of wash solution and supernatant after immobilization; and A1 and A0 are the total activity of the immobilized and free enzymes, respectively.

Characterization of Magnetic Fe3O4 and Fe3O4@Chitosan Nanoparticles

The FTIR spectra of the Fe3O4 and Fe3O4@chitosan nanoparticles were obtained using an FTIR spectrophotometer (PARAGON 500; Perkin Elmer). The diameter and morphology of magnetic chitosan nanoparticles were observed by transmission electron microscopy (TEM) (JEM-200CX, FEI, USA).

Characterization of the Immobilized PGA

The activities of free and immobilized PGAs were measured at different pHs ranging from 6 to 11 at 40℃ to determine the optimal pH. The pH buffers used were 0.1 M phosphate (pH 6 and 7), 0.1 M Tris-HCl (pH 8 and 9), and 0.1 M sodium carbonate (pH 10 and 11). The effect of temperature on the activities of the free and immobilized PGAs was also investigated at temperatures ranging from 30℃ to 60℃. Enzyme activity was measured and calculated according to the above-mentioned methods. All the experiments were repeated three times and the standard deviation was calculated.

Thermal stability was evaluated by incubating the free and immobilized enzymes in a water bath at 50℃ and 55℃ for different times. The samples were withdrawn and subjected to enzyme assay for residual activity. The initial activity was set as 100%.

The reusability was examined by repeated utilization of immobilized PGA to catalyze the hydrolysis of PG. The immobilized enzyme was recovered by magnetic separation after each round and washed with phosphate buffer (50 mM, pH 7.0). The original activity of the immobilized PGA was set as 100%. The residual activity after each round was compared with the initial activity to calculate the relative activity.

The effect of substrate concentration on the activities of PGA was studied at 45°C and pH 9.0. The maximum reaction rate of the enzymatic reaction (Vmax) and the Michaelis–Menten constant (Km) were calculated from a Lineweaver–Burk plot.

Synthesis of Amoxicillin with the Immobilized PGA

Enzymatic syntheses of amoxicillin with the prepared immobilized PGA were executed by using 6-APA and D-p-hydroxyl-phenylglycine methyl ester hydrochloride (HPGME) as substrates. The conditions of the synthesis were as follows: 5 ml of 40% ethylene glycol, 0.08 M 6-APA, 0.16 M HPGME, 0.6 M ZnSO4, and 100 mg of immobilized PGA. The enzymatic reactions were performed at 25℃ with continuous shaking at 180 rpm for various times. The samples were withdrawn and analyzed for the concentration of 6-APA. The conversions were calculated on the basis of the amount of 6-APA converted to amoxicillin.

 

Results and Discussion

Characterization of the Prepared Nanoparticles

To confirm the success of the surface coating, FTIR spectra of the pure Fe3O4, chitosan, and magnetic chitosan were examined and are shown in Fig. 1. The band at 598 cm-1 (1b and 1c) corresponded to the vibration of the Fe-O bonds in the spectrum of Fe3O4. For chitosan, the characteristic adsorption bands appeared at 3,438, 2,899, and 1,069 cm1 (1a and 1c) corresponding to the bending vibration of N-H and partially OH group, aliphatic C-H, and bending vibrations of C-O in the chitosan, respectively. The IR spectra indicated that chitosan and Fe3O4 were both present in the magnetic chitosan nanoparticles, and the Fe3O4 magnetic nanoparticles were coated by chitosan [43].

Fig. 1.FTIR spectra of chitosan (a, blue dot), Fe3O4 (b, black dash), and Fe3O4@chitosan nanoparticles (c, red line).

The TEM images for Fe3O4 nanoparticles and Fe3O4@chitosan nanoparticles are shown in Fig. 2. The size of the particles was in the range of 20-30 nm (Fig. 2A). Owing to the magnetostatic interactions between the nanoparticles [23], the Fe3O4 nanoparticles were physically aggregated (Fig. 2B). After the coating of chitosan, the size of Fe3O4@chitiosan was approximately 5 nm larger than that of Fe3O4 nanoparticles, and the magnetic chitosan nanospheres were monodispersed. These results indicated that the preparation of magnetic chitosan nanoparticles was successful.

Fig. 2.TEM images of Fe3O4 (A) and Fe3O4@chitosan (B) nanoparticles.

Immobilization of PGA onto Fe3O4@Chitosan Nanoparticles

PGA was immobilized onto magnetic chitosan nanoparticles with EDC as the cross-linker through the Schiff base reaction [44]. In the reaction, the –NH2 of EDC will react with the –COOH of aspartic acid, glutamic acid, or C-terminal to form the covalent C=N bonds, and release water molecules. In order to achieve the highest immobilization efficiency and enzyme loading, the effects of several factors, including enzyme/support ratio, and time and pH on the immobilization were studied.

The effect of enzyme/support ratio on the immobilization was investigated. As shown in Fig. 3A, the highest immobilization yield of 77% was obtained at the enzyme:support ratio of 8.8 mg/g. The immobilization efficiency had the same tendency. It might be because the high enzyme/support ratio increased the access chances of PGA molecules onto the surface of Fe3O4@chitosan nanoparticles, resulting in improvement of immobilization yield. However, a higher enzyme/support ratio would result in the aggregation of enzyme onto the support to hide some active sites [21], which could decrease the enzyme activity. Therefore, the optimal enzyme/support ratio of immobilization was 8.8 mg/g.

Fig. 3.Effect of (A) enzyme concentration, (B) immobilization time, and (C) pH on the immobilization of PGA. The reaction conditions were (A) pH 6.0, immobilization time 12 h, 25 ℃ (B) pH 6.0, enzyme concentration 2.7 mg/ml, 25 ℃ and (C) immobilization time 10 h, enzyme concentration 2.7 mg/ml, 25 ℃.

To determine the optimal immobilization time, the immobilization was carried out at varied time from 20 min to 12 h. According to Fig. 3B, the immobilization yield reached 70% after 1.5 h and there was not much change when the immobilization time was increased. However, the efficiency showed the maximal value of 68% at 40 min, and decreased subsequently. Therefore, the optimal immobilization time was 40 min.

The pH influences the adsorption amount of enzyme onto the carrier and the activity of the immobilized enzyme, so the pH of the immobilization reaction ranging from 4 to 9 was examined. In the present work, the maximal immobilization yield of 91% was obtained at pH 4.0, and the immobilization efficiency had the highest value at pH 6.0 (Fig. 3C). The enzyme molecules were adsorbed onto the surface of nanoparticles and linked by EDC. Based on our previous report, the immobilization reaction is a two-step process [32]. First, enzyme was adsorbed onto the nanoparticles. Then, EDC reacted with the enzyme and chitosan as a cross-linker to form covalent bonds. The chitosan layer on the surface exhibited a net positive charge at acidic condition, attracting more enzyme molecules by the electrostatic force. Moreover, there are many hydroxyl groups on the surface of the prepared Fe3O4@chitosan nanoparticles to form hydrogen bonds between nanoparticles and enzyme molecules [10]. However, the poor stability of PGA at acidic condition decreased the activity of the immobilized PGA. Considering the immobilization yield and efficiency, pH 6.0 was selected as the optimal pH for immobilization.

Characterization of the Immobilized PGA

The effect of pH on the activities of the free and immobilized PGAs were investigated by varying the pH of the reaction solution from 6 to 11 and the results are shown in Fig. 4A. Generally, the binding of enzymes to solid supports occasionally results in a shift of the optimal pH [42]. In the present work, the optimal pH of the immobilized PGA was 10.0, shifting from 9.0 for the free enzyme. A broader working pH of the immobilized PGA was achieved compared with the free one. The immobilized enzyme retained more than 70% relative activity in the pH range 7.0-11.0. The free enzyme had only 8% relative activity at pH 11. The higher relative activity might be attributed to the multipoint attachment of PGA with Fe3O4@chitosan nanoparticles [13], which buffered the variability of enzyme molecules under varying pH.

Fig. 4.Effects of temperature (A) and pH (B) on the activities of the free and immobilized PGAs.

The effect of temperature on the activity of free and immobilized PGAs is shown in Fig. 4B. Immobilization did not change the optimal temperature (45℃) of PGA. Compared with the free enzyme, the immobilized PGA exhibited a higher relative activity in the investigated temperature range. Higher activity under varying temperature for the immobilized PGA could be explained in that binding of the support might limit the conformational mobility of the enzyme molecules at high temperature, protecting it from inactivation [27]. Therefore, free PGA could easily undergo denaturation, whereas immobilized PGA is protected in terms of rigid conformation [31], and the immobilized PGA could retain its high catalytic activity.

The thermal stability of the free and immobilized PGAs was investigated by measuring the relative residual activity after incubating them at 50℃ and 55℃ for different times. As illustrated in Fig. 5, the immobilized PGA attained a smaller rate of thermal inactivation relative to the free PGA. The immobilized PGA retained 80% and 45% activity after incubation for 180 min at 50℃ and 55℃, respectively. Comparatively, the free one only retained 50% at 50℃ for 180 min and lost all the activity at 55℃ for 90 min. This means the immobilization of PGA onto Fe3O4@chitosan nanoparticles improved the thermal stability. The increased stability of immobilized PGA could be ascribed to the enhancement of enzyme rigidity and conformational flexibility by immobilization, preventing the conformation change of PGA at high temperature [27].

Fig. 5.Effect of temperature on the stability of free and immobilized PGAs.

The reusability of immobilized enzyme is one of the most important properties in industrial applications. The immobilized PGA was employed in repeated uses to test the reusablity. According to Fig. 6, the immobilized PGA showed good reusablity, retaining 77% of its initial activity after 10 reuses. The activity loss could be attributed to enzyme deactivation and protein leakage during washing and in the repeated uses [3].

Fig. 6.Reusability of the immobilized PGA.

The kinetic parameters Km and Vmax were calculated from double reciprocal plots in Fig. 7. The Vmax value of free PGA (0.113 mmol/mg/min-protein) was higher than that of immobilized PGA (0.059 mmol/min/mg-protein). The Km for the immobilized PGA (1.19 mM) was higher than that of the free PGA (0.68 mM), indicating a lower affinity of PGA on Fe3O4@chitosan towards the substrate. The increase of the Km value may be due to either the structural changes of enzyme under the immobilization procedure [33], or the lower accessibility to the substrate for approaching the active sites caused by steric hindrance of the Fe3O4@chitosan nanoparticles [1].

Fig. 7.Lineweaver-Burk plot of the free and immobilized PGAs.

Although many papers regarding immobilization of PGA have been published in the past years, there is still a lot of work pending to do for the improvement. In the previous work, the biocompatible material chitosan was coated onto magnetic core nanoparticles, and PGA was immobilized onto the modified nanoparticles. It is a simple way to prepare the immobilized enzyme, compared with other covalent immobilization processes, which is shown in Table 1. The high immobilization yield of 77% was achieved, and it was 1.14-, 1.42-, and 2.21-fold of that immobilized on mesostructured cellular foam (MCF) [27], aminopropyl-functionalized silica (APFS) [25], and Fe3O4@SiO2 [45] with physical adsorption. For covalent coupling, the immobilization yield (77%) of this work was also much higher than that of the previous reports. There were only 16.2% of the PGA from Bacillus megaterium on Fe3O4@SiO2-NH2 [26] and 75.5%, 56.9%, and 34.8% of PGA from Escherichia coli on MCF [38], Co-MCF-48 [37], Fe3O4@SiO2, respectively. The Km of the immobilized PGA of the present work was 1.19 mM, which is much lower than that on MCF [38]. After five cycles, 87% residual activity of the immobilized PGA was retained, higher than that of other nanomaterials such as 73% of MCF [38], 81.7% of Co-MCM-48 [38], and 60% of Fe3O4@SiO2 [45]. Hence, the prepared Fe3O4@chitosan magnetic nanomaterials are ideal materials for the immobilization of PGA.

Table 1.aThe residual activity after five cycles. NA: Not available.

Synthesis of Amoxicillin with Immobilized PGA

Based on the previous reports [1,3,13,27], PGA has been reported as a suitable catalyst for amoxicillin synthesis at appropriate reaction conditions. Amoxicillin synthesis was selected as a model reaction to evaluate the catalytic efficiency of the prepared immobilized PGA. The catalytic reactions were carried out in the presence of Zn2+ because it could complex with amoxicillin to enhance the production of amoxicillin [14]. The time course of conversion process is shown in Fig. 8. A yield of 72.2% was obtained after 12 h reaction. Then the reaction rate decreased, possibly because of the simultaneous decrease in the concentration of the reacting species and deactivation of enzyme. The conversion was only improved to 73% after 18 h of reaction. Compared with the previous report [35], there was 7% improvement. In consideration of the improved thermal stability, excellent reusability, and easy recovery, the prepared immobilized PGA has potential applications in the industrial enzymatic synthesis of β-lactam antibiotics.

Fig. 8.Time course of the enzymatic synthesis of amoxicillin with the immobilized PGA.

In conclusion, in this work, Fe3O4@chitosan nanoparticles were prepared for the immobilization of PGA. The immobilized PGA on the prepared magnetic nanoparticles has higher activity, good reusability, and higher thermal stability over the range of tested pH and temperature. The Vmax and Km were determined to be 0.059 mmol/min/mg-protein and 1.19 mM, respectively. The immobilized PGA was employed to synthesize amoxicillin with 6-APA and HPGME in 40% ethylene glycol, and a conversion of 72.2% was achieved after 12 h reaction. On the basis of these results, it was concluded that the prepared magnetic nanoparticles could be excellent support for immobilization of PGA and the immobilized enzyme has promising industrial applications.

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