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Preparation and enzymatic activity of penicillin G acylase immobilized on core–shell porous glass beads
Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Preparation and enzymatic activity of penicillin G acylase immobilized on core–shell porous glass beads Hang Shi, Yujun Wang ∗ , Guangsheng Luo State Key Laboratory of Chemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 3 February 2014 Received in revised form 17 April 2014 Accepted 17 April 2014 Available online 28 April 2014 Keywords: Porous glass beads Core–shell structure Penicillin G acylase Enzyme immobilization Fixed bed reactor
a b s t r a c t In this work, core–shell porous glass beads were used to immobilize the PGA based on the theory that diffusion is the rate-determining step of a reaction with fast kinetics. To improve the loading amount of PGA, chitosan was grafted on the support to increase the interaction between PGA and the support. The effects of flow rate, chitosan concentration and initial concentration of Pen G on immobilized PGA were investigated. The core–shell structure of the support resulted in a higher efficiency of the diffusion of substrate and products, which improved the activity of immobilized PGA. The conversation rate of Pen G (26.9 mmol/L) reached 91.1% when the residential time was about 30 s in the fixed bed reactor. Meanwhile, the conversation rate of 100 mL of Pen G (134.3 mmol/L) reached 89.1% after 8 h in the reactor with recirculation. Moreover, 97.5% activity was retained after seven days of continuous reaction. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Enzyme biocatalysts have been widely used in the fine chemicals industry, including the food and pharmaceutical industries [1]. Enzyme immobilization solves the common problems of free enzyme, including the lack of long-term stability, low tolerance to organic solvents, and difficult recovery and reuse [2–8]. Among all kinds of enzymes, penicillin G acylase (PGA, E.C. 3.5.1.11) is an important enzyme that catalyzes the hydrolysis of Penicillin G potassium (Pen G) to produce 6-aminopenicillanic acid (6-APA), which is the precursor for manufacturing -lactum antibiotics [9]. Considering the hydrolysis of Pen G, which is a fast reaction, diffusions of substrate and products are the key factors that control the reaction. Therefore, the property of the support remarkably influences the diffusion rates of substrate and products, and then determines the catalytic performance of immobilized PGA. Organic materials are widely used as the supports of immobilized PGA because of the presence of rich functional groups, which provide essential interactions with enzymes [1]. For instance, Eupergit C is commercially used as a support for immobilization. PGA is covalently attached to Eupergit C and used in industrial production [10]. However, the major shortcoming of Eupergit C is its diffusion limitations, the effects of which are more pronounced in kinetically controlled processes [11]. Researchers have also
∗ Corresponding author. Tel.: +86 10 62783870; fax: +86 10 62770304. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.molcatb.2014.04.013 1381-1177/© 2014 Elsevier B.V. All rights reserved.
immobilized PGA onto other organic materials, including aminefunctionalized PVC membranes and glutaraldehyde-activated chitosan [12,13]. Inorganic materials are applied to enzyme immobilization because of their special characteristics, including thermal and mechanical stability, high resistance against organic solvents, and nontoxicity. For example, PGA molecules were immobilized on hollow silica nanotubes [14], in which the thin shell structure of the immobilized enzyme effectively reduced mass transfer resistance that led to a high activity yield. Macro-mesoporous silica spheres were prepared with a micro channel and were used as the support for PGA immobilization [15]. The introduction of macropores increased the enzyme loading amount and decreased the internal mass transfer resistance. This condition resulted in a high apparent enzymatic activity. In addition, PGA was also immobilized on mesostructured cellular foams, macrocellular heterogeneous silica-based monoliths, aminoalkylated polyacrylic supports, and oxirane-modifiedmesoporous silicas [16–20]. These studies strongly illustrated the importance of diffusion rate to catalytic performance. Materials with core–shell structure are also good choices for PGA immobilization. In our previous work, a one-step subcritical water treatment method was developed to prepare porous glass beads which have core–shell structure [21]. Ion exchange properties were further investigated [22], and chitosan was supported on this kind of inorganic material, which was used as a green adsorbent for heavy metal adsorption [23]. Porous glass beads have several advantages, which are as follows: (1) can effectively eliminate the influence of inner diffusion because the distance of mass diffusion is
H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
Fig. 1. The process of hydrolysis reaction catalyzed by PGA immobilized on the porous glass beads.
the thickness of the porous shell, (2) can be easily modified because of the numerous hydroxyl groups on the surface, (3) contain good chemical and thermal stabilities, and (4) can be easily filled and separated based on the appropriate particle size (75–150 m). In this work, PGA immobilized on the chitosan-treated porous glass beads was investigated. As shown in Fig. 1, the thickness of the porous shell was about 4 m. Thus, the process of hydrolysis reaction is considered the contact between the shell layer and the aqueous phase layer. The short distance of mass transfer improves the efficiency of the diffusion of substrate and products. PGA molecules are centralized in a small shell area, which means that the density of the enzyme is larger than those of any other uniform sphere supports. Hence, the contact probability of enzymes and substrates could be increased. To achieve continuous production, the obtained immobilized PGA was filled in a fixed bed reactor. This process could avoid the damage of mechanical stirring and the direct contact of enzyme and alkaline, which ensures good stability of the enzyme. The physical properties of the immobilized PGA were characterized, the effects of flow rate, chitosan concentration, initial concentration of Pen G, and the accumulation of 6-APA on immobilized PGA were studied. 2. Materials and methods 2.1. Materials Glass microbeads with diameters ranging from 75 m to 150 m were obtained from Hebei Chiye Corporation, with the composition of 59.7 wt% SiO2 , 25.1 wt% Na2 O, 9.8 wt% MgO, and 4.9 wt% CaO. Hydrochloric acid (HCl), acetic acid, and sodium hydroxide (NaOH) were produced by Beijing Chemical Company (Beijing, China). Chitosan (DD 85.1%) was purchased from Sinopharm Chemical Reagent Company (Beijing, China). Sodium dihydrogen phosphate (NaH2 PO4 ), dibasic sodium phosphate (Na2 HPO4 ), potassium dihydrogen phosphate (KH2 PO4 ), and glutaraldehyde (GA) were obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). PGA (E.C. 3.5.1.11) was purchased from Shunfeng Haideer Co., Ltd. (Zhejiang, China). Pen G was purchased from Dalian Meilun Biology Technology Co, Ltd. (Dalian, China). 6-APA was obtained from Tokyo Chemical Industry Co., Ltd. (Japan). HPLC-grade methanol was produced by Anhui Fulltime Specialized Solvents & Reagents Co., Ltd. (Anhui, China). All chemicals were used as received without any further treatment. 2.2. Synthesis of porous glass beads grafted by chitosan The porous glass beads were prepared via the subcritical water treatment method based on the reference [22]. In this method, 5.0 g of glass beads and 200 g of water were placed in a tank reactor. The temperature of the reactor was gradually increased to 300 ◦ C, and the pressure was increased to 8 MPa. The subcritical state was maintained for 1 h, after which the reactor was cooled to room temperature. The porous glass beads were separated from water via
41
filtration and were washed several times with water. To replace the metal ions contained in the shell part with hydrogen ions, the obtained porous glass beads were mixed with 500 mL of HCl solution with a concentration of 0.5 M. The mixture was shaken in the SHA-B Incubator Shaker at 160 rpm at 30 ◦ C for 12 h. The obtained modified glass beads were filtered, washed with plenty of water, and dried at 80 ◦ C for 24 h. To obtain a larger specific surface area and a stronger connection with PGA molecules, the surface of the porous glass beads was coated with chitosan before the immobilization of PGA. Chitosan was dissolved in acetic acid (1 wt.%) to obtain the chitosan solution with a concentration that ranged from 0.5 wt.% to 3.0 wt.%. Up to 1 g of the modified glass beads was mixed with 30 g of chitosan solution, and the mixture was then shaken at 160 rpm at 30 ◦ C for 8 h. After removing the supernatant, the porous glass beads that adsorbed chitosan was mixed with 15 g of 1.0 wt.% GA and was shaken for another 12 h at 160 rpm at 30 ◦ C. After filtration, washing, and drying, porous glass beads coated with chitosan were obtained. 2.3. Immobilization of PGA Up to 0.5 g of porous glass beads coated with chitosan were incubated with 30 mL of PGA solution (5.0 mg/ml), 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. After that, the mixture was centrifuged to separate the PGA solution and the porous glass beads with the immobilized PGA. The protein concentration of the supernatant was measured to calculate the PGA loading amount. The PGA loading amount (qE (mg/g)), was calculated according to the following formula: qE (mg/g) =
q1 − q2 m
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 To investigate the activity of immobilized PGA, the hydrolysis of Pen G was performed as follows: the immobilized PGA obtained in Section 2.3 was placed in a fixed bed reactor with a length of 50 mm and an inner diameter of 4 mm. Pen G solution in phosphate buffer (pH 7.9, 0.05 M) was allowed to flow through the fixed bed reactor in a water bath. The substrate and products of the hydrolysis reaction were analyzed via high-performance liquid chromatography (HP series 1200 HPLC system equipped with a wavelength-variable UV detector). An Agilent ZORBAX SB-C18 column with dimensions of 250 mm × 4.6 mm was used at 30 ◦ C. The mobile phase was composed of 55% (v/v) potassium phosphate buffer (pH 3.5, 0.02 M) and 45% (v/v) HPLC-grade methanol. The flow rate of the mobile phase was 1.0 mL/min, and the detector was set at a wavelength of 225 nm. The apparent activity (A, U/g), conversion rate of Pen G (X, %), and specific activity (SA, U/mg-enzyme) were calculated according to the following formulas: A=
C6−APA × F W
X = (1 −
CPen G,out ) × 100% CPen G,in
SA (U/mg) =
A qE
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H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
where C6-APA (mmol/L) is the concentration of 6-APA in the outlet solution, F (mL/min) is the flow rate of the substrate solution, W (g) is the dry weight of the support. CPen G , in and CPen G , out (mmol/L) are the concentrations of Pen G in the inlet and outlet solution respectively.
2.5. Characterization A scanning electron microscopy (SEM) system (JEOL JSM7401F) operated at 1.0 kV was used to characterize the samples. Thermogravimetry (TG) measurement was performed using a TG 2050 analyzer. Infrared spectroscopic analysis was performed using a Shimadzu FTIR-8100 Fourier infrared spectroscopy. The enzyme concentration was analyzed using an Agilent 8453 Ultraviolet–visible (UV–vis) Spectrophotometer.
3. Results and discussion 3.1. Characterization of the immobilized PGA The SEM images of porous glass beads (Fig. 2) were obtained to observe the surface structure of the supports. Fig. 2(a) shows the whole photo of porous glass beads, Fig. 2(b) shows that the surface of the blank porous glass beads after treated with subcritical water became rough and was covered with a uniform nano-flake array. As shown in Fig. 2(c), the nano-flake array, after the modification of chitosan, was covered with an amorphous coating which is believed to be chitosan. After enzyme immobilization, the main structure of the porous shell layer was still retained (Fig. 2(d)). The FTIR spectroscopic analysis of immobilized PGA was conducted from 400 cm−1 to 4000 cm−1 (Fig. 3(a)). Characteristic absorption was displayed at 1634 cm−1 , which corresponded to the amide group, indicating the presence of chitosan. The peak
at 1047 cm−1 corresponded to the stretching vibration of Si–O–Si groups contained in the support. The TG measurements of porous glass beads treated with different concentration of chitosan are shown in Fig. 3(b). The different slopes of the four curves indicated that chitosan in the support slowly decomposed with the increase in temperature. When the concentration of chitosan was increased, the amount of chitosan that was coated onto the surface of the porous glass beads also increased remarkably. The amount of chitosan had a significant influence on the performance of enzyme catalysis. This matter is discussed in the succeeding section.
3.2. Influence of the flow rate and chitosan concentration on immobilized PGA The flow rate of the substrate solution had a significant influence on the performance of the immobilized PGA. As shown in Fig. 4 (red line as an example), the apparent activity of the enzyme rapidly increased from 10.4 U/g to 130.6 U/g when the flow rate was increased from 0.2 mL/min to 5.0 mL/min at the same concentration of the substrate solution. Meanwhile, the conversation rate of Pen G decreased from 98.4% to 51.5%. The high efficiency of immobilized PGA allowed the conversation rate to reach 91.1% when the residential time was only about 30 s (at a flow rate of 1 mL/min). When the flow rate was further increased from 5.0 mL/min to 9.0 mL/min, the apparent rate did not show much change in a specific range. Other lines in the figure also show the same trend, which indicates the influence of the external mass transfer. When the flow rate was low, the external diffusion of the substrate was considered the ratedetermining step. Thus, the apparent activity rapidly increased. Once the external influence was eliminated, the apparent activity remained constant. The concentration of chitosan is also an important factor that must be given attention. The deacetylation degree of the chitosan
Fig. 2. SEM images of porous glass beads (a) blank beads, (b) surface of blank beads, (c) surface of beads grafted with chitosan, (d) surface of beads after immobilization.
H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
Fig. 3. (a) FTIR spectra of immobilized PGA, (b) TG curves of porous glass beads treated with different concentration of chitosan. Table 1 Influence of chitosan concentration on immobilized PGA. Chitosan concentration (wt.%)
0.5
1.0
2.0
3.0
qE (mg/g) Amax (U/g) SA (U/mg enzyme)
40.1 109.1 2.72
52.7 116 2.20
65.1 120.4 1.85
94.2 133.8 1.42
used in the experiment is 85.1%. The chitosan concentrations ranged from 0.5 wt.% to 3.0 wt.% were investigated in this section, higher chitosan concentration was not investigated because of the solubility limit of chitosan in the acetic acid (1 wt.%). As shown in Table 1, the PGA loading amount increased with the increase in chitosan concentration. Combined with the conclusions of the previous section, such as the enzyme concentration as the determinant of the chitosan amount coated on the porous glass beads and the numerous amine groups on chitosan as the cause of the formation of link points to the PGA enzyme, the chitosan concentration
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Fig. 4. Influence of flow rate and chitosan concentration on (a) apparent activity, (b) conversion rate of Pen G. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
is deduced to influence the loading amount of PGA. Four samples with different enzyme loading amounts were investigated at the same concentration of the substrate solution. As shown in Fig. 4, both the apparent activity and conversation rate of Pen G increased at the same flow rate when the loading amount was increased, whereas the specific activity decreased (Table 1). This result is due to the fact that increasing the chitosan amount limited the contact between the substrate and the active site, which led to the decrease in the specific activity. Based on the result, the concentration of chitosan can be adjusted to a proper value to obtain a balance between the apparent and specific activities. Compared with other reported spherical supports, as shown in Table 2, the apparent activity of the porous glass beads is not very high since it is limited by the loading amount, but the specific activity shows a relatively high competitiveness. In addition, the immobilized PGA could be filled in the fixed bed reactor due to the appropriate size and the mechanical stability of the support, this process could avoid the damage of mechanical stirring and the direct contact of enzyme and
Table 2 Comparisons of PGA immobilized on other spherical supports. Support type
DS (m)
qE (mg/g)
Amax (U/g)
SA (U/mg enzyme)
Reactor
Amberlite XAD7 beads [19] Macro–mesoporous silica spheres [15] Porous glass beads
100–200 700 75–150
145.4 639 40.1
400 1033 109.1
2.75 1.62 2.72
Stirred tank Packed bed Fixed bed
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H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
(the cycle symbol) to keep the pH value at 7.9, the conversation rate of Pen G would continuously increase as the reaction went on. The conversation rate reached 89.1% after 8 h, which showed a good performance in the hydrolysis of a high-concentration substrate. In the contrast group without any NaOH, the pH value decreased because of the accumulation of 6-APA. The conversation rate stopped increasing after 6 h and remained at 38%. 6-APA shows competitive inhibition to PGA and can decrease the activity of the enzyme. In addition, the enzyme can only maintain the activity within a specific range of pH value. The accumulation of 6-APA significantly influences the pH value, which can lead to the inactivation of the enzyme. 3.5. Stability of the immobilized PGA
Fig. 5. Influence of initial concentration of Pen G on immobilized PGA.
alkaline, the good stability of the immobilized PGA in presented in Section 3.5. 3.3. Influence of initial concentration of Pen G on immobilized PGA To investigate the inhibition of PGA activity by the substrate, immobilized PGA was studied at substrate concentrations in the range of 13.4–268.5 mmol/L. As shown in Fig. 5, the conversion rate of Pen G continuously decreased with the increase in initial concentration. The apparent activity of the immobilized PGA increased with substrate concentration until a concentration of 80.5 mmol/L was reached. The apparent activity of PGA immobilized on porous glass beads was up to 140.3 U/g (dry support). However, with further increase in the substrate concentration, the apparent activity sharply decreased because of the noncompetitive enzyme inhibition by Pen G.
Evaluating the stability of immobilized PGA is very important because obtaining a stable enzyme is one of the main purposes of enzyme immobilization. As shown in Fig. 7, the percentage enzyme activity of immobilized PGA after normalization was determined at different temperatures compared with free enzyme. The free enzyme is quite sensitive to temperature change, especially at high temperatures. The percentage activity of free enzyme was reduced to 7.5% at 67 ◦ C. Meanwhile, the percentage activity after immobilization to porous glass beads increased when the temperature was increased from 37 ◦ C to 67 ◦ C because a higher temperature accelerated the hydrolysis of Pen G. When the temperature was higher than 77 ◦ C, the immobilized PGA started to be inactive. The immobilized PGA showed a better thermal stability than the free
3.4. Influence of the accumulation of 6-APA in reactor with recirculation The reaction influenced by the accumulation of 6-APA was further studied in the fixed bed reactor with recirculation. Up to 100 mL of Pen G (134.3 mmol/L) was recirculated after it passed through the fixed bed reactor at a flow rate of 5 mL/min. As shown in Fig. 6, if NaOH solution was placed into the substrate solution
Fig. 6. Influence of the accumulation of 6-APA in reactor with recirculation.
Fig. 7. (a) Thermal stability of immobilized PGA, (b) reusability of immobilized PGA.
H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
enzyme. According to some reported research results [24,25], this behavior can be related to the formation of multi-covalent linkage with the enzyme. The intermolecular forces between PGA and chitosan could restrict the enzyme molecule to avoid structure conformational changes at high temperature. Reusability is also a crucial factor in industrial production economics. Continuous experiments of the hydrolysis of Pen G were performed in the fixed bed reactor. The initial concentration of Pen G was 26.9 mmol/L and the flow rate was 1 mL/min. The results showed that the immobilized PGA retained 97.5% activity after seven days, which indicated good stability of reusability. 4. Conclusion Porous glass beads with core–shell structure were used to immobilize PGA. The enzyme performance in the fixed bed reactor was investigated. The effects of flow rate, chitosan concentration, and initial concentration of Pen G on immobilized PGA were also analyzed. The conversation rate of the substrate (26.9 mmol/L) reached 91.1% when the residential time was about 30 s in the fixed bed reactor. Meanwhile, the conversation rate of 100 mL of the substrate (134.3 mmol/L) reached 89.1% after 8 h in the reactor with recirculation. In addition, 97.5% activity was retained after seven days of continuous reaction. Acknowledgements 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
45
No. 2013CB733600), and the Innovative Science and Technology Foundation of PetroChina (Grant No. 2011D-5006-0407). References [1] Chong, X. Zhao, Catal. Today 93 (2004) 293–299. [2] A. Petri, T. Gambicorti, P. Salvadori, J. Mol. Catal. B: Enzym. 27 (2004) 103–106. [3] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. FernandezLafuente, Enzyme Microb. Technol. 40 (2007) 1451–1463. [4] X. Huang, A. Yu, Z. Xu, Bioresour. Technol. 99 (2008) 5459–5465. [5] Y. Hung, C. Peng, J. Tzen, M. Chen, J. Liu, Bioresour. Technol. 99 (2008) 8662–8666. [6] S. Pahujani, S.S. Kanwar, G. Chauhan, R. Gupta, Bioresour. Technol. 99 (2008) 2566–2570. [7] B.K. Vaidya, G.C. Ingavle, S. Ponrathnam, B. Kulkarni, S.N. Nene, Bioresour. Technol. 99 (2008) 3623–3629. [8] X. Hu, P. Wang, H.-M. Hwang, Bioresour. Technol. 100 (2009) 4963–4968. [9] H. Duggleby, S. Tolley, C. Hill, E. Dodson, G. Dodson, P. Moody, Nature 373 (1995) 264–268. [10] E. Katchalski-Katzir, D.M. Kraemer, J. Mol. Catal. B: Enzym. 10 (2000) 157–176. [11] R.A. Sheldon, Adv. Synth. Catal. 349 (2007) 1289–1307. [12] M. Eldin, H. El Enshasy, M. Hassan, B. Haroun, E. Hassan, J. Appl. Polym. Sci. 125 (2012) 3820–3828. [13] W. Adriano, J. Silva, R. Giordano, L. Gonc¸alves, Braz. J. Chem. Eng. 22 (2005) 529–538. [14] Q.-G. Xiao, X. Tao, J.-P. Zhang, J.-F. Chen, J. Mol. Catal. B: Enzym. 42 (2006) 14–19. [15] J. Zhao, Y. Wang, G. Luo, S. Zhu, Bioresour. Technol. 102 (2011) 529–535. [16] J. Zhao, Y. Wang, G. Luo, S. Zhu, Bioresour. Technol. 101 (2010) 7211–7217. [17] H. Shi, Y. Wang, G.S. Luo, Ind. Eng. Chem. Res. 53 (2014) 1947–1953. [18] H. Wang, Y. Jiang, L. Zhou, Y. He, J. Gao, J. Mol. Catal. B: Enzym. 96 (2013) 1–5. [19] D. Bianchi, P. Golini, R. Bortolo, P. Cesti, Enzyme Microb. Technol. 18 (1996) 592–596. [20] H. Sun, X.Y. Bao, X. Zhao, Langmuir 25 (2009) 1807–1812. [21] Y. Sun, Y. Wang, Y. Lu, T. Wang, G. Luo, J. Am. Ceram. Soc. 91 (2008) 103–109. [22] C. Shen, Y. Wang, J. Xu, Y. Lu, G. Luo, Particuology 10 (2012) 317–326. [23] C. Shen, Y. Wang, J. Xu, G. Luo, Chem. Eng. J. 229 (2013) 217–224. [24] L. Lei, Y. Bai, Y. Li, L. Yi, Y. Yang, C. Xia, J. Magn. Magn. Mater. 321 (2009) 252–258. [25] X. Luo, L. Zhang, Biomacromolecules 11 (2010) 2896–2903.
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Preparation and enzymatic activity of penicillin G acylase immobilized on core–shell porous glass beads Hang Shi, Yujun Wang ∗ , Guangsheng Luo State Key Laboratory of Chemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 3 February 2014 Received in revised form 17 April 2014 Accepted 17 April 2014 Available online 28 April 2014 Keywords: Porous glass beads Core–shell structure Penicillin G acylase Enzyme immobilization Fixed bed reactor
a b s t r a c t In this work, core–shell porous glass beads were used to immobilize the PGA based on the theory that diffusion is the rate-determining step of a reaction with fast kinetics. To improve the loading amount of PGA, chitosan was grafted on the support to increase the interaction between PGA and the support. The effects of flow rate, chitosan concentration and initial concentration of Pen G on immobilized PGA were investigated. The core–shell structure of the support resulted in a higher efficiency of the diffusion of substrate and products, which improved the activity of immobilized PGA. The conversation rate of Pen G (26.9 mmol/L) reached 91.1% when the residential time was about 30 s in the fixed bed reactor. Meanwhile, the conversation rate of 100 mL of Pen G (134.3 mmol/L) reached 89.1% after 8 h in the reactor with recirculation. Moreover, 97.5% activity was retained after seven days of continuous reaction. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Enzyme biocatalysts have been widely used in the fine chemicals industry, including the food and pharmaceutical industries [1]. Enzyme immobilization solves the common problems of free enzyme, including the lack of long-term stability, low tolerance to organic solvents, and difficult recovery and reuse [2–8]. Among all kinds of enzymes, penicillin G acylase (PGA, E.C. 3.5.1.11) is an important enzyme that catalyzes the hydrolysis of Penicillin G potassium (Pen G) to produce 6-aminopenicillanic acid (6-APA), which is the precursor for manufacturing -lactum antibiotics [9]. Considering the hydrolysis of Pen G, which is a fast reaction, diffusions of substrate and products are the key factors that control the reaction. Therefore, the property of the support remarkably influences the diffusion rates of substrate and products, and then determines the catalytic performance of immobilized PGA. Organic materials are widely used as the supports of immobilized PGA because of the presence of rich functional groups, which provide essential interactions with enzymes [1]. For instance, Eupergit C is commercially used as a support for immobilization. PGA is covalently attached to Eupergit C and used in industrial production [10]. However, the major shortcoming of Eupergit C is its diffusion limitations, the effects of which are more pronounced in kinetically controlled processes [11]. Researchers have also
∗ Corresponding author. Tel.: +86 10 62783870; fax: +86 10 62770304. E-mail address: [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.molcatb.2014.04.013 1381-1177/© 2014 Elsevier B.V. All rights reserved.
immobilized PGA onto other organic materials, including aminefunctionalized PVC membranes and glutaraldehyde-activated chitosan [12,13]. Inorganic materials are applied to enzyme immobilization because of their special characteristics, including thermal and mechanical stability, high resistance against organic solvents, and nontoxicity. For example, PGA molecules were immobilized on hollow silica nanotubes [14], in which the thin shell structure of the immobilized enzyme effectively reduced mass transfer resistance that led to a high activity yield. Macro-mesoporous silica spheres were prepared with a micro channel and were used as the support for PGA immobilization [15]. The introduction of macropores increased the enzyme loading amount and decreased the internal mass transfer resistance. This condition resulted in a high apparent enzymatic activity. In addition, PGA was also immobilized on mesostructured cellular foams, macrocellular heterogeneous silica-based monoliths, aminoalkylated polyacrylic supports, and oxirane-modifiedmesoporous silicas [16–20]. These studies strongly illustrated the importance of diffusion rate to catalytic performance. Materials with core–shell structure are also good choices for PGA immobilization. In our previous work, a one-step subcritical water treatment method was developed to prepare porous glass beads which have core–shell structure [21]. Ion exchange properties were further investigated [22], and chitosan was supported on this kind of inorganic material, which was used as a green adsorbent for heavy metal adsorption [23]. Porous glass beads have several advantages, which are as follows: (1) can effectively eliminate the influence of inner diffusion because the distance of mass diffusion is
H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
Fig. 1. The process of hydrolysis reaction catalyzed by PGA immobilized on the porous glass beads.
the thickness of the porous shell, (2) can be easily modified because of the numerous hydroxyl groups on the surface, (3) contain good chemical and thermal stabilities, and (4) can be easily filled and separated based on the appropriate particle size (75–150 m). In this work, PGA immobilized on the chitosan-treated porous glass beads was investigated. As shown in Fig. 1, the thickness of the porous shell was about 4 m. Thus, the process of hydrolysis reaction is considered the contact between the shell layer and the aqueous phase layer. The short distance of mass transfer improves the efficiency of the diffusion of substrate and products. PGA molecules are centralized in a small shell area, which means that the density of the enzyme is larger than those of any other uniform sphere supports. Hence, the contact probability of enzymes and substrates could be increased. To achieve continuous production, the obtained immobilized PGA was filled in a fixed bed reactor. This process could avoid the damage of mechanical stirring and the direct contact of enzyme and alkaline, which ensures good stability of the enzyme. The physical properties of the immobilized PGA were characterized, the effects of flow rate, chitosan concentration, initial concentration of Pen G, and the accumulation of 6-APA on immobilized PGA were studied. 2. Materials and methods 2.1. Materials Glass microbeads with diameters ranging from 75 m to 150 m were obtained from Hebei Chiye Corporation, with the composition of 59.7 wt% SiO2 , 25.1 wt% Na2 O, 9.8 wt% MgO, and 4.9 wt% CaO. Hydrochloric acid (HCl), acetic acid, and sodium hydroxide (NaOH) were produced by Beijing Chemical Company (Beijing, China). Chitosan (DD 85.1%) was purchased from Sinopharm Chemical Reagent Company (Beijing, China). Sodium dihydrogen phosphate (NaH2 PO4 ), dibasic sodium phosphate (Na2 HPO4 ), potassium dihydrogen phosphate (KH2 PO4 ), and glutaraldehyde (GA) were obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). PGA (E.C. 3.5.1.11) was purchased from Shunfeng Haideer Co., Ltd. (Zhejiang, China). Pen G was purchased from Dalian Meilun Biology Technology Co, Ltd. (Dalian, China). 6-APA was obtained from Tokyo Chemical Industry Co., Ltd. (Japan). HPLC-grade methanol was produced by Anhui Fulltime Specialized Solvents & Reagents Co., Ltd. (Anhui, China). All chemicals were used as received without any further treatment. 2.2. Synthesis of porous glass beads grafted by chitosan The porous glass beads were prepared via the subcritical water treatment method based on the reference [22]. In this method, 5.0 g of glass beads and 200 g of water were placed in a tank reactor. The temperature of the reactor was gradually increased to 300 ◦ C, and the pressure was increased to 8 MPa. The subcritical state was maintained for 1 h, after which the reactor was cooled to room temperature. The porous glass beads were separated from water via
41
filtration and were washed several times with water. To replace the metal ions contained in the shell part with hydrogen ions, the obtained porous glass beads were mixed with 500 mL of HCl solution with a concentration of 0.5 M. The mixture was shaken in the SHA-B Incubator Shaker at 160 rpm at 30 ◦ C for 12 h. The obtained modified glass beads were filtered, washed with plenty of water, and dried at 80 ◦ C for 24 h. To obtain a larger specific surface area and a stronger connection with PGA molecules, the surface of the porous glass beads was coated with chitosan before the immobilization of PGA. Chitosan was dissolved in acetic acid (1 wt.%) to obtain the chitosan solution with a concentration that ranged from 0.5 wt.% to 3.0 wt.%. Up to 1 g of the modified glass beads was mixed with 30 g of chitosan solution, and the mixture was then shaken at 160 rpm at 30 ◦ C for 8 h. After removing the supernatant, the porous glass beads that adsorbed chitosan was mixed with 15 g of 1.0 wt.% GA and was shaken for another 12 h at 160 rpm at 30 ◦ C. After filtration, washing, and drying, porous glass beads coated with chitosan were obtained. 2.3. Immobilization of PGA Up to 0.5 g of porous glass beads coated with chitosan were incubated with 30 mL of PGA solution (5.0 mg/ml), 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. After that, the mixture was centrifuged to separate the PGA solution and the porous glass beads with the immobilized PGA. The protein concentration of the supernatant was measured to calculate the PGA loading amount. The PGA loading amount (qE (mg/g)), was calculated according to the following formula: qE (mg/g) =
q1 − q2 m
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 To investigate the activity of immobilized PGA, the hydrolysis of Pen G was performed as follows: the immobilized PGA obtained in Section 2.3 was placed in a fixed bed reactor with a length of 50 mm and an inner diameter of 4 mm. Pen G solution in phosphate buffer (pH 7.9, 0.05 M) was allowed to flow through the fixed bed reactor in a water bath. The substrate and products of the hydrolysis reaction were analyzed via high-performance liquid chromatography (HP series 1200 HPLC system equipped with a wavelength-variable UV detector). An Agilent ZORBAX SB-C18 column with dimensions of 250 mm × 4.6 mm was used at 30 ◦ C. The mobile phase was composed of 55% (v/v) potassium phosphate buffer (pH 3.5, 0.02 M) and 45% (v/v) HPLC-grade methanol. The flow rate of the mobile phase was 1.0 mL/min, and the detector was set at a wavelength of 225 nm. The apparent activity (A, U/g), conversion rate of Pen G (X, %), and specific activity (SA, U/mg-enzyme) were calculated according to the following formulas: A=
C6−APA × F W
X = (1 −
CPen G,out ) × 100% CPen G,in
SA (U/mg) =
A qE
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H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
where C6-APA (mmol/L) is the concentration of 6-APA in the outlet solution, F (mL/min) is the flow rate of the substrate solution, W (g) is the dry weight of the support. CPen G , in and CPen G , out (mmol/L) are the concentrations of Pen G in the inlet and outlet solution respectively.
2.5. Characterization A scanning electron microscopy (SEM) system (JEOL JSM7401F) operated at 1.0 kV was used to characterize the samples. Thermogravimetry (TG) measurement was performed using a TG 2050 analyzer. Infrared spectroscopic analysis was performed using a Shimadzu FTIR-8100 Fourier infrared spectroscopy. The enzyme concentration was analyzed using an Agilent 8453 Ultraviolet–visible (UV–vis) Spectrophotometer.
3. Results and discussion 3.1. Characterization of the immobilized PGA The SEM images of porous glass beads (Fig. 2) were obtained to observe the surface structure of the supports. Fig. 2(a) shows the whole photo of porous glass beads, Fig. 2(b) shows that the surface of the blank porous glass beads after treated with subcritical water became rough and was covered with a uniform nano-flake array. As shown in Fig. 2(c), the nano-flake array, after the modification of chitosan, was covered with an amorphous coating which is believed to be chitosan. After enzyme immobilization, the main structure of the porous shell layer was still retained (Fig. 2(d)). The FTIR spectroscopic analysis of immobilized PGA was conducted from 400 cm−1 to 4000 cm−1 (Fig. 3(a)). Characteristic absorption was displayed at 1634 cm−1 , which corresponded to the amide group, indicating the presence of chitosan. The peak
at 1047 cm−1 corresponded to the stretching vibration of Si–O–Si groups contained in the support. The TG measurements of porous glass beads treated with different concentration of chitosan are shown in Fig. 3(b). The different slopes of the four curves indicated that chitosan in the support slowly decomposed with the increase in temperature. When the concentration of chitosan was increased, the amount of chitosan that was coated onto the surface of the porous glass beads also increased remarkably. The amount of chitosan had a significant influence on the performance of enzyme catalysis. This matter is discussed in the succeeding section.
3.2. Influence of the flow rate and chitosan concentration on immobilized PGA The flow rate of the substrate solution had a significant influence on the performance of the immobilized PGA. As shown in Fig. 4 (red line as an example), the apparent activity of the enzyme rapidly increased from 10.4 U/g to 130.6 U/g when the flow rate was increased from 0.2 mL/min to 5.0 mL/min at the same concentration of the substrate solution. Meanwhile, the conversation rate of Pen G decreased from 98.4% to 51.5%. The high efficiency of immobilized PGA allowed the conversation rate to reach 91.1% when the residential time was only about 30 s (at a flow rate of 1 mL/min). When the flow rate was further increased from 5.0 mL/min to 9.0 mL/min, the apparent rate did not show much change in a specific range. Other lines in the figure also show the same trend, which indicates the influence of the external mass transfer. When the flow rate was low, the external diffusion of the substrate was considered the ratedetermining step. Thus, the apparent activity rapidly increased. Once the external influence was eliminated, the apparent activity remained constant. The concentration of chitosan is also an important factor that must be given attention. The deacetylation degree of the chitosan
Fig. 2. SEM images of porous glass beads (a) blank beads, (b) surface of blank beads, (c) surface of beads grafted with chitosan, (d) surface of beads after immobilization.
H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
Fig. 3. (a) FTIR spectra of immobilized PGA, (b) TG curves of porous glass beads treated with different concentration of chitosan. Table 1 Influence of chitosan concentration on immobilized PGA. Chitosan concentration (wt.%)
0.5
1.0
2.0
3.0
qE (mg/g) Amax (U/g) SA (U/mg enzyme)
40.1 109.1 2.72
52.7 116 2.20
65.1 120.4 1.85
94.2 133.8 1.42
used in the experiment is 85.1%. The chitosan concentrations ranged from 0.5 wt.% to 3.0 wt.% were investigated in this section, higher chitosan concentration was not investigated because of the solubility limit of chitosan in the acetic acid (1 wt.%). As shown in Table 1, the PGA loading amount increased with the increase in chitosan concentration. Combined with the conclusions of the previous section, such as the enzyme concentration as the determinant of the chitosan amount coated on the porous glass beads and the numerous amine groups on chitosan as the cause of the formation of link points to the PGA enzyme, the chitosan concentration
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Fig. 4. Influence of flow rate and chitosan concentration on (a) apparent activity, (b) conversion rate of Pen G. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
is deduced to influence the loading amount of PGA. Four samples with different enzyme loading amounts were investigated at the same concentration of the substrate solution. As shown in Fig. 4, both the apparent activity and conversation rate of Pen G increased at the same flow rate when the loading amount was increased, whereas the specific activity decreased (Table 1). This result is due to the fact that increasing the chitosan amount limited the contact between the substrate and the active site, which led to the decrease in the specific activity. Based on the result, the concentration of chitosan can be adjusted to a proper value to obtain a balance between the apparent and specific activities. Compared with other reported spherical supports, as shown in Table 2, the apparent activity of the porous glass beads is not very high since it is limited by the loading amount, but the specific activity shows a relatively high competitiveness. In addition, the immobilized PGA could be filled in the fixed bed reactor due to the appropriate size and the mechanical stability of the support, this process could avoid the damage of mechanical stirring and the direct contact of enzyme and
Table 2 Comparisons of PGA immobilized on other spherical supports. Support type
DS (m)
qE (mg/g)
Amax (U/g)
SA (U/mg enzyme)
Reactor
Amberlite XAD7 beads [19] Macro–mesoporous silica spheres [15] Porous glass beads
100–200 700 75–150
145.4 639 40.1
400 1033 109.1
2.75 1.62 2.72
Stirred tank Packed bed Fixed bed
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H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
(the cycle symbol) to keep the pH value at 7.9, the conversation rate of Pen G would continuously increase as the reaction went on. The conversation rate reached 89.1% after 8 h, which showed a good performance in the hydrolysis of a high-concentration substrate. In the contrast group without any NaOH, the pH value decreased because of the accumulation of 6-APA. The conversation rate stopped increasing after 6 h and remained at 38%. 6-APA shows competitive inhibition to PGA and can decrease the activity of the enzyme. In addition, the enzyme can only maintain the activity within a specific range of pH value. The accumulation of 6-APA significantly influences the pH value, which can lead to the inactivation of the enzyme. 3.5. Stability of the immobilized PGA
Fig. 5. Influence of initial concentration of Pen G on immobilized PGA.
alkaline, the good stability of the immobilized PGA in presented in Section 3.5. 3.3. Influence of initial concentration of Pen G on immobilized PGA To investigate the inhibition of PGA activity by the substrate, immobilized PGA was studied at substrate concentrations in the range of 13.4–268.5 mmol/L. As shown in Fig. 5, the conversion rate of Pen G continuously decreased with the increase in initial concentration. The apparent activity of the immobilized PGA increased with substrate concentration until a concentration of 80.5 mmol/L was reached. The apparent activity of PGA immobilized on porous glass beads was up to 140.3 U/g (dry support). However, with further increase in the substrate concentration, the apparent activity sharply decreased because of the noncompetitive enzyme inhibition by Pen G.
Evaluating the stability of immobilized PGA is very important because obtaining a stable enzyme is one of the main purposes of enzyme immobilization. As shown in Fig. 7, the percentage enzyme activity of immobilized PGA after normalization was determined at different temperatures compared with free enzyme. The free enzyme is quite sensitive to temperature change, especially at high temperatures. The percentage activity of free enzyme was reduced to 7.5% at 67 ◦ C. Meanwhile, the percentage activity after immobilization to porous glass beads increased when the temperature was increased from 37 ◦ C to 67 ◦ C because a higher temperature accelerated the hydrolysis of Pen G. When the temperature was higher than 77 ◦ C, the immobilized PGA started to be inactive. The immobilized PGA showed a better thermal stability than the free
3.4. Influence of the accumulation of 6-APA in reactor with recirculation The reaction influenced by the accumulation of 6-APA was further studied in the fixed bed reactor with recirculation. Up to 100 mL of Pen G (134.3 mmol/L) was recirculated after it passed through the fixed bed reactor at a flow rate of 5 mL/min. As shown in Fig. 6, if NaOH solution was placed into the substrate solution
Fig. 6. Influence of the accumulation of 6-APA in reactor with recirculation.
Fig. 7. (a) Thermal stability of immobilized PGA, (b) reusability of immobilized PGA.
H. Shi et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 40–45
enzyme. According to some reported research results [24,25], this behavior can be related to the formation of multi-covalent linkage with the enzyme. The intermolecular forces between PGA and chitosan could restrict the enzyme molecule to avoid structure conformational changes at high temperature. Reusability is also a crucial factor in industrial production economics. Continuous experiments of the hydrolysis of Pen G were performed in the fixed bed reactor. The initial concentration of Pen G was 26.9 mmol/L and the flow rate was 1 mL/min. The results showed that the immobilized PGA retained 97.5% activity after seven days, which indicated good stability of reusability. 4. Conclusion Porous glass beads with core–shell structure were used to immobilize PGA. The enzyme performance in the fixed bed reactor was investigated. The effects of flow rate, chitosan concentration, and initial concentration of Pen G on immobilized PGA were also analyzed. The conversation rate of the substrate (26.9 mmol/L) reached 91.1% when the residential time was about 30 s in the fixed bed reactor. Meanwhile, the conversation rate of 100 mL of the substrate (134.3 mmol/L) reached 89.1% after 8 h in the reactor with recirculation. In addition, 97.5% activity was retained after seven days of continuous reaction. Acknowledgements 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
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