Entrapment of Methyl Parathion Hydrolase in Cross-Linked Poly(γ

Article pubs.acs.org/Biomac

Entrapment of Methyl Parathion Hydrolase in Cross-Linked Poly(γglutamic acid)/Gelatin Hydrogel Jianfei Xie,†,‡ Huiwen Zhang,*,† Xu Li,† and Yuanliang Shi† †

Institute of Applied Ecology, Chinese Academy of Sciences, No. 72, Wenhua Road, Shenhe District, Shenyang, Liaoning, China University of Chinese Academy of Sciences, No. 19A, Yuquan Road, Beijing, China

‡

ABSTRACT: Methyl parathion hydrolase (MPH) is an important enzyme in hydrolyzing toxic organophosphorus (OP) compounds. However, MPH is easily deactivated when subjected to extreme environmental conditions and is difficult to recover from the reaction system for reuse, thereby limiting its practical application. To address these shortcomings, we examined the entrapment of MPH in an environment-friendly, biocompatible and biodegradable cross-linked poly(γ-glutamic acid)/gelatin hydrogel. The cross-linked poly(γ-glutamic acid)/gelatin hydrogels were prepared with different gelatin/poly(γ-glutamic acid) mass ratios using water-soluble carbodiimide as the cross-linking agent. The MPH-entrapped cross-linked poly(γ-glutamic acid)/ gelatin hydrogel (CPE−MPH) not only possessed improved thermostability, pH stability, and reusability but also exhibited enhanced efficiency in hydrolyzing OP compounds. Furthermore, CPE−MPH possesses high water-absorbing and waterretaining capabilities. We believe that the cross-linked poly(γ-glutamic acid)/gelatin hydrogels are an attractive carrier for the entrapment of diverse enzymes, affording a new approach for enzyme entrapment.

1. INTRODUCTION Organophosphorus (OP) compounds are highly toxic because they inhibit acetylcholinesterase in the nervous system, leading to nerve function loss and potential death.1 OP compounds account for over 34% of the total pesticides used worldwide.2 Recent studies indicate severe contamination of OP compounds in soil, drinking water, and agricultural products.3−5 Thus, effective approaches to address OP compounds contamination are essential. Methyl parathion hydrolase (MPH, E.C.3.1.8.1), which is isolated from Plesiomonas sp. strain M6,6 is capable of hydrolyzing a broad spectrum of OP compounds such as chlorpyrifos, malathion, fenitrothion.7 However, exposure of MPH to varying detrimental environmental conditions, such as extreme temperatures, extreme pHs, and microbial-degradation, and the difficulty to separate MPH from the reaction system for reuse, limit its practical application. Entrapment technology is an effective approach to address these issues. Entrapment has been used widely to entrap enzymes in a carrier to impart protection against extreme temperatures, extreme pHs, and biodegradation during application. A number of carriers have been used such as calcium alginate, polyacrylamide, and gelatin gels.8,9 Each carrier has advantages and disadvantages relating to the biocompati© 2014 American Chemical Society

bility, biodegradation, and entrapment efficiency. For instance, calcium alginate beads are one of the most commonly used carriers because of their high biocompatibility and low cost. However, their shortcomings include low mechanical strength, large pore size, and leakage of enzyme from the beads.10 On the other hand, gel-forming polymerization of acrylamide monomer (to produce polyacrylamide), which is neurotoxic and reactive toward enzymes, often destroys enzymatic activity; furthermore, polyacrylamide has low mechanical properties.11 Hence, this study investigates the use of an environment-friendly, highly effective, biocompatible, and biodegradable carrier with increased resistance to stabilize the entrapped MPH. Poly(γ-glutamic acid), γ-PGA, is an unusual anionic, naturally occurring homopolyamide that consists of D- and L- glutamic acid units connected by amide linkages between the α-amino and γcarboxylic acid groups.12 γ-PGA is water-soluble, biodegradable, edible, and nontoxic to humans and the environment. Hence, it is a good candidate for various industrial applications, including sustained release material, drug carrier,13,14 nanoparticles for drug controlled release,15 curable biological adhesives,16 Received: December 5, 2013 Published: January 14, 2014 690

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biodegradable fibers, highly water absorbing hydrogels, and biopolymer flocculants.17 Furthermore, γ-PGA features extremely high absorption capacity for heavy metals.18−20 In agriculture, γ-PGA is used as a fertilizer synergist21 to improve the fertilizer absorption efficiency and increase crop yield. Additionally, γ-PGA has been used as a carrier for immobilization and encapsulation. Chang et al. successfully immobilized Candida rugosa lipase on γ-PGA and studied the optimum conditions for immobilization.22 Chiu et al. reported the encapsulation of lycopene using an emulsion system consisting of gelatin, γ-PGA, and lycopene extract.23 Unlike these above-mentioned methods, in this study we investigate the entrapment of MPH in a three-dimensional network of cross-linked γ-PGA/gelatin hydrogel using watersoluble carbodiimide as the cross-linking agent. MPH-entrapped cross-linked γ-PGA/gelatin hydrogel (CPE−MPH) displayed advantages over the free enzyme such as improved thermostability, pH stability, and reusability. To our knowledge, crosslinked γ-PGA/gelatin hydrogels, which are generally used as a biological glue,24−29 have not been explored as a carrier for enzyme entrapment.

2.6. Activity Assay of CPE−MPH and Release Activity of MPH. A 0.02 g of freeze-dried CPE−MPH hydrogel powder was soaked in 10 mL of PBS buffer in a tube until release activity from the hydrogel network was no longer detected. After centrifugation at 4000 rpm for 1 min, 50 μL of hydrogels at the bottom of the tube and 50 μL of supernatant were respectively added to the MPH assay buffer solution to evaluate the activity of the hydrogels and release activity of the MPH, respectively. All assays were performed in 1 mL of MPH assay buffer (50 mmol L−1 phosphate buffer, pH 7.0) containing methyl parathion (final concentration: 0.4 mmol L−1) as the substrate. All assays were performed in triplicate. The activity was defined as the amount of enzyme required to hydrolyze 1 μmol of methyl parathion per minute under the specified conditions (pH 7.0 and 30 °C). The hydrolysis of methyl parathion was measured by monitoring the appearance of pnitrophenol at 410 nm (ε410 nm = 16 500 mol−1 L cm−1), using a PerkinElmer Lambda 25 spectrophotometer. For the catalytic experiments, the detected activity was defined as the measured activity of the MPH-entrapped hydrogel. The released activity was defined as the activity of the enzyme released from the hydrogel network. The entrapped activity was defined as the theoretical activity of the enzyme entrapped in the network, which was the difference between the initial activity (6000 U) and released activity. The kinetic constants of the CPE−MPH hydrogels and free MPH were obtained by measuring the initial hydrolysis rate at different substrate concentrations at a given enzyme concentration. The Lineweaver−Burk reciprocal plots were used to determine Km and Vmax of the CPE−MPH hydrogels and free MPH. 2.7. Thermostability and pH Stability of CPE−MPH. For the thermostability study, CPE−MPH hydrogels and free MPH were placed in the assay buffer and incubated at varying temperatures between 35 and 70 °C for 10 min. Following heating, the CPE−MPH hydrogels were promptly removed and allowed to cool to room temperature before methyl parathion was added. The residual enzyme activities were then measured by the method described above. The sample incubated at 35 °C was used as the control (100%). The pH stability was evaluated by incubating the CPE−MPH hydrogels and free MPH at 25 °C for 1 h at pH 4.0−6.0 using 100 mmol L−1 citrate buffer, pH 7.0 using 100 mmol L−1 phosphate buffer, pH 8.0− 9.0 using 100 mmol L−1 tris/HCl buffer, and pH 10.0 using 100 mmol L−1 glycine−NaOH buffer. The residual enzyme activities were then measured. The nontreated sample was used as the control (100%). 2.8. Reusability of CPE−MPH. The CPE−MPH hydrogels were placed in the assay buffer to measure the enzyme activity by the method described above. Following the assay, the CPE−MPH hydrogels were washed with PBS buffer to remove the reaction product, p-nitrophenol, and recovered by centrifugation at 4000 rpm for 1 min. The recovered hydrogels were then placed in a fresh assay buffer, and enzyme activity was measured again. The procedure was repeated to assess the reusability of the CPE−MPH hydrogels over five cycles. The enzyme activity in the first cycle was used as the control (100%). 2.9. OP Compound Degradation by CPE−MPH. The decomposition of OP compounds by CPE−MPH and free MPH in contaminated water and soil was evaluated. For the water studies, CPE−MPH (total enzyme activity in water, 15 U L−1) and free MPH (enzyme activity in water, 15 U L−1) were respectively introduced to the contaminated tap water containing methyl parathion (100 mg L−1) and chlorpyrifos (50 mg L−1). The contaminated water (no added enzyme) was used as the control (100%). The water samples were incubated at 25 °C in the dark. At corresponding time points, 5 mL of the water samples was taken for pesticide concentration determination. The residual pesticide content in the contaminated water was extracted with 25 mL of acetonitrile under sonication for 30 min followed by filtration, drying, reconstitution by acetone, and finally analyzed by gas chromatography (GC). A Shimadzu GC-2014 Series gas chromatograph with a flame photometric detector and a fused silica capillary column (length, 30 m; internal diameter, 0.53 mm; and film thickness, 1 μm; RESTEX, U.S.A.) was used for analysis of the pesticides. Nitrogen was used as the carrier gas at a flow rate of 1 mL min−1. The air and hydrogen flow rates were 81.8 and 3.2 mL min−1, respectively. The injector, column, and detector temperatures were set at 250, 150, and 250 °C, respectively. The column

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(γ-glutamic acid) (γ-PGA, average Mw 120 kDa) was supplied by Fruida Co., Jinan, Shandong, China. N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), a water-soluble carbodiimide, was purchased from J&K, Beijing, China. Gelatin (porcine skin, 300 Bloom) was purchased from Sigma (St. Louis, MO, U.S.A.). 2.2. Expression and Purification of MPH. The coding sequence of the mpd gene (GenBank Accession No. JQ686087) without signal peptide sequence30 was subcloned into the NdeI/XhoI (TaKaRa, Dalian, China) site of pET-30a expression plasmid (Novagen, Darmstadt, Germany) with a C-terminal hexahistidine (6×His) tag and transformed into E. coli BL21 (DE3) cells (Tiangen, Beijing, China) under kanamycin selection. Expression of the 6×His-MPH was induced with 1 mmol L−1 IPTG for 16 h at 28 °C. The induced bacteria were then lysed by incubation with 100 μg mL−1 lysozyme followed by sonication. After centrifugation, the expressed 6×His-MPH was purified using NiNTA agarose (QIAGEN, Hilden, Germany). Enzyme concentration was determined by the Bradford method, using bovine serum albumin as the standard. 2.3. CPE−MPH Fabrication. The purified MPH was entrapped in the cross-linked γ-PGA/gelatin hydrogels, prepared with different gelatin/γ-PGA mass ratios (from 1:10 to 5:10). Briefly, 1 gof γ-PGA and 0.1−0.5 gof gelatin were added to 20 mL of PBS buffer to form an aqueous solution to which the purified MPH (6000 U, 175 U mg−1) and EDC (final concentration: 2 mg mL−1) were added. The mixture was homogenized immediately and placed at 25 °C for 20 min for crosslinking, freeze-dried, and powdered (particle size: 20 μm). The CPE− MPH hydrogels prepared with gelatin/γ-PGA mass ratios of 1:10, 2:10, 3:10, 4:10, and 5:10 are denoted as CPE−MPH-1, CPE−MPH-2, CPE−MPH-3, CPE−MPH-4, and CPE−MPH-5, respectively. 2.4. Fourier Transform Infrared (FTIR) Spectroscopy. The cross-linking degree between γ-PGA and gelatin was monitored by FTIR spectroscopy. For the sample preparation, the sample and KBR standard (sample/KBr ratio of 1:100) was mixed and compressed into a pellet. Spectra were recorded on a Nicolet 6700 FT−IR spectrometer (Nicolet Instrument Co., Madison, WI, U.S.A.), in the range of 4000−400 cm−1 at 25 °C. 2.5. Scanning Electron Microscopy (SEM). The CPE−MPH hydrogels that were swollen in distilled water at 25 °C for 24 h were frozen in liquid nitrogen, fractured immediately, and freeze-dried. The fractured surface of the hydrogels was sputtered with gold prior to SEM analysis on a Quanta 250 SEM (FEI Co., Hillsboro, OR, U.S.A.). The morphology of the swollen CPE−MPH hydrogels before freeze-drying was also observed. 691

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temperature was initially stabilized at 150 °C for 3 min, heated to 250 °C at a rate of 8 °C min−1, and maintained for 8 min. For the soil studies, sandy soil (pH 6.5) from farmland at Shenyang, Liaoning, China was examined. CPE−MPH (total enzyme activity in the soil, 48 U kg−1) and free MPH (enzyme activity in the soil, 48 U kg−1) were respectively introduced to the contaminated soil comprising methyl parathion (80 mg kg−1) and chlorpyrifos (40 mg kg−1). The enzymes were thoroughly mixed with the soil samples. The contaminated soil (no added enzyme) was used as the control (100%). The soil samples with a 40% water retention capacity were incubated at 25 °C in the dark. At corresponding time points, 5 g of the soil samples was taken for pesticide concentration determination. The residual pesticide content in the soil samples was analyzed by gas chromatography, as described above. 2.10. Biodegradation of CPE−MPH. Biodegradation of CPE− MPH was investigated in the soil extract by monitoring the weight loss of the swollen CPE−MPH hydrogel. A total of 100 g of sandy soil was added to a 1 L phosphate buffer (50 mmol L−1, pH 7.0). The resulting mixture was agitated vigorously and then allowed to stand for 6 h to collect the soil extract in the supernatant. CPE−MPH and free MPH were respectively introduced in separate tubes containing the soil extract and incubated at 25 °C in the dark. At corresponding time points, the CPE−MPH hydrogel was collected by centrifugation at 4000 rpm for 1 min and weighed. The residual activities of CPE−MPH and free MPH were measured by the method described above. 2.11. Swelling and Water Retention Property of CPE−MPH. An accurately weighed amount (0.01 g) of the CPE−MPH hydrogel was immersed in distilled water overnight to reach swelling equilibrium. The swelling ratio (R) of the CPE−MPH hydrogel was calculated as follows

R=

Figure 1. CPE-MPH hydrogel fabrication. (a) Photograph of γ-PGA/ gelatin aqueous solution before cross-linking. (b,c) Photograph of crosslinked γ-PGA/gelatin hydrogel. (d) Photograph of freeze-dried CPEMPH hydrogel. (e) Photograph of freeze-dried powder of CPE-MPH hydrogel.

The cross-linking of the resulting hydrogel was evaluated by FTIR. The spectrum, Figure 3, features a broad band at 3433.1 cm−1 that can be assigned to OH stretching of the COOH group in the γ-PGA backbone. The band became narrower with increase of the gelatin/γ-PGA mass ratio. Cross-linking between γ-PGA and gelatin resulted in a decrease in the number of hydrogen bonds formed between the COOH groups in the γPGA backbone, leading to a narrow FTIR band. The signal around 1636.3 cm−1 can be attributed to the tretching band of CO of the COOH groups in the γ-PGA backbone. The peak at 1267.97 cm−1 corresponds to the characteristic stretching peak of C−O of the COOH group. The OH bending vibration is observed at 1405.69 cm−1. The intensity of these three bands (1636.3, 1267.97, and 1405.69 cm−1) decreased with increasing gelatin/γ-PGA mass ratio. On the basis of SEM and FTIR analyses, the cross-linked network structure is formed by amide linkages between the α-carboxylic acid groups in γ-PGA and amino groups in gelatin; also, cross-linking degree increased with increasing gelatin/γ-PGA mass ratio. 3.2. Activity of CPE−MPH and Release Activity of MPH. Because of the restricted diffusion of MPH, entrapped in crosslinked poly(γ-glutamic acid)/gelatin from the network to the solution, the enzymatic reaction was carried out by the access of the substrate into the network, following diffusion of the reaction product, ρ-nitrophenol, into the solution, as shown in Figure 4. Methyl parathion has a water solubility of 55−60 mg L−1 at 25 °C and a low octanol/water coefficient of Kow = 3.5−3.8, which is considered relatively hydrophilic.33−35 In this study, the concentration of methyl parathion in the assay buffer was 0.4 mmol L−1 (100 mg L−1), 55−60% of which was dissolved in the assay buffer. Thus, the reactants accessed the hydrogel matrix mainly through molecular diffusion. In our work, both the detected activity of CPE-MPH hydrogels and released MPH activity decreased with increasing crosslinking degree (Table 1). The highest MPH activity, 1620.9 ± 254.9 U (efficiency 33.5%), was detected in CPE−MPH-2 (gelatin/γ-PGA mass ratio of 2:10), whereas the lowest activity, 1020.2 ± 116.5 U (efficiency 20%), was detected in CPE−MPH5 (gelatin/γ-PGA mass ratio of 5:10), Table 1. Compared with a previous report,36 the efficiency of the CPE−MPH hydrogels was considerably higher than that of polyacrylamide (i.e., 10%), and only slightly lower than that of calcium alginate (i.e., 36.3%). The cross-linked poly(γ-glutamic acid)/gelatin hydrogels with a

(Ws − Wd) Wd

where Wd and Ws are the weights of the dry and swollen CPE−MPH hydrogels, respectively. To evaluate the water retention property of CPE−MPH, the sandy soil was mixed with CPE−MPH at different concentrations (0.2 and 0.4%, w/w). The soil mixture (100 g) was introduced in a 200 cm3 plastic pot containing a filter paper with holes, placed at the base of the pot. The soil mixture-containing pots were saturated with tap water by immersion in containers for 24 h. The pots were then raised to drain out excess water. The pots were subsequently subjected to laboratory conditions at 25 °C and relative air humidity of 35%. The mass of the pots was recorded every 2 days until no weight loss was observed, and the water content of the soil was calculated.31 The experiments were conducted in triplicate.

3. RESULTS AND DISCUSSION 3.1. Cross-Linking between γ-PGA and Gelatin. To date, two methods have been commonly used for γ-PGA cross-linking. In the first method, polyglycidyl ether is used as the cross-linking agent, producing stable three-dimensional cross-linked hydrogels.32 In the second method, water-soluble carbodiimide is used as the cross-linking agent, enabling a three-dimensional crosslinked hydrogel that is typically used as a biological glue.24−29 Because of the loss of MPH activity owing to the use of polyglycidyl ether, we adopted the second approach for MPH entrapment. The CPE−MPH hydrogels, formed upon addition of the purified MPH and EDC to γ-PGA/gelatin aqueous solutions, are shown in Figure 1. The morphology of the hydrogels prepared at different gelatin/γ-PGA mass ratios was evaluated by SEM. Unlike the structures of CPE−MPH-1 and CPE−MPH-2 (Figure 2a,b, respectively), those of CPE−MPH-4 and CPE− MPH-5 (Figure 2d,e, respectively) were more compact and homogeneous. The morphology of the swollen CPE−MPH hydrogels, as shown in Figure 2f−h, is characterized by a distinct three-dimensional network. 692

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Figure 2. SEM images of the fractured surface of CPE-MPH-1 (a), CPE-MPH-2 (b), CPE-MPH-3 (c), CPE-MPH-4 (d), CPE-MPH-5 (e), and the surface morphology of swollen CPE-MPH-2 (f,g) and CPE-MPH-4 (h) before freeze-drying.

Figure 3. FTIR spectra of CPE-MPH hydrogels with different gelatin/γPGA ratio. (a) CPE-MPH-5 with gelatin/γ-PGA ratio of 5:10. (b) CPEMPH-1 with gelatin/γ-PGA ratio of 1:10.

high cross-linking degree featured a compact three-dimensional structure that enabled rigid entrapment of MPH. In contrast, the hydrogels with a low cross-linking degree featured a loose structure, leading to a high release activity of MPH, Table 1. However, the compact structure might also exert a stronger steric effect that inhibited the dispersion of the substrate into the network, leading to a low detected activity, Table 1. A previous study suggested that the low activity of the entrapped enzyme may be due to the accessibility constraints of the immobilized enzyme molecules to the substrate molecules.36 To investigate the effect of cross-linked poly(γ-glutamic acid)/ gelatin entrapment on the kinetic parameters of MPH, Michaelis−Menten kinetic analysis was performed on CPE− MPH and free MPH. As shown in Table 2, both Km and Vmax of the CPE−MPH hydrogels toward methyl parathion decreased with increasing degree of cross-linking. The highest Km value was 12.4 ± 2.3 μmol L−1, which was lower than that of free MPH (23.9 ± 1.4 μmol L−1). Free MPH displayed Vmax, that is, 176.3 ± 6.1 μmol min−1, 71.1% of which corresponded to CPE−MPH-1 (125.3 ± 2.6 μmol min−1), Table 2. The extent of decrease in Vmax following enzyme entrapment in this study was relatively low compared with that observed for organophosphorus hydrolase (OPH) entrapped in silk fibroin (i.e., 10-fold drop in Vmax was observed).37 This indicates the good biocompatibility

Figure 4. The hydrolysis process of substrate (methyl parathion) by CPE-MPH hydrogel. (a) The freeze-dried powder of CPE-MPH hydrogel. (b) The swollen CPE-MPH hydrogel after water-absorbing. (c) The enzymatic reaction was carried out by the access of methyl parathion into the CPE-MPH hydrogel network. (d) The reaction product ρ-nitrophenol was released from the CPE-MPH hydrogel.

and high entrapment efficiency characteristics of the cross-linked poly(γ-glutamic acid)/gelatin. 3.3. Thermostability, pH Stability, and Reusability of CPE−MPH. Entrapment can provide significant benefits to the free enzyme such as enhanced thermostability, pH stability, reusability, and sensitivity. Enzyme entrapment has been widely used for OP compounds degradation and detection.37−45 For instance, Dennis et al. entrapped OPH in silk fibroin.37 Walker et al. developed an intelligent polymerized crystalline colloidal array photonic crystal sensing material using OPH enzyme, capable of sensing organophosphate compound methyl paraoxon at micromolar concentrations in aqueous solutions.39 All the entrapped enzymes in these studies possessed significant advantages and were more suitable for practical application. To assess the performance of CPE−MPH for practical purposes, we examine its thermostability, pH stability, and 693

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Table 1. Activity Assay of CPE-MPH Hydrogels and Released MPHa

a

name

gelatin/γ-PGA (w/w)

(A) activity detected (U)

(B) activity released (U)

(C) activity entrapped (U)

efficiency (A/C) (%)

CPE-MPH-1 CPE-MPH-2 CPE-MPH-3 CPE-MPH-4 CPE-MPH-5

1:10 2:10 3:10 4:10 5:10

1441.5 ± 172.8 1620.9 ± 254.9 1180.3 ± 79.4 1173.9 ± 76.1 1020.2 ± 116.5

1610.2 ± 126.2 1161.4 ± 11.8 963.4 ± 43.2 933.3 ± 80.1 894.4 ± 84.3

4389.8 ± 126.2 4838.6 ± 11.8 5036.6 ± 43.2 5066.7 ± 80.1 5105.6 ± 84.3

32.8 ± 3.0 33.5 ± 5.3 23.4 ± 1.7 23.2 ± 1.9 20.0 ± 2.6

All values are expressed as mean ± SD, based on three separate experiments.

More specifically, CPE−MPH-5 displayed an increased enzyme activity up to 130% following treatment at pHs 7.0, 8.0, and 9.0 for 1 h, Figure 6. CPE−MPH-5 had the highest cross-linking

Table 2. Kinetic Parameters for CPE-MPH Hydrogels and Free MPH in Hydrolyzing Methyl Parathiona name

gelatin/γ-PGA (w/w)

Km (μmol−1)

Vmax (μmol−1 min−1)

free MPH CPE-MPH-1 CPE-MPH-2 CPE-MPH-3 CPE-MPH-4 CPE-MPH-5

1:10 2:10 3:10 4:10 5:10

23.9 ± 1.4 12.4 ± 2.3 10.0 ± 0.9 9.1 ± 0.8 6.1 ± 0.5 4.3 ± 0.6

176.3 ± 6.1 125.3 ± 2.6 113.2 ± 1.0 95.0 ± 1.4 87.9 ± 0.7 78.7 ± 1

All values are expressed as mean ± SD, based on three separate experiments.

a

reusability. As shown in Figure 5, the activity of free MPH steadily decreased at temperatures above 35 °C; the residual

Figure 6. pH stability of CPE-MPH and free MPH. The pH stability of CPE-MPH and free MPH was determined by monitoring residual enzymatic activity after incubation for 1 h at pH 4.0−10.0. Data points correspond to the mean values of three independent experiments.

degree that effectively protected MPH against extreme pH conditions. The increase in enzyme activity observed for CPE− MPH-5 was most likely the result of increased enzyme kinetics under pH 7−9 conditions that was not offset by the pH-induced instability as observed for the other CPE−MPH hydrogels and free MPH. As shown in Figure 7, the reusability of CPE−MPH hydrogels was enhanced with increasing degree of cross-linking. CPE−

Figure 5. Thermostability of CPE-MPH and free MPH. The thermostability of CPE-MPH and free MPH was determined by monitoring residual enzymatic activity after incubation at varying temperatures between 35 and 70 °C for 10 min. Data points correspond to the mean values of three independent experiments.

activity amounted to 50% of its initial activity at 50 °C. In contrast, the CPE−MPH hydrogels exhibited enhanced thermostability, in particular CPE−MPH-5 that featured a 100% increase in activity following heating at 50 °C for 10 min. The latter increase in activity was most likely because of increased enzyme kinetics at the higher temperature that was not offset by the temperature-induced instability that was otherwise observed for the free enzyme.37 However, at temperatures above 50 °C, the activity of the CPE−MPH hydrogels decreased steadily. This result was consistent with those of previous reports.37,46,47 All of the CPE−MPH hydrogels exhibited better stability at pH 6.0−10.0 when compared with that of free MPH. After preincubation for 1 h at pH 6.0−9.0, the CPE−MPH hydrogels retained most of the enzyme activity as opposed to the free MPH that rapidly lost enzyme activity under the same conditions.

Figure 7. Reusability of CPE-MPH. 694

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Figure 8. OP compound degradation by CPE-MPH and free MPH in water. (a) Methyl parathion. (b) Chlorpyrifos.

Figure 9. OP compound degradation by CPE-MPH and free MPH in soil. (a) Methyl parathion. (b) Chlorpyrifos.

decomposition within 100 h that remained unchanged after prolonged reaction time, Figure 8b. The treatment of the soil samples contaminated with methyl parathion (80 mg kg−1) and chlorpyrifos (40 mg kg−1) by CPE− MPH-4 and free MPH was also examined. As shown in Figure 9, both methyl parathion and chlorpyrifos can be degraded naturally in soil. The catalytic action of the added free MPH was not observed, as confirmed by the similarity of the measured OP compounds concentrations in the soil in the presence and absence of the free MPH enzyme. This was likely because of the deactivation of the free MPH. Conversely, CPE−MPH exhibited enhanced efficiency in hydrolyzing OP compounds in soil. Approximately 80% of methyl parathion was degraded within 12 h in the presence of CPE−MPH (Figure 9a). Likewise, the decomposition efficiency of chlorpyrifos, relative to that of methyl parathion, by CPE−MPH was significantly lower. Approximately 70% of the chlorpyrifos in soil was decomposed by CPE−MPH-4 within 140 h, whereas only 30% of the chlorpyrifos in soil was degraded in the presence of free MPH; the same degradation efficiency was obtained in the absence of MPH, Figure 9b. The degradation performance of OP compounds in contaminated soil by the CPE−MPH was similar to that achieved by Sirotkina et al. who examined OPH enzyme immobilized in wheat straw.49 3.5. Biodegradation of CPE−MPH. The biodegradability of CPE−MPH is important for potential application in the

MPH-5 that was prepared with a gelatin/γ-PGA mass ratio of 5:10 featured the best reusability, as demonstrated by its sustained activity over the five cycles studied; only a slight loss of 10% was observed at the fifth cycle. The reduced activity might be due to the release of the MPH from the cross-linked network or deactivation of the enzyme. Regardless, compared with the reported materials in previous studies10,47,48 the herein prepared CPE−MPH hydrogels had higher reusability. 3.4. OP Compound Degradation by CPE−MPH. The treatment of water samples contaminated with methyl parathion (100 mg L−1) and chlorpyrifos (50 mg L−1) by CPE−MPH (CPE−MPH-4) and free MPH was investigated. The decomposition efficiency of the OP compounds by CPE−MPH-4 and free MPH is shown in Figure 8. Both CPE−MPH-4 and free MPH showed rapid hydrolysis of methyl parathion (Figure 8a). Free MPH exhibited higher degrading efficiency relative to CPE−MPH-4 at the early stages of the decomposition process. The large difference in the decomposition efficiencies of CPE− MPH-4 and free MPH became less significant following six hours of decomposition until the difference became negligible. Approximately 95% of the methyl parathion was degraded within 10 h. In contrast, the chlorpyrifos degrading efficiency of CPE−MPH-4 and free MPH was significantly lower, Figure 8b; Chlorpyrifos is a poor hydrolyzing substrate for MPH. Chlorpyrifos was completely degraded by CPE−MPH-4 within 200 h, whereas the free MPH only achieved a 50% 695

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Figure 10. Biodegradation of CPE-MPH. Biodegradation of CPE-MPH was investigated in soil extract by monitoring the weight loss of the swollen CPE-MPH hydrogel, the residual activities of CPE-MPH and free MPH were also measured. (a) Nonsterilized soil extract. (b) Sterilized soil extract.

Figure 11. Swelling and water retention property of CPE-MPH. (a) Swelling ratio of CPE-MPH. (b) Water content of sandy soil with 0.2 and 0.4% (w/ w) CPE-MPH-4.

3.6. Swelling and Water Retention of CPE−MPH. The water retention of the CPE−MPH hydrogels was examined. As observed in Figure 11a, the CPE−MPH hydrogel absorbed a considerable amount of water that was thousands times higher than the weight of the hydrogel itself; the water retention capacity was significantly higher than that of reported hydrogels.51−54 The sandy soil that comprised 0.2 and 0.4% CPE− MPH-4 featured a high water retention capacity; the highest value was achieved with the addition of 0.4% CPE−MPH-4, Figure 11b. This remarkable increase in soil water retention might be attributed to the three-dimensional network of CPE− MPH.

environment. Biodegradation evaluation studies of CPE−MPH4 were conducted in nonsterilized soil extracts. As observed in Figure 10a, CPE−MPH-4 was completely decomposed in the nonsterilized soil extract within 100 h. The residual enzyme activities of the CPE−MPH-4 and free MPH in the nonsterilized soil extract were also measured. Deactivation of free MPH was observed within 40 h after its incorporation in the soil extract, whereas CPE−MPH-4 effectively displayed delayed enzyme deactivation. The half-life of the enzyme activity of CPE−MPH-4 and free MPH under the specified condition was 80 and 15 h, respectively, Figure 10a. The increased activity observed for CPE−MPH-4 within the 20−50 h biodegradation period was most likely because of the collapse of the hydrogel network during the biodegradation process, resulting in a reduced steric effect, consequently leading to an increased reactant diffusion. The same experiment was carried out in a sterilized soil extract. As shown in Figure 10b, under these conditions the biodegradation of CPE−MPH-4 was prolonged, indicating that soil microbe played an important role in the biodegradation process. The cross-linked poly(γ-glutamic acid)/gelatin hydrogel had better biodegradability in soil compared with that of other entrapment carriers; for instance, alginate−starch−bentonite bead only exhibited a 12% weight loss after incubation in soil for 30 days.50

4. CONCLUSION We successfully entrapped MPH in an environment-friendly, biocompatible, and biodegradable cross-linked γ-PGA/gelatin hydrogel. The CPE−MPH hydrogels not only possessed improved thermostability, pH stability, and reusability but also displayed enhanced efficiency in degrading OP compounds in soil and water. The CPE−MPH hydrogels featured more advantages when compared with the free enzyme, thereby enabling stronger practical applicability. The commercialization of γ-PGA and nontoxic and low-cost of gelatin enable facile 696

dx.doi.org/10.1021/bm401784r | Biomacromolecules 2014, 15, 690−697

Biomacromolecules

Article

fabrication of the cross-linked γ-PGA/gelatin hydrogel that offers a new approach for enzyme entrapment.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 024 83970380. Fax: +86 024 83970381. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 863 hi-tech research and development program of Ministry of Science and Technology of The People’s Republic of China (Project No. 2012AA101403).

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