Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation
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Enzymatic biodegradation of organophosphate pesticides (OPs) is a promising technology to remove these toxic compounds. However, its application in industrial washing was restricted by the lack of efficient immobilized enzymes that can work at high temperatures and high pHs in the presence of various detergents. Therefore, it is necessary to develop a simple method to prepare a robust immobilized enzyme for efficient degradation of OPs.
An organophosphate hydrolase (OPH), PoOPHM9, was conjugated and immobilized with a commercially available polymer, Pluronic F127. The prepared cross-linked enzyme-polymer conjugate (CLEPC) displayed higher pH stability in the range from 7.0 to 11.0 and a higher optimal temperature (50 °C) than that of free PoOPHM9 (30 °C). Its half-life and apparent kcat/KM reached 12.8 h at 50 °C and 390.3 ± 7.8 mM−1 s−1, respectively, which were even better than that of the traditional cross-linked enzyme aggregates (CLEA, 7.2 h and 10.9 ± 1.7 mM−1 s−1). The activity of PoOPHM9 CLEPC was further enhanced up to 2.5-fold by the anionic, nonionic and biocompatible detergents, which was first observed. 0.15 mM Malathion was degraded completely by PoOPHM9 CLEPC after activation within 10 min in the presence of 0.1% (w/w) detergents of all types at pH 9.0 and 25 °C, demonstrating its capability in degrading OPs at practically relevant conditions.
KeywordsOrganophosphate Organophosphate hydrolase Immobilization Biodegradation Enzyme-polymer conjugates Pluronic F127
cross-linked enzyme-polymer conjugate
cross-linked enzyme aggregate
sodium dodecyl sulphate
coconut oil derivative
The accumulation of toxic organophosphate pesticides (OPs) in food and environment has caused an increasing threat to public health (Mostafalou and Abdollahi 2017; Cycon et al. 2017; Hernandez et al. 2017). OPH based biodegradation, which can efficiently hydrolyze OPs to benign molecules, is one of the most attractive technologies for removing OPs (Schenk et al. 2016; Kim et al. 2014a, b; Ramalho et al. 2016; Theriot and Grunden 2011). Many naturally occurring or engineered OPHs have been developed so far (Cherny et al. 2013; Khare et al. 2012; Luo et al. 2014; Abe et al. 2014; Bigley et al. 2016; Chen et al. 2015; Jackson et al. 2009). However, in spite of high activity, free OPHs are difficult to be recovered from aqueous solution, and are often denatured quickly under harsh environments such as high temperatures and high pHs that are usually used in industrial washing and water treatment, hindering its usage in practical applications (Giudice et al. 2016; Bai et al. 2017). Therefore, it is essential to develop a simple and cost-effective method for fully exploiting the potential of OPHs in biodegradation.
Currently, various enzyme immobilization methods to improve the stability and recyclability of OPHs have been extensively explored, including the immobilization of enzymes on nanoparticles (Breger et al. 2015; Hondred et al. 2017), mesoporous silica (Singh et al. 1999), membranes (Yan et al. 2015), textiles (Gao et al. 2014), and encapsulation or complexation of enzymes with amyloid fibrils (Raynes et al. 2011), metal–organic frameworks (Li et al. 2016), polyurethane foam (LeJeune et al. 1997) and polymers (Suthiwangcharoen and Nagarajan 2014; Wei et al. 2013). Carrier-free method, such as cross-linked enzyme aggregate (Zheng et al. 2011; Pan et al. 2011), has been used in the production of value-added chemicals in large quantities (Pan et al. 2014). Although enzyme immobilization gives rise to a better thermostability, it is usually achieved at the expense of the decreased catalytic activity of an enzyme. In addition, the immobilization method should face challenges from practical applications. For example, the immobilized OPHs need to be stable and active at very harsh conditions such as high temperatures and alkaline pH in industrial washing of OP contaminated food. Particularly, they should bear various detergents that can easily denature proteins. Therefore, this special application requirement makes the immobilization of OPHs an extremely difficult task, compared with other immobilized enzymes for traditional biocatalysis.
Recently, a triblock amphiphilic copolymer, poly (ethylene oxide-b-propylene oxide-b-ethylene oxide) F127 (known as Pluronic F127), has been demonstrated to enhance the stability and activity of free OPHs through the interaction between the hydrophobic segments of polymers and the hydrophobic regions of enzyme surface (Suthiwangcharoen and Nagarajan 2014; Kim et al. 2014b). In addition to OPHs, various enzymes can be modified by direct conjugation or covalent binding with Pluronic F127 (Wu et al. 2014, 2015; Zhang et al. 2013). Although advances in the conjugation of Pluronic F127 and free enzymes have been made so far, the application of Pluronic F127 in immobilized enzymes has not been achieved. One possible reason is that the current methods of preparing enzyme-polymer conjugates are performed in aqueous phase (Suthiwangcharoen and Nagarajan 2014; Kim et al. 2014b) and the carrier-immobilized enzymes are not soluble in aqueous solution. More importantly, the improving effect of Pluronic F127 in the catalytic properties of free OPH has not been demonstrated in immobilized OPH yet. Thus, it is of interest to develop a simple method of preparing immobilized OPH-Pluronic F127 conjugates with the expectation of maintaining the high stability and activity of OPH with Pluronic F127 simultaneously.
In the previous study, we engineered a newly discovered phosphotriesterase (PoOPHM9, Luo et al. 2016) with improved catalytic activity and thermostability for efficient malathion degradation, and demonstrated its effectiveness in removing malathion using free PoOPHM9 in the presence of various detergents (Bai et al. 2017), which prompted us to further explore the possibility of preparing immobilized enzymes for potential industrial applications. Herein, we reported a simple method to prepare cross-linked enzyme-polymer conjugates (CLEPC) by directly incorporating Pluronic F127 in the process of carrier-free PoOPHM9 immobilization. The catalytic efficiency and stability of PoOPHM9 CLEPC were systematically investigated, and its capability in degrading malathion in the presence of various detergents under practically relevant conditions were demonstrated.
Materials and method
All chemicals of reagent grade were purchased from Sigma (St. Louis, MO). Malathion (99% purity) was purchased from Shanghai Pesticide Research Center. The P. pastoris strain X33 (Mut+His+) bearing the plasmid of pPICZαA containing PoOPHM9 gene was constructed in our lab. Detergents containing coconut oil derivatives (COD, Komi, Lot 6907974981509), Sodium dodecyl sulphate (SDS, Mama Lemon, Lot 6903624600158) and alkyl polyglycoside (APG, Diaopai, Lot 6910019001841) were purchased from a local supermarket without further treatment.
Preparation of PoOPHM9 CLEPC
PoOPHM9 was produced extracellularly by high-density fermentation of an engineered Pichia pastoris strain described previously (Bai et al. 2017). After fermentation, the broth was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated and freeze-dried to form lyophilized enzyme powders. The lyophilized enzyme powders were dissolved in a Tris–HCl buffer (pH 7.0) to 20 mg/mL, to which ammonium sulfate was slowly added to 0.5 g/mL. The solution was then stirred at 4 °C for 30 min to precipitate enzymes. Subsequently, Pluronic F127 was slowly added into the suspension to 80 mg/mL and the mixture was continuously stirred at 4 °C for 30 min. Glutaraldehyde of 40 mM was added to cross-link the aggregates and the suspension was stirred at 4 °C for 3 h. Then, the suspension was centrifuged at 12,000 rpm for 3 min, and the precipitate was washed three times and re-suspended in 0.5 mL Tris–HCl buffer (pH 7.0).
Activity assays of PoOPHM9 CLEPC
Enzymatic characterization of PoOPHM9 CLEPC
The relative activity of immobilized and free enzymes in the presence of various detergents at different time was calculated according to Eq. (3). To determine the reusability, the reaction mixture was centrifuged at 12,000 rpm for 3 min to recover the immobilized enzymes. The collected enzymes were washed for 3 times and the relative activity of PoOPHM9 CLEPC after each round was measured and calculated against the initial activity detected in the first batch.
Degradation of malathion by PoOPHM9 CLEPC in the presence of detergents
Typically, 980 μL of 50 mM Tris–HCl buffer (pH 9.0) containing 0.3 mM DNTB and 0.1% (w/w) of COD, APG and SDS was mixed with 10 μL of 15 mM malathion and 10 μL of 420 U/mL PoOPHM9 CLEPC. The mixture was stirred at 25 °C or 50 °C for 30 min and the absorbance readings were taken every minute. The hydrolysis percentage was calculated by comparing the absorbance at different times with that of the completely hydrolyzed product.
Results and discussion
Preparation of PoOPHM9 CLEPC
Kinetic parameters of the free and immobilized enzymes toward malathion
kcat/KM (mM−1 s−1)
24.2 ± 0.3
91.2 ± 5.3
265.4 ± 2.0
Luo et al. (2016)
49.1 ± 1.2
125.7 ± 7.7
390.3 ± 7.8
1.1 ± 0.1
103.8 ± 1.7
10.9 ± 1.7
Enzymatic properties of PoOPHM9 CLEPC
Effect of detergents on the activity of PoOPHM9 CLEPC
Previously, we found that various commercially available detergents can influence the activity of PoOPHM9 (Bai et al. 2017). For example, the anionic and non-ionic detergents, such as sodium dodecyl sulphate (SDS) and alkyl polyglycoside (APG), decreased the enzyme activity while the biocompatible detergent, coconut oil derivatives (COD), showed a less negative impact on the enzyme activity. In this work, we compared the catalytic activity of PoOPHM9 CLEPC and free PoOPHM9 incubated in the presence of 0.1% (w/w) detergents at 25 °C (Fig. 3d). As observed previously, the activity of free PoOPHM9 decreased to various extent in the presence of COD, APG and SDS. In contrast, the activity of PoOPHM9 CLEPC were all enhanced by 2- to 2.5-fold in the presence of the three detergents after 2 h incubation. It has been reported that SDS can activate phosphotriesterase (PTE) and enhance its activity (Giudice et al. 2016), but inactivate PoOPHM9 (Bai et al. 2017). However, the phenomena that all kinds of detergents can enhance the activity of immobilized enzyme-polymer conjugates was first observed. To confirm this, we compared the activity of PoOPHM9 CLEPC in the absence of detergents under the same condition (red symbols, Fig. 3d). The result showed that the activity of PoOPHM9 CLEPC increased to approximately 154% after 2 h, which was lower than that of CLEPC in the presence of detergents (2- and 2.5-fold). The result suggested that detergents indeed activated and further enhanced the catalytic activity of PoOPHM9 CLEPC.
Effect of Pluronic F127 on the activity of PoOPHM9 CLEPC
First, we prepared and incubated PoOPHM9 CLEA in the absence or presence of detergents. However, the activity in all cases decreased (Additional file 1: Figure S2), indicating that only CLEA cannot enhance the activity of PoOPHM9. Compared with the result in Fig. 3d, it can be concluded that Pluronic F127 is essential in improving the activity of immobilized enzymes. Second, we prepared the conjugate PoOPHM9-PF127 formed between the free PoOPHM9 and Pluronic F127 and measured their activity in the absence of detergents after 2 h incubation (Additional file 1: Figure S3a). The activity of PoOPHM9-PF127 was slightly increased to 105% by 1% and 10% (w/w) Pluronic F127 after 2 h incubation (Additional file 1: Figure S3a). Interestingly, when 0.1% (w/w) detergents were added, the activity of PoOPHM9-PF127 conjugates was all enhanced to approximately 120% relative to that of the free enzyme (Additional file 1: Figure S3b). This result suggested that the activity improvement was achieved by the cooperation between Pluronic F127 and detergents. However, this activity improvement was lower than that of PoOPHM9 CLEPC incubated with detergents (2- and 2.5-fold), indicating that the enzyme aggregation and cross-linkage in PoOPHM9 CLEPC are very important in the activity enhancement (Akbar et al. 2007; Perzon et al. 2017; Wang et al. 2011). In summary, the significant enhancement in the activity of PoOPHM9 CLEPC was caused by the synergic effect of Pluronic F127, enzyme immobilization and detergents.
Degradation of malathion by PoOPHM9 CLEPC
In conclusion, we developed a simple method to prepare an immobilized organophosphate hydrolase (PoOPHM9) conjugated with Pluronic F127 for efficient malathion degradation. Compared to the traditional PoOPHM9-Pluronic F127 CLEA, PoOPHM9-Pluronic F127 CLEPC has a 2.2 times higher activity recovery. It features excellent thermostability with a half-life of 12.8 h at 50 °C, which is 25.6 and 1.7 times higher than that of the free PoOPHM9 and PoOPHM9-Pluronic F127 CLEA CLEA. Particularly, PoOPHM9 CLEPC was not deactivated by detergents; instead, its activity can even be further enhanced by up to 2.5-fold in the presence of detergents such as coconut oil derivatives, sodium dodecyl sulphate and alkyl polyglycoside. Degradation of 0.15 mM malathion with PoOPHM9 CLEPC at 50 °C or 25 °C in the presence of detergents was demonstrated, showing the potential of this new immobilized OPH in practical industrial applications. We believe this technology can be extended easily to produce other immobilized polymer-enzyme conjugates, owing to the simplicity of this technology.
XYZ, JG and YPB designed the experiments; HC, YLZ and XJL performed the research experiments; DSX, QD and XC helped in the experiments. XYZ, JG and YPB analyzed the data; JHX helped in manuscript preparation; HC, XYZ, JG and YPB wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Consent for publication
All of the authors have read and approved to submit it to bioresources and bioprocessing.
Ethics approval and consent to participate
This work was financially sponsored by the National Key Research and Development Program of China (2016YFA0204300), National Natural Science Foundation of China (Nos. 21505044, 21536004 and 21506055) and Natural Science Foundation of Shanghai (18ZR1409900).
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