doi:10.1016/j.procbio.2004.04.009 
Copyright © 2004 Elsevier Ltd. All rights reserved.
Application of immobilized horseradish peroxidase for the removal of p-chlorophenol from aqueous solution
Yi-Chen Lai and Sung-Chyr Lin
, 
Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan ROC
Received 17 October 2003;
Revised 25 March 2004;
accepted 4 April 2004.
Available online 24 June 2004.
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Abstract
The development and application of an immobilized horseradish peroxidase (HRP) system with porous aminopropyl glass (APG) beads are reported. The optimal condition for the immobilization of HRP on APG with glutaraladhyde, which leads to a specific activity of 50.7 U/g APG, was identified. Upon immobilization, the operation pH range was broadened downward by one pH unit from 7.5–9.5 to 6.5–9.5 and optimal operation temperature was increased by at least 10 °C. Thermostability of HRP was also improved by approximately 20% upon immobilization. The application of such HRP immobilized APG for the removal of p-chlorophenol from water was investigated. An HPLC method for the quantification of p-chlorophenol was developed to substitute for spectrophotometric methods that are subject to interferences from proteins and other UV-absrobing compounds in the reaction mixtures. The polymerization of p-chlorophenol into insoluble precipitate was completed within 3 h after the initiation of reaction with the addition of hydrogen peroxide with a maximal removal efficiency of 25%, observed at pH 7.5. The addition of poly(ethylene glycol) significantly enhanced the removal efficiency presumably via the formation of a protective shield in the vicinity of the active site of HRP from the free radicals formed during polymerization and can thus reduce the amount of enzymes needed to achieve the desired removal efficiency. The reusability of the HRP immobilized APG was also demonstrated.
Author Keywords: Enzyme immobilization; Horseradish peroxidase; Chlorophenol
Fig. 1. Effect of activation time on immobilization percentage (•) and specific activity (○) of HRP immobilized APG. Porous aminopropyl glass beads were activated with 2.5% glutaraldehyde at 25 °C for the prescribed period of time. The activated APG was then conjugated with HRP at 4 °C for 12 h.
Fig. 2. Effect of conjugation time on immobilization percentage of APG upon 8 h (•) and 20 h (○) activation. Activated APG was conjugated with HRP solution at 4 °C.
Fig. 3. Effect of HRP concentration on the enzyme load of HRP immobilized APG (•), specific activity of the HRP immobilized APG (○), and specific activity of HRP molecules immobilized on APG (
). APG was activated with 2.5% glutaraldehyde at 25 °C for 8 h and subsequently conjugated with HRP at 4 °C for 12 h.
Fig. 4. Effect of pH on the relative activity of soluble HRP (•) and HRP immobilized APG (○). HRP activity at 30 °C was accessed spectrophotometrically at 420 nm.
Fig. 5. Effect of temperature on the relative activity of soluble HRP (•) and HRP immobilized APG (○). HRP activity at pH 8.0 was accessed spectrophotometrically at 420 nm.
Fig. 6. Thermostability of soluble HRP (•) and HRP immobilized APG (○). HRP activity was accessed at 25 °C spectrophotometrically at 420 nm after incubation at the prescribed temperature for 30 min.
Fig. 7. Typical HPLC chromatogram of clarified p-chlorophenol solution after HRP-catalyzed polymerization. The analysis was conducted with a C18 reverse phase column eluted isocratically with a mobile phase of 35% acetonitrile in 0.01% phosphoric acid solution at a flow rate of 1 ml/min. The peak, monitored at 426 nm, with a retention time of 10.8 min was identified as that of p-chlorophenol.
Fig. 8. Reaction profile of p-chlorophenol removal with HRP immobilized APG. HRP immobilized APG, at an amount of 0.5 U/ml, was added to aqueous solution containing 50 mg/l p-chlorophenol at 25 °C, pH 7.5. The residual p-chlorophenol concentration was determined by HPLC method.
Fig. 9. Effect of HRP activity on the efficiency of p-chlorophenol removal. HRP immobilized APG was added to aqueous solution containing 50 mg/l p-chlorophenol. The reaction was allow to proceed at 25 °C, pH 7.5 for 3 h before the reaction mixture was clarified for HPLC analysis. The efficiency of p-chlorophenol removal was defined as the percentage of p-chlorophenol removed from the aqueous solution upon filtration.
Fig. 10. Effect of pH on the efficiency of p-chlorophenol removal. HRP immobilized APG, at an amount of 0.5 U/ml, was added to aqueous solution containing 50 mg/l p-chlorophenol. The reaction was allow to proceed at 25 °C for 3 h before the reaction mixture was clarified for HPLC analysis.
Fig. 11. Effect of PEG on the efficiency of p-chlorophenol removal. PEG with a molecular weight of 6000 was included in p-chlorophenol solution containing 0.5 U HRP/ml (•) and 0.2 U HRP/ml (○). The reaction was allow to proceed at 25 °C, pH 7.5 for 3 h before the reaction mixture was clarified for HPLC analysis.
Fig. 12. Performance of the HRP immobilized APG in repeated-batch operation for p-chlorophenol removal. HRP immobilized APG was used for p-chlorophenol removal. After each batch, HRP immobilized APG was recovered by centrifugation and rinsed for the subsequently batch. For each batch, the reaction was allow to proceed at 25 °C, pH 7.5 for 3 h before the reaction mixture was clarified for HPLC analysis.
Abstract
Purchase PDF (109 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (10)
Purchase PDF (219 K)
