Polyethylene glycol improves phenol removal by immobilized turnip peroxidase
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Bioresource Technology 99 (2008) 8605–8611
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Polyethylene glycol improves phenol removal by immobilized turnip peroxidase F. Quintanilla-Guerrero a, M.A. Duarte-Vázquez b, B.E. García-Almendarez a, R. Tinoco c, R. Vazquez-Duhalt c, C. Regalado a,* a
Departamento de Investigación y Posgrado en Alimentos PROPAC, Universidad Autónoma de Querétaro, Facultad de Química, C.U. Cerro de las Campanas s/n, Querétaro, Qro 76010, Mexico b NUCITEC S.A. de C.V. Comerciantes 15-3, Peñuelas, Querétaro, Qro 76148, Mexico c Instituto de Biotecnología UNAM Av. Universidad 2001 Col. Chamilpa Cuernavaca Morelos 62215, Mexico
a r t i c l e
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Article history: Received 25 September 2007 Received in revised form 3 April 2008 Accepted 3 April 2008 Available online 23 May 2008 Keywords: Peroxidase Phenolic compounds Immobilization Polyethylene glycol
a b s t r a c t Puriﬁed peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Afﬁ-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxiﬁcation of a synthetic phenolic solution and a real wastewater efﬂuent from a local paints factory. The effectiveness of phenolic compounds (PC’s) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3 h to 10 min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial efﬂuent showing PC’s removals >90% PC’s during the ﬁrst 10 reaction cycles. High PC’s removal efﬁciencies (>95%) were obtained using both immobilized preparations at PC’s concentrations 50% removal, reported 10 phenol removal cycles, using the same reaction time. Thus, the protective effect of PEG during phenol removal by peroxidase activity is clearly demonstrated. 3.7. Thermal stability of free and immobilized TP The thermal stability of the free, covalent immobilized, and alginate-entrapped peroxidase was monitored after incubating at 50, 60 and 70 °C for various time intervals. Heat inactivation rate increased with the temperature and the length of heat exposure. Free enzyme retained 50% of the original activity within the ﬁrst ﬁvemin incubation at 70 °C, while the covalently and alginate-immobilized peroxidase exhibited close to 85% and 70%, respectively, of their original activity at the same conditions. Arrhenius plots were prepared and the activation energy for peroxidase destruction, DG and DH were calculated (Table 3). Free native turnip peroxidase showed lower values of the thermodynamic parameters evaluated (Ea, DG and DH), when compared to those obtained for both immobilized TP preparations. Results from Table 3 demonstrated that the covalently immobilized was signif-
Residual activity (%) Phenol removal (%)
100 80 60 40 20
Covalently immobilized 100
Residual activity (%) Phenolremoval (%)
80 60 40 20 0 0
9 10 11 12 13 14 15
Reaction cycle Fig. 7. Effect of reaction cycles number on the efﬁciency of phenolic compounds removal from a paints factory wastewater, and on residual enzyme activity, using covalently immobilized TP through column operation. Cycle contact time = 10 min, [PEG] = 100 mg l1. TP activity 1.2 U ml1, 1.09 mM total phenols concentration, 1.7 mM H2O2. Free TP removed 99 ± 1% phenols, and showed a residual activity of 60 ± 3%, in a single cycle process.
Table 3 Thermodynamic values of thermal inactivation of native and immobilized preparations of turnip peroxidase (± standard deviation) TP preparation Native Alginate-entrapped Covalently immobilized
Ea (kJ mol1) a
113.9 ± 6.2 159.5 ± 8.3b 170.4 ± 9.5b
DG (kJ mol1) a
103.2 ± 4.8 106.4 ± 5.3a 107.0 ± 5.1a
DH (kJ mol1) 111.0 ± 5.9a 156.6 ± 8.8b 167.5 ± 9.3b
Different letters within the same column are signiﬁcantly different (p < 0.05).
icantly more thermally stable than the alginate-entrapped TP. This higher thermostability and catalytic efﬁciency of covalent immobilized peroxidase, make it a good alternative for many biotechnological processes in which a thermally stable enzyme is required. 4. Conclusion We can conclude so far that the presence of PEG in the reaction mixture protects TP from inactivation during phenol polymerization. This protection allows to extend the enzyme lifetime when it is immobilized, both covalently or entrapped, increasing the number of reaction cycles with high removal efﬁciency. Phenol removal from a real wastewater efﬂuent was efﬁciently performed by immobilized TP in presence of PEG. The alginate-entrapped TP showed an unexpectedly higher efﬁciency in phenols removal than the covalently immobilized enzyme, despite peroxidase activity losses due to enzyme diffusion out of the matrix, which was attributed to the PEG-TP conﬁnement within the alginate matrix. This intimate PEG-TP contact was not achieved by the recycle system used for phenols removal by covalently immobilized peroxidase. A large-scale enzymatic process for industrial efﬂuent treatment is expected to be developed with immobilized TP that should be stable enough to make the process economically feasible. Acknowledgement
9 10 11 12 13 14 15
We are grateful to CONACYT for a Ph.D. scholarship to FQG.
Reaction cycle References Fig. 6. Effect of reaction cycles number on the efﬁciency of phenolic compounds removal from a paints factory wastewater, and on residual enzyme activity, using calcium alginate-entrapped TP. Cycle contact time = 10 min, [PEG] = 100 mg l1, TP activity (free and immobilized) 1.2 U ml1, 1.09 mM total phenols concentration, 1.7 mM H2O2. Free TP removed 99 ± 1% phenols, and showed a residual activity of 60 ± 3%, in a single cycle process.
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