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Enzymatic oxidation of phenols by immobilized oxidoreductases
10th International Symposium “Scientific Bases for the Preparation of Heterogeneous Catalysts” E.M. Gaigneaux, M. Devillers, S. Hermans, P. Jacobs, J. Martens and P. Ruiz (Editors) © 2010 Elsevier B.V. All rights reserved.
Enzymatic oxidation of phenols by immobilized oxidoreductases B. Tikhonov, A. Sidorov, E. Sulman, V. Matveeva Tver Technical University, A. Nikitina str., 22, Tver, 170026, Russia
Abstract 7 various cation-exchange resins on the basis of styrenedivynilbenzene were used as the carriers for immobilization of oxidoreductases (horseradish peroxidase and musroom tyrosinase). Ion exchangers were treated with sodium alginate, chitosan, glutaric dialdehyde and N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide hydrochloride. Synthesized biocatalytic systems on the basis of oxidoreductases were found to be highly active and stable in catalytic oxidation of phenols including sewage treatment and industrial waste products to harmless melanin-type polymers. Keywords: immobilization, oxidoreductases, waste water, phenol, oxidation
1. Introduction The use of oxidoreductases in the industrial catalysis has considerably increased recently . Their efficiency is proved in reactions of homogeneous oxidation of aromatic components, in particular, aniline and phenol in modeling solutions and waste waters [1,2]. Worldwide application of this method is limited to the high cost and poor stability of purified enzymes. These problems can be solved by the immobilization of enzymes from the aqueous extracts on inorganic or organic carriers with the obtaining as a result of a heterogeneous system [3,4]. One of the most prospective methods of enzymes immobilization is the covalent cross-linking of enzymes with the modified carrier which should be mechanically strong, water-insoluble, has high chemical and biological stability and low cost. Experimental selective oxidation of monosaccharides: L-sorpbose and D-glucose.
2. Experimental 2.1. Materials and methods In this work various biocatalytic systems were investigated. 7 various cation-exchange resins (Dowex 50WX, Dowex 50WX2, Lewatit CNP-105, Amberlite 200, Amberlite IR-120, Amberlite IRC-86, Ku 2-8) on the basis of styrenedivynilbenzene with SO3H or COOH active groups were used as the carriers. Ion exchangers were treated with sodium alginat, chitosan, glutaric dialdehyde and N-(3-dimethyl-aminopropyl)-Nethylcarbodiimide hydrochloride. Two methods of chitosan and activating agent deposition on ion exchange resin were studied. They are consecutive deposition and deposition of components mixture. The schemes of biocatalyst synthesis: (i) with primary support activation S + M Æ С-M;
S-M + A Æ S-M-A;
S-M-A + Е Æ S-M-A-Е;
326
B. Tikhonov et al. (ii) with primary activation of the enzyme S + MÆ S-M;
A + Е Æ A-Е;
S-M + A-Е Æ S-M-A-Е.
where S – support; M – modifier; A – crosslinking agent; E – enzyme. Also catalytic efficiency of peroxidase and tyrosinase from various sources were investigated. The activity of biocatalysts in reactions of phenol and catechol oxidation to melanin-type polymers was found as a change of optical density of reaction mixture at 440 nm. Besides, to determine the kinetic parameters of the catalysts the chronometric method was used [5].
3. Results and discussion Experiments showed that the most efficient carriers are Ku 2-8 and Amberlite 200, which functional groups have high reactivity. Besides, they can be applied for biocatalysis by surface characteristics. It has been revealed that the scheme (i) is optimal for crosslinking agent glutaric dialdehyde, while the scheme (ii) – for N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide hydrochloride. During the measurements it was determined that consecutive deposition of chitosan and glutaric aldehyde on cation-exchanger provides the strongest and more stable bounding of enzyme with the carrier. It was shown that glutaric dialdehyde provides better stabilization of enzyme on the carrier to compare with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride. For the investigated biocatalysts the optimal conditions of phenols oxidation process with the achievement of high degree of conversion (more than 95%) were found: temperature - 25°C, intensity of mixing - 300 min-1, pH 6.5 and 7 - for peroxidase and tyrosinase, respectively. The optimal ratio of the biocatalyst components was determined (see Table 1). Table 1. The optimal ratio of the components of the biocatalysts. Biocatalyst
Concentration of chitosan solution, %
Concentration of m(E)/m(S), % glutaric dialdehyde solution, % S-М-А-E1 0,1 25 8 S-М-А-E2 0,2 25 10 where S – cation exchanger; M – chitosan; A – glutaric dialdehyde; E1 – peroxidase; E2 tyrosinase.
Physicochemical investigations (FTIR spectroscopy, XPS, nitrogen physisorption) of optimal biocatalytic systems were carried out. The result of nitrogen physisorption for the biocatalyst components is shown in Fig. 1. Kinetic and physicochemical investigations showed that biopolymer (chitosan) is distributed on the surface of the carrier as separate molecules or bidimentional clasters without formation of 3D structures. Such distribution promotes minimization of intradiffusive limitation during the oxidation. The scheme of the optimal biocatalyst is shown in Fig. 2.
Enzymatic oxidation of phenols by immobilized oxidoreductases
327
Figure 1. The result of nitrogen physisorption.
Figure 2. The scheme of the optimal biocatalyst formation.
The representation of the surface of a biocatalytic system is shown in Fig. 3. Kinetic parameters of synthesized biocatalysts are shown in Table 2. Table 2. Kinetic parameters of synthesized biocatalysts. Biocatalyst Native Peroxidase S–C–A–E1
Substrate Phenol Catechol Phenol Catechol
Vm, mM s-1 0.069 0.156 0.024
Km, M 3.791 11.439 29.79
0.023
54.23
Native Tyrosinase Catechol 0.022 18.99 S–C–A–E2 Catechol 0.009 85.6 S – cation exchange resin Ku 2-8; C – chitosan; A – glutaric dialdehyde; E1 – horseradish peroxidise, E2 – mushroom tyrosinase
The decrease of immobilized peroxidase and tyrosinase activities are the consequence of heterogenization of enzymes and the influence of intradiffusive factors.
328
B. Tikhonov et al.
Figure 3. Representation of a biocatalytic system surface (S – carrier, A – activator, E – enzyme).
However, biocatalysts are stable in more than 10 cycles, and heterogenization makes the system more technological. One more advantage of the developed biocatalytic systems is an essential depreciation of catalyst due to the obtaining of enzymes from vegetative raw material without expensive purification.
4. Conclusions Synthesized biocatalytic systems on the basis of horseradish peroxidase and mushroom tyrosinase were found to be highly active and stable in catalytic oxidations of phenols including sewage treatment and industrial waste products. The catalysts obtained can be used for sewage and industrial waste biocatalytic treatment as they allow transfering dangerous phenolic compounds to harmless melanin-type polymers.
Acknowledgements We sincerely thank Federal Education Agency of Russian Federation (contracts P 257 and P 1196) for the financial support.
References 1. 2. 3. 4. 5.
S. Ibrahim, H. I. Ali, K. E. Taylor., N. Biswas, J. K. Bewtra. Enzyme-catalyzed removal of phenol from refinery wastewater: Feasibility studies. Water Environ. Res. 73 (2001) 165172. M. A. Gilabert, L. G. Fenoll, F. Garcia-Molina, J. Tudela, F. Garcia-Canovas, J. N RodryguezLopez. Kinetic characterization of phenol and aniline derivates as substrates of peroxidase Biol. Chem., Vol. 385, (2004) 795–800. L. V. Bindhu, E.T. Abraham. Immobilization of Horseradish Peroxidase on Chitosan for Use in Nonaqueous Media. Inc. J. Appl. Polym. Sci. 88 (2003) 1456-1464. W. Tischer, F. Wedekind. Immobilized enzymes: methods and applications. Top. Curr. Chem., Vol. 200 (1999) 95–126. D. C. Goodwin, I. Yamazaki, S. D. Aust, and T. A. Grover, Determination of Rate Constants for Rapid Peroxidase Reactions, Anal. Bioch, 231, 333–338 (1995).
Enzymatic oxidation of phenols by immobilized oxidoreductases B. Tikhonov, A. Sidorov, E. Sulman, V. Matveeva Tver Technical University, A. Nikitina str., 22, Tver, 170026, Russia
Abstract 7 various cation-exchange resins on the basis of styrenedivynilbenzene were used as the carriers for immobilization of oxidoreductases (horseradish peroxidase and musroom tyrosinase). Ion exchangers were treated with sodium alginate, chitosan, glutaric dialdehyde and N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide hydrochloride. Synthesized biocatalytic systems on the basis of oxidoreductases were found to be highly active and stable in catalytic oxidation of phenols including sewage treatment and industrial waste products to harmless melanin-type polymers. Keywords: immobilization, oxidoreductases, waste water, phenol, oxidation
1. Introduction The use of oxidoreductases in the industrial catalysis has considerably increased recently . Their efficiency is proved in reactions of homogeneous oxidation of aromatic components, in particular, aniline and phenol in modeling solutions and waste waters [1,2]. Worldwide application of this method is limited to the high cost and poor stability of purified enzymes. These problems can be solved by the immobilization of enzymes from the aqueous extracts on inorganic or organic carriers with the obtaining as a result of a heterogeneous system [3,4]. One of the most prospective methods of enzymes immobilization is the covalent cross-linking of enzymes with the modified carrier which should be mechanically strong, water-insoluble, has high chemical and biological stability and low cost. Experimental selective oxidation of monosaccharides: L-sorpbose and D-glucose.
2. Experimental 2.1. Materials and methods In this work various biocatalytic systems were investigated. 7 various cation-exchange resins (Dowex 50WX, Dowex 50WX2, Lewatit CNP-105, Amberlite 200, Amberlite IR-120, Amberlite IRC-86, Ku 2-8) on the basis of styrenedivynilbenzene with SO3H or COOH active groups were used as the carriers. Ion exchangers were treated with sodium alginat, chitosan, glutaric dialdehyde and N-(3-dimethyl-aminopropyl)-Nethylcarbodiimide hydrochloride. Two methods of chitosan and activating agent deposition on ion exchange resin were studied. They are consecutive deposition and deposition of components mixture. The schemes of biocatalyst synthesis: (i) with primary support activation S + M Æ С-M;
S-M + A Æ S-M-A;
S-M-A + Е Æ S-M-A-Е;
326
B. Tikhonov et al. (ii) with primary activation of the enzyme S + MÆ S-M;
A + Е Æ A-Е;
S-M + A-Е Æ S-M-A-Е.
where S – support; M – modifier; A – crosslinking agent; E – enzyme. Also catalytic efficiency of peroxidase and tyrosinase from various sources were investigated. The activity of biocatalysts in reactions of phenol and catechol oxidation to melanin-type polymers was found as a change of optical density of reaction mixture at 440 nm. Besides, to determine the kinetic parameters of the catalysts the chronometric method was used [5].
3. Results and discussion Experiments showed that the most efficient carriers are Ku 2-8 and Amberlite 200, which functional groups have high reactivity. Besides, they can be applied for biocatalysis by surface characteristics. It has been revealed that the scheme (i) is optimal for crosslinking agent glutaric dialdehyde, while the scheme (ii) – for N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimide hydrochloride. During the measurements it was determined that consecutive deposition of chitosan and glutaric aldehyde on cation-exchanger provides the strongest and more stable bounding of enzyme with the carrier. It was shown that glutaric dialdehyde provides better stabilization of enzyme on the carrier to compare with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride. For the investigated biocatalysts the optimal conditions of phenols oxidation process with the achievement of high degree of conversion (more than 95%) were found: temperature - 25°C, intensity of mixing - 300 min-1, pH 6.5 and 7 - for peroxidase and tyrosinase, respectively. The optimal ratio of the biocatalyst components was determined (see Table 1). Table 1. The optimal ratio of the components of the biocatalysts. Biocatalyst
Concentration of chitosan solution, %
Concentration of m(E)/m(S), % glutaric dialdehyde solution, % S-М-А-E1 0,1 25 8 S-М-А-E2 0,2 25 10 where S – cation exchanger; M – chitosan; A – glutaric dialdehyde; E1 – peroxidase; E2 tyrosinase.
Physicochemical investigations (FTIR spectroscopy, XPS, nitrogen physisorption) of optimal biocatalytic systems were carried out. The result of nitrogen physisorption for the biocatalyst components is shown in Fig. 1. Kinetic and physicochemical investigations showed that biopolymer (chitosan) is distributed on the surface of the carrier as separate molecules or bidimentional clasters without formation of 3D structures. Such distribution promotes minimization of intradiffusive limitation during the oxidation. The scheme of the optimal biocatalyst is shown in Fig. 2.
Enzymatic oxidation of phenols by immobilized oxidoreductases
327
Figure 1. The result of nitrogen physisorption.
Figure 2. The scheme of the optimal biocatalyst formation.
The representation of the surface of a biocatalytic system is shown in Fig. 3. Kinetic parameters of synthesized biocatalysts are shown in Table 2. Table 2. Kinetic parameters of synthesized biocatalysts. Biocatalyst Native Peroxidase S–C–A–E1
Substrate Phenol Catechol Phenol Catechol
Vm, mM s-1 0.069 0.156 0.024
Km, M 3.791 11.439 29.79
0.023
54.23
Native Tyrosinase Catechol 0.022 18.99 S–C–A–E2 Catechol 0.009 85.6 S – cation exchange resin Ku 2-8; C – chitosan; A – glutaric dialdehyde; E1 – horseradish peroxidise, E2 – mushroom tyrosinase
The decrease of immobilized peroxidase and tyrosinase activities are the consequence of heterogenization of enzymes and the influence of intradiffusive factors.
328
B. Tikhonov et al.
Figure 3. Representation of a biocatalytic system surface (S – carrier, A – activator, E – enzyme).
However, biocatalysts are stable in more than 10 cycles, and heterogenization makes the system more technological. One more advantage of the developed biocatalytic systems is an essential depreciation of catalyst due to the obtaining of enzymes from vegetative raw material without expensive purification.
4. Conclusions Synthesized biocatalytic systems on the basis of horseradish peroxidase and mushroom tyrosinase were found to be highly active and stable in catalytic oxidations of phenols including sewage treatment and industrial waste products. The catalysts obtained can be used for sewage and industrial waste biocatalytic treatment as they allow transfering dangerous phenolic compounds to harmless melanin-type polymers.
Acknowledgements We sincerely thank Federal Education Agency of Russian Federation (contracts P 257 and P 1196) for the financial support.
References 1. 2. 3. 4. 5.
S. Ibrahim, H. I. Ali, K. E. Taylor., N. Biswas, J. K. Bewtra. Enzyme-catalyzed removal of phenol from refinery wastewater: Feasibility studies. Water Environ. Res. 73 (2001) 165172. M. A. Gilabert, L. G. Fenoll, F. Garcia-Molina, J. Tudela, F. Garcia-Canovas, J. N RodryguezLopez. Kinetic characterization of phenol and aniline derivates as substrates of peroxidase Biol. Chem., Vol. 385, (2004) 795–800. L. V. Bindhu, E.T. Abraham. Immobilization of Horseradish Peroxidase on Chitosan for Use in Nonaqueous Media. Inc. J. Appl. Polym. Sci. 88 (2003) 1456-1464. W. Tischer, F. Wedekind. Immobilized enzymes: methods and applications. Top. Curr. Chem., Vol. 200 (1999) 95–126. D. C. Goodwin, I. Yamazaki, S. D. Aust, and T. A. Grover, Determination of Rate Constants for Rapid Peroxidase Reactions, Anal. Bioch, 231, 333–338 (1995).