Top Catal (2011) 54:1309–1317 DOI 10.1007/s11244-011-9753-3
ORIGINAL PAPER
Biocatalitic Oxidation of 2,3,6-Trimethylphenol Over Immobilized Horseradish Peroxidase in Nonaqueous Media O. Matveeva • N. Lakina • V. Matveeva M. Sulman • E. Sulman • P. Valetsky • V. Doluda
•
Published online: 7 September 2011 Ó Springer Science+Business Media, LLC 2011
Abstract In this work various samples of horseradish peroxidase immobilized over organic and inorganic supports were synthesized and studied in 2,3,6-trimethylphenol biocatalitic oxidation. Silica, alumina and commercial samples of polymers MN-100, Sepabeads EC-HA were used as the carriers for horseradish peroxidase. Different methods of immobilization including treatment with chitosan, glutaric dialdehyde and carbodiimide were used to enhance catalytic activity. For synthesized catalysts optimal conditions for 2,3,6-trimethylphenol (main intermediates in vitamin E synthesis) oxidation providing high degree of conversion (more than 95%) and high selectivity to target products (more then 98%) were found. Physicochemical investigations (FTIR spectroscopy, XPS, nitrogen physosorption) of optimal biocatalytic systems were provided. Keywords Horseradish peroxidase 2,3,6-Trimethylphenol Oxidation Immobilization
1 Introduction In recent years the interest to the possible use of the immobilized enzymes has considerably increased [1–7]. There are the following major reasons for attaching enzymes to various
O. Matveeva N. Lakina V. Matveeva M. Sulman E. Sulman V. Doluda (&) Department of Biotechnology and Chemistry, Tver State Technical University, 22 A.Nikitin Emb, Tver, Russia 170026 e-mail:
[email protected] P. Valetsky A.N.Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov str, Moscow, Russia 119991
supports: multiple or repetitive use of a single batch of enzymes; the ability to stop the reaction rapidly by removing the enzyme from the reaction solution; enzymes are usually stabilized by bounding and therefore it is possible to use properly immobilized enzymes in nonaqueous media [1–4]. While creating the reusable catalytic systems on the basis of enzymes one of the main problems is the choice of the carrier for the immobilization of enzyme, which will provide its best stabilization, and the method of immobilization [1, 5]. Horseradish peroxidase (EC 1.11.1.7, HRP) is one of the most frequently studied enzymes and it is widely used as liquid clinical chemistry reagent, dry powder reagent and test strip, it is also used for labeling in immunoassay, in blotting and histochemistry [7–16]. In spite of its high potential as a very efficient oxidation catalyst HRP was not applied to the synthesis of valuable chemical substances. Enzymatic oxidation of 2,3,6-trimethylphenol by HRP can be possible alternative to chemical reagent oxidation processes in vitamin E synthesis (Scheme 1). Here we report on the synthesis of HRP immobilized catalysts on the base of silica, alumina and commercial samples of polymers MN-100 and Sepabeads EC-HA supports treated with chitosan, glutaric dialdehyde and carbodiimide. Several methods of chitosan and activating agent deposition on the support were studied. Physicochemical investigations (FTIR spectroscopy, XPS, nitrogen physosorption) for the optimization of biocatalytic systems were carried out.
2 Materials and Methods 2.1 Materials The aminated hypercrosslinked polystyrene was purchased from Purolite Int. (U.K.), as Macronet MN 100 (designated as
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Scheme 1 Vitamin E synthesis route
CH3
CH3
HO
[ O]
+ H3C
H3C CH3
CH3
CH3
HO
H3C (CH CH 2 C H2 CH2)3 C CH CH2CI
OH CH3
2,3,6- trimethylphenol
2,3,5- trimethylhydroquinone CH3
CH3 CH3COO
HO
CH3
H3C
O
(CH2 CH2 CH2 CH)3 CH3
CH3
CH3
(CH3CO)2O CH3 H3C
O
(CH2 CH2 CH2 CH)3 CH3 CH3
CH3 Vitamin E
MN-100). It was washed with water twice and 1 M solution of ammonia and purified under vacuum. Sepabeads EC-HA was purchased from Residinol SRL and used as received. Silica (SiO2), alumina (c-Al2O3), sodium hydrogen carbonate (NaHCO3), chitosan (molecular weight *3,00,000, degree of deacetylation *75–80%) sodium hydroxide (NaOH), hydrochloric acid (HCl) and hydrogenperoxide (H2O2), ethanol were obtained from Reakhim (Moscow, Russia). HRP of 250 units of activity per mg, reagent-grade 2,3,6-trimethylphenol and 2,3,5-trimethylhydroquinone, glutardialdehyde, carbodiimide, sodium polystyrene sulfonate (PSS molecular weight *70,000) acid were purchased from Aldrich and were used as received. Distilled water was purified with Elsi-Aqua water purification system. 2.2 Catalysts Synthesis 2.2.1 Immobilization of HRP Over Silica and Alumina c-Al2O3 and SiO2 were used as an enzyme support, preliminary samples of c-Al2O3 and SiO2 were calcinated at 300 °C for 3 h (particle fraction 45–60 lm, surface area of 108 m2 for c-Al2O3 and surface area of 11 m2 SiO2). For the synthesis of the support capable for intermolecular cross-linking, inorganic materials were modified and activated. For that, 1 g of silica or alumina was stirred in 10 mL of 0.1 M NaOH for 60 min. Than 10 mL of PSS (0.1–0.75 g/L, stirred for 60 min) was added to the suspension. The synthesized polyanion was washed by deionized water to pH * 7 and dried in a vacuum at 60 °C for 24 h. Then the polyanion samples were stirred for 12 h in 50 mL of 0.1–0.25 g/L chitosan (polycation) solution in an acetic acid, filtered, washed with distilled water and dried in vacuum at 60 °C. This modified support is covered with polyelectrolyte complex containing easily accessible amino groups which can be used for intermolecular cross-linking with HRP. However, the major part of the amino groups are poorly
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accessible for HRP attaching so linker was used to provide better accessible amino groups. In a typical experiment, 1 g of support modified with polyelectrolites is slowly stirred for 24 h with 50 mL of a polyfunctional reagent, glutaraldehyde (0.1–0.4 g/L) or carbodiimide (0.1–0.4 g/L) in water. The activated support was shaken gently in a shaker for 24 h with 50 mL of 0.15 g of HRP (*250 units/mg), filtered, washed and dried at 25 °C and vacuumed. The synthesized catalyst was stored under argon at 2 °C. Catalysts synthesis is presented in Scheme 2. 2.2.2 Immobilization of HRP over Sepabeads EC-HA and Hypercrosslinked Polystyrene MN 100 Samples of aminofunctionalized carriers Sepabeads ECHA and MN 100 were used for HRP immobilization. Amino functionalized carriers are activated by glutardialdehyde. Immobilization scheme for this type of carrier is presented in Scheme 3. In a typical experiment, 1 g of support is slowly stirred for 24 h with 50 mL of a glutaraldehyde (0.1–1.30 g/L) in phosphate buffer. The activated support is filtrated and washed with deionized water then activated support is stored in a shaker for 24 h with 50 mL of 0.15 g of HRP (*250 units/mg), filtered, washed and dried at 25 °C and vacuumed. The synthesized catalyst was stored under argon at 2 °C. 2.3 2,3,6-Trimethylphenol Oxidation Methodology The oxidation reaction was conducted batchwise in a glass reactor (Fig. 1), which provides independent control over parameters such as substrate concentration and hydrogen peroxide concentration, biocatalyst concentration, temperature, hydrogen peroxide feed rate, and stirring rate. A suspension of the catalyst, substrate solution (20 mL) in 25 vol% of ethanol prepared at a predetermined concentration were placed in the reactor. The rate of hydrogen peroxide feed was controlled by a HPLC-pump (Maraphon
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Scheme 2 HRP immobilization procedure over silica and alumina
4 5
7
8 6 1
22
3 3 76 54
Scheme 3 HRP immobilization procedure over polymer carriers
Bio). Different pH rates were maintained by phosphates buffers. The high stirring rates employed here ensured good mixing without diffusion limitation. Samples of the reaction mixture were periodically removed for the analysis. At the end of each experiment, the catalyst was separated by filtration. 2.4 HPLC Analysis The analysis of the reaction media for residual and 2,3,6trimethylphenol concentrations and forming 2,3,5-trimethylhydroquinone was performed using a Ultimate 3000 HPLC
Fig. 1 Reactor for 2,3,6-trimethylphenol oxidation. 1 Flask with hydrogen peroxide, 2 HPLC-pump, 3 Glass reactor, 4 magnetic stirrer, 5 Condenser 6 Reflux, 7 Thermostat, 8 pH-meter
(Dionex) chromatograph equipped with UV detector and mass spectrometer-API-2000 (Applied Biosystems). Reverse phase chromatography using a 25 cm tungsten column characterized by a theoretical plate number of 45,000 was chosen for sample analysis. Luna C18 (7 lm) served as a stationary phase, whereas, acetonitrile/water solution in gradient regime was used as a mobile phase. The flow rate was held constant as 2 mL min-1 at 70 bar and 30 °C. The UV detector wavelength was maintained at 254 nm. The concentration of the standards varied from 0.001 to 100 mmol L-1. LC–MS spectrograms of molecular
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ions of 2,3,6-trimethylphenol and 2,3,5-trimethylhydroquinone, methylnaphthol and methylnaphthoquinone were collected in negative ionization regime. Additional product or reaction intermediates were not found in the reaction media that can be explained by high substrate specificity of HRP.
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reflectance attachment. The samples were placed in an ampoule supplied with a KBr window and 400 scans were collected from 400 to 6000 cm-1 with a 2 cm-1 resolution.
3 Results and Discussions 2.5 Liquid Nitrogen Physisorption 3.1 Influence of the Solvent on HRP Oxidation Rate Liquid nitrogen physosorption for perform to determine enzyme adsorption over supports. Nitrogen physisorption was conducted at the normal boiling point of liquid nitrogen using BECMAN COULTER SA 3100 apparatus (COULTER CORPORATION, Miami, Florida). Samples were degassed in BECMAN COULTER SA-PREP apparatus for sample preparation (COULTER CORPORATION, Miami, Florida), at 120 °C in vacuum for 1 h, prior to the analysis. 2.6 X-Ray Photoelectron Spectroscopy Analysis XPS analysis was performed to determined formation of covalent bonds between enzyme and support. X-ray photoelectron data were obtained using Mg Ka (hm = 1253.6 eV) monochromatized radiation with a modified ES-2403 spectrometer (provided by the Institute for Analytic Instrumentation of the Russian Academy of Sciences, St. Petersburg, Russia). All the data were obtained at an X-ray power of 200 W and an energy step of 0.1 eV. The electron-flood-gun accessory was used to neutralize the current of the total emitted electron flux from the floodgun. Samples were allowed to outgas for 30 min before the analysis and were sufficiently stable during the examination. Data analysis was performed using the standard XPSset with Resolver program. 2.7 Diffuse Reflectance Infrared Fourier Transform (DRIFT) Spectroscopy DRIFT analysis was performed to evaluate formation of covalent bonds between enzyme and support. DRIFT spectra were recorded at an ambient temperature with a Nicolet 460 Prote´ge´ spectrometer equipped with a diffuse Fig. 2 a Solvent influence on HRP oxidation rate for 2,3,6trimethylphenol, b Ethanol concentration influence on HRP oxidation rate for 2,3,6trimethylphenol (t = 36 °C, C(2,3,6-trimethyphenol) = 0.5 mg/ml, C(HRP) = 0,01 mg/ml, pH = 7.5)
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2,3,6-trimethylphenol is slightly soluble in water (1 g/L) therefore the investigations of its oxidation catalyzed by pure HRP were provided in conventional solvents (Fig. 2a). Oxidation reaction rate is drastically decrease with the use of alcohols as solvents, the highest activity for HRP catalyzed oxidation of 2,3,6-trimethylphenol was found in case of ethanol but it is more than one thousand times lower compared to water. To increase HRP activity and achieve sufficient 2,3,6trimethylphenol solubility the influence of ethanol concentration on the reaction activity was studied (Fig. 2b). Ethanol volume concentration of 25% providing 0.045 mg(substrate)/(mg(HRP)*s) reaction rate and 2,3,6-trimethylphenol solubility up to 150 g/L were chosen for further experiments. But 2,3,6-trimethylphenol oxidation rate is still very low compared to water as a solvent therefore conventional immobilization is needed to increase it by proper enzyme stabilization. 3.2 Influence of HRP Immobilization on the Reaction Rate for SiO2 and Al2O3 3.2.1 PSS Concentration Influence Methods of the catalysts synthesis have significant influence on their activity. For the evaluation of PSS concentration influence on the biocatalysts activity samples of silica and alumina impregnated with 10 mL of PSS (0.1, 0.25, 0.50, 0.75 g/L), 50 mL of 0.1 g/L chitosan 50 mL of glutaraldehyde (0.1 g/L) were prepared and tested. Silica immobilized HRP biocatalysts (Fig. 3a) appeared to be more active compared to alumina based samples
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(Fig. 2b). It is explained by higher degree of HRP immobilization over silica (25% of immobilization for most active samples saturated with 0.25 g/L PSS solution) compared to alumina (21% for most active samples saturated with 0.5 g/L PSS solution and 18% for samples saturated with 0.25 g/L PSS).
appropriate decrease of the substrate conversion rate because of HRP overlinking with extra amino groups of chitosan appeared on the support surface. The found optimal concentrations of chitosan solutions were used for further optimization of biocatalysts. 3.2.3 Linker Type and its Concentration Influence
3.2.2 Chitosan Concentration Influence Linker type and its concentration play significant role in efficient biocatalyst synthesis. To evaluate the influence of the linker type on the activity of HRP immobilized over SiO2 and Al2O3 samples with optimal concentration of PSS(0.25 g/L for silica and 0.5 g/L for alumina) and chitosan (0.15 g/L for silica and 0.2 g/L for alumina) were impregnated with 50 ml of glutaraldehyde or carbodiimide solution (0.1, 0.2, 0.3, 0.4 g/L). The most active silica based biocatalyst (Fig. 5a) prepared using 0.2 g/L solution of glutaraldehyde was almost two times more active compared to samples prepared with carbodiimide (Fig. 5c). It is explained by low immobilization degree of the samples prepared with carbodiimide (12% of immobilization for the most active sample synthesized using 0.2 g/L carbodiimide solution).
a 0,10 0,08 0,06 0,04 C(PSS)=0.1 g/l C(PSS)=0.25 g/l C(PSS)=0.5 g/l C(PSS)=0.75 g/l
0,02 0,00 0
5
10
15
20
C mol/l 2,3,5-trimethylhydroquinone
Fig. 3 Kinetic curves of 2,3,5timethylhydroquinone catalyzed by HRP immobilized over a silica and b alumina with different concentration of PPS solution. Initial reaction volume 20 mL, catalyst concentration 5 g/L, initial 2,3,6trimethylphenol concentration 0.1 mol/L, pH = 6.5–7.2, temperature 36 °C, hydrogen peroxide feed rate 0.5 mL/min (C(H2O2) = 0.2 mol/L)
C mol/l 2,3,5-trimethylhydroquinone
For the evaluation of chitosan concentration influence on the activity of HRP immobilized over SiO2 and Al2O3 samples with optimal concentration of PSS(0.25 g/L for silica and 0.5 for alumina) were impregnated with 50 mL of 0.1, 0.15, 0.2, 0,25 g/L chitosan solutions and 50 mL of glutaraldehyde (0.1 g/L). The most active silica based biocatalyst (Fig. 4a) was prepared by saturation of modified support with 0.15 g/L solution of chitosan, while most active alumina based catalyst (Fig. 4b) was prepared using 0.2 g/L chitosan solution. The increase of chitosan concentration on the biocatalytic support surface results in the appropriate increase of immobilization degree up to 25% for most active silica and 22% for alumina based samples. Further increase of chitosan concentration results in the
b 0,10 0,08 0,06 0,04 C(PSS)=0.1 g/l C(PSS)=0.25 g/l C(PSS)=0.5 g/l C(PSS)=0.75 g/l
0,02 0,00 0
5
10
a 0,10 0,08 0,06 0,04 C(Chit)=0.1 g/l C(Chit)=0.15 g/l C(Chit)=0.2 g/l C(Chit)=0.25 g/l
0,02 0,00 0
5
10
15
20
Time, min
Fig. 4 Kinetic curves of 2,3,5-timethylhydroquinone formation for HRP immobilized on a SiO2 and b Al2O3 saturated with different concentration of chitosan solution. Initial reaction volume 20 mL,
15
20
Time, min
C mol/l 2,3,5-trimethylhydroquinone
C mol/l 2,3,5-trimethylhydroquinone
Time, min
b 0,10 0,08 0,06 0,04 C(Chit)=0.1 g/l C(Chit)=0.15 g/l C(Chit)=0.2 g/l C(Chit)=0.25 g/l
0,02 0,00 0
5
10
15
20
Time, min
catalyst concentration 5 g/L, initial 2,3,6-trimethylphenol concentration 0.1 mol/L, pH = 6.5–7.2, temperature 36 °C, hydrogen peroxide feed rate 0.5 mL/min (C(H2O2) = 0.2 mol/L
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a 0,10 0,08 0,06 0,04 C(Glut)=0.1 g/l C(Glut)=0.2 g/l C(Glut)=0.3 g/l C(Glut)=0.4 g/l
0,02 0,00 0
2
4
6
8
10
C mol/l 2,3,5-trimethylhydroquinone
Fig. 5 Kinetic curves of 2,3,5timethylhydroquinone formation for HRP immobilized on a, c SiO2 and b, d Al2O3 saturated with different concentration of a, b glutaraldehyde and c, d carbodiimide solution
Top Catal (2011) 54:1309–1317 C mol/l 2,3,5-trimethylhydroquinone
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b 0,10 0,08 0,06 0,04 C(Glut)=0.1 g/l C(Glut)=0.2 g/l C(Glut)=0.3 g/l C(Glut)=0.4 g/l
0,02 0,00
0
2
4
0,03
0,02
0,01
C(Carb)=0.1 g/l C(Carb)=0.2 g/l C(Carb)=0.3 g/l C(Carb)=0.4 g/l
0,00 0
5
10
Time, min
Biocatalysts prepared with alumina have the same correlation of oxidation activity to the method of HRP immobilization on the modified support. On the basis of the experiments performed glutaraldehyde was the most suitable linker for further experimental work. The optimized catalysts synthesis procedure was the following: a) 1 g of SiO2 was modified with 0,25 g/L PSS solution, 0.15 g/L chitosan solution, 0.2 g/L glutaraldehyde solution and was immobilized by 0.15 g of HRP (*250 units/mg), the degree of HRP immobilization was found to be 32%; b) 1 g of Al2O3 was modified with 0.5 g/L PSS solution, 0.2 g/L chitosan solution, 0.3 g/L glutaraldehyde solution and was immobilized by 0.15 g of HRP (*250 units/mg), the degree of HRP immobilization was 24%.
15
20
C mol/l 2,3,5-trimethylhydroquinone
C mol/l 2,3,5-trimethylhydroquinone
c 0,04
6
8
10
Time, min
Time, min
d 0,030 C(Carb)=0.1 g/l C(Carb)=0.2 g/l C(Carb)=0.3 g/l C(Carb)=0.4 g/l
0,025 0,020 0,015 0,010 0,005 0,000 0
5
10
15
20
Time, min
glutaraldehyde solution are presented in Fig. 6. For both supports optimal concentration of glutaraldehyde was found to be 0.2 g/L, but HPS samples were more active in the formation of 2,3,5-timethylhydroquinone compared to Sepabeads EC-HA. The degree of HRP immobilization for most active MN-100 sample was found to be 20% while for Sepabeads EC-HA degree of immobilization was 17%. Low activity and degree of HRP immobilization for organic supports compared to silica and alumina based samples can be explained by enzyme overshifting on polymer supports. Further experiments were carried out using Sepabeads EC-HA and MN-100 synthesized samples saturated with 0.2 g/L solution of glutaraldehyde. 3.4 Effect of Biocatalyst and Substrates Loading
3.3 Influence of HRP Immobilization on the Reaction Rate for MN-100, Sepabeads EC-HA Supports Commercial supports already have functional amino groups for enzymes immobilization therefore only optimization of glutaraldehyde saturation solution concentration was carried out. To evaluate the influence of this parameter on HRP activity immobilized on MN-100, Sepabeads EC-HA supports, samples impregnated with 50 mL of (0.1, 0.2, 0.3, 0.4 g/L) glutaraldehyde solutions were synthesized. Kinetic curves of 2,3,5-timethylhydroquinone formation for HRP immobilized on MN-100 and Sepabeads EC-HA saturated with different concentration of
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The influence of the biocatalyst and substrates loading was examined by varying catalysts concentration (Cc) from 0.1 to 15 g L-1 and substrate concentration (Co) from 1 to 150 g(Co) L-1. The reaction curves for the three catalysts are presented in Fig. 7 as a function of the substrate-to-catalyst ratio q = C0/Cc [g/g]. The decrease of q results in the appropriate increase of the reaction rate (Fig. 7a) up to substrate to catalyst ratio 6 g/g for biocatalysts synthesized on silica and alumina supports, further increase of substrate to catalyst ratio leads to the inhibiting of the reaction rate that can be explained by HRP deactivation with the substrate excess. For the samples synthesized using commercial support it is
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C mol/l 2,3,5-trimethylhydroquinone
Fig. 6 Kinetic curves of 2,3,5trimethylhydroquinone formation for HRP immobilized on a MN-100 and b Sepabeads EC-HA saturated with different concentration of glutaraldehyde solution
a 0,08 C(Glut)=0.1 g/l C(Glut)=0.2 g/l C(Glut)=0.3 g/l C(Glut)=0.4 g/l
0,06
0,04
0,02
0,00 0
5
10
15
20
C mol/l 2,3,5-trimethylhydroquinone
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b C(Glut)=0.1 g/l C(Glut)=0.2 g/l C(Glut)=0.3 g/l C(Glut)=0.4 g/l
0,03
0,02
0,01
0,00 0
2
4
8
10
b
3,0 HRP/SiO2 HRP/Al2O3
2,5
HRP/MN100 HRP/Sepabeads EC HA
2,0 1,5 1,0 0,5 0,0 0
2
4
6
8
10
12
14
16
q, g(2,3,6-trimethylphenol)/g(Catalyst)
W20%, g(2,3,6-trimethylphenol) /(g(Catalyst)*s)
W20%, g(2,3,6-trimethylphenol) /(g(Catalyst)*s)
a
6
Time, min
Time, min
3,0 HRP/SiO2 HRP/Al2O3
2,5
HRP/MN100 HRP/Sepabeads EC HA
2,0 1,5 1,0 0,5 0,0 4
5
6
7
8
9
10
pH
Fig. 7 a Influence of substrate to catalyst ratio on substrate oxidation rate for 20% substrate conversion, b Influence of pH over substrate oxidation rate on 20% substrate conversion (W20% = m20%(substrate)/ (m(catalyst)*t20%, were m20%(substrate) is weight of substrate react on
20% conversion, m(catalyst) initial weight of the catalyst, t20%—time of 20% substrate conversion) for oxidation of a 2,3,6-trimethylphenol and b methylnaphthol
possible to see that the reaction (Fig. 7a) rate increases up to 11 g/g substrate to catalyst ratio that can be explained by better immobilization of the enzyme on the support surface. All further experiments were carried out with the following substrate to catalyst ration 6 g/g for HRP immobilized over silica and alumina and 11 g/g for HRP immobilized over Sepabeads EC-HA and MN-100.
3.6 Effect of Reaction Temperature
3.5 Effect of pH In order to investigate pH influence on the activity and selectivity of 2,3,6-trimethylphenol, the process was carried out at pH ranging from 1.4 to 11. Phosphate buffer solutions were used to reach extreme pH. Maximum selectivity is observed when the reaction is performed at pH 6.5–7.2 (Fig. 7 b). At pH lower than 6 and higher than 8, oxidation rate strongly diminishes. 7.2 pH providing highest biocatalyst activity was used in all the experiments. The samples HRP immobilized on silica and alumina supports are more sensitive to pH changes compared to the samples immobilized on the commercial supports, this can be explained by better immobilization of HRP on preliminary formed covalent linked amino groups.
Figure 8f shows that the reaction temperature has no strong effect on the activity of immobilized HRP up to 40 °C, after this temperature reaction rate is strongly diminishes. Optimal temperature range for substrate oxidation was found to be 35–40 °C, it is possible to see that HRP immobilized on silica and alumina supports are more sensitive to temperature changes compared to the samples immobilized on the commercial supports, this can be again explained by better immobilization of HRP over preliminary bounded amino groups. 3.7 Long-Term Biocatalyst Activity Assessment For the evaluation of the synthesized biocatalysts longterm activity 10 consecutive oxidation experiments with the same samples were carried out (Fig. 8b). It is possible to notice strong deactivation of silica and alumina based biocatalysts in the repeated experiments while MN100 and Sepabeads EC-HA practically have no deactivation that can be explained by strong bonding of HRP over the commercial support.
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b 3,0
HRP/SiO2 HRP/Al2O3
2,5
HRP/MN100 HRP/Sepabeads EC HA
2,0 1,5 1,0 0,5 0,0 20
30
40
50
W20%, g(2,3,6-trimethylphenol) /(g(Catalyst)*s)
W20%, g(2,3,6-trimethylphenol) /(g(Catalyst)*s)
a
HRP/Al2O3 HRP/MN100 HRP/Sepabeads EC HA
2,0 1,5 1,0 0,5 0,0 2
4
6
8
10
Batch
Fig. 8 a Temperature influence on substrate oxidation rate at 20% substrate conversion, b Long-term biocatalyst activity (W20% = m20%(substrate)/(m(catalyst)*t20%, were m20%(substrate) is weight of
substrate react on 20% conversion, m(catalyst) initial weight of the catalyst, t20%—time of 20% substrate conversion) for oxidation of a 2,3,6-trimethylphenol and b methylnaphthol
Sample
BET surface area (sq m/g)
DRIFT lines (cm-1)
XPS N 1 s bonding energy (eV ± 0.1 eV)
Initial activity (mg) (2,3,6trimethylphenol)/ (mg(HRP)*s)
HRPin
water
–
–
–
0.124
HRPin
25% ethanol
–
–
–
0.045
SiO2 HRP/SiO2
31 25
– 1650; 2450
– 400.2
– 0.081
Al2O3
56
–
–
–
HRP/Al2O3
10
1655; 2452
400.4
0.074
MN100
920
–
–
–
HRP/MN100
750
1660; 2457(strong
400.2
0.061
lines)
Sepabeads EC-HA
74
–
–
–
HRP/Sepabeads EC-HA
54
1630; 2420(strong
400.3
0.052
3.8 Physicochemical Characterization of Biocatalysts BET surface areas of the synthesized biocatalyst are presented in Table 1. Silica, alumina and commercial Sepabeads EC-HA samples are characterized by macropore structure and low BET surface area compare to MN-100 that has a lot of micro and mesopores and high BET surface area. Support macropore structure is favorable for formation of easily assessable enzyme complexes, but such structure can lead to easily enzyme leaching and deactivation. Micromesoporous MN100 polymer structure is favorable for sorption of enzyme and formation of strong covalent bonds with enzyme that can prevent leaching and deactivation. The values of surface area obtained for biocatalysts samples are always lower compare to parent support that mean that enzyme was linked to the surface of the supports. The spectra recorded for HRP immobilized over inorganic and organic supports exhibits two bands at 1630–1660 and 2420–2457 cm-1, in the area attributed to stable –N=CH– bond that is also confirmed by XPS data (Table 1).
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HRP/SiO2
2,5
0
Temperature, °C
Table 1 Physicochemical and activity characteristics of synthesized biocatalysts
3,0
lines)
Strong lines attributed to stable –N=CH– bond for commercial supports can explain high stability of polymer immobilized catalytic systems compare to inorganic supports.
4 Conclusions HRP-containing silica, alumina, commercial MN-100 and Sepabeads supports with different type of synthesis were described in this paper. The immobilization of enzyme with the formation of covalent bonds occurs on the surface of chitosan incorporated over silica, alumina supports and in aminated macropores of the commercial organic polymers used. Catalytic activity and selectivity of the catalysts towards the formation of 2,3,5-trimethylhydroquinone under a wide variety of reaction conditions are described. The developed immobilized HRP catalysts have higher activity in 25% ethanol media compared to pure HRP. The most active catalyst contained HRP immobilized on
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modified silica, the selectivity of this system to the target product formation was 98.5–99.5% at 99% substrates conversion and the calculated activity of the catalyst was 2.61 g(substrate) g-1(catalyst) s-1. Silica and alumina based biocatalysts synthesized showed deactivation in the repeated experiments while commercial samples MN-100 and Sepabeads EC-HA samples practically did not show any deactivation. The high catalytic stability is attributed to the high sorption capacity of commercial samples MN-100 and Sepabeads EC-HA samples and formation of strong covalent bonds between enzyme and carrier. Acknowledgment This work has been supported by the Russian ministry of science and education Grand P-1000, grand MK7390.2010.4 (Contract number 02.120.11.7390MK).
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