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Characterization of soybean peroxidase for the treatment of aqueous phenols
Bioresource Technology 70 (1999) 69±79
Characterization of soybean peroxidase for the treatment of aqueous phenols Harold Wright, James A. Nicell* Department of Civil Engineering & Applied Mechanics, McGill University, 817 Sherbrooke Street West, Montreal PQ,Canada H3A 2K6 Received 24 July 1998; revised 12 January 1999; accepted 18 January 1999
Abstract The application of soybean peroxidase (SBP) to catalyze the polymerization and precipitation of aqueous phenols by hydrogen peroxide is potentially promising because this peroxidase is less expensive than horseradish peroxidase (HRP), which has been the focus of most wastewater research. SBP can act on a broad range of compounds and retains its catalytic ability over wide ranges of temperature and pH. Activity was optimal at pH 6.4, with signi®cant activity observed between pH 3 and 9. SBP was very stable at 25°C at neutral and alkaline conditions but experienced rapid inactivation below pH 3. SBP underwent biphasic inactivation by hydrogen peroxide in the absence of a reductant substrate. SBP was most eective when used to treat phenolic solutions between pH 6 and 9. In comparison with HRP, the activity of SBP was only slightly more sensitive to pH, was more stable at elevated temperatures, and was less susceptible to permanent inactivation by hydrogen peroxide. However, SBP was catalytically slower than HRP and a larger molar quantity of SBP was usually required to remove a given quantity of phenolic substrate. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Soybean peroxidase; Wastewater treatment; Inactivation; Stability; Temperature; pH
1. Introduction The catalytic removal of phenolic and other aromatic compounds from wastewaters using peroxidase enzyme and hydrogen peroxide has been the focus of extensive research since the initial work of Klibanov et al. (1980). Treatment involves the catalytic oxidation of the aromatic contaminants by hydrogen peroxide to form aromatic free radicals that subsequently combine to create high molecular weight products. These products precipitate from solution due to their low solubility. The method presents a possible alternative for the treatment of industrial wastewaters when conventional methods such as biological treatment, activated carbon and advanced oxidation may be ineective due to the nature of the wastewater stream (Aitken, 1993; Nicell et al., 1993). While a number of peroxidase enzymes obtained from plant, animal and microbial sources have been investigated for their ability to catalyze the removal of aromatic compounds from wastewaters, the majority of studies have focused on using horseradish peroxidase *
Corresponding author. E-mail: [email protected].
(HRP). HRP is commercially available in puri®ed form and has a proven ability to remove a variety of phenolic contaminants from aqueous solution over relatively wide ranges of pH and temperature (Nicell et al., 1993). However, one of the major challenges associated with peroxidase catalyzed phenol removal is the prohibitive cost of the enzyme (Cooper and Nicell, 1996). Enzyme costs are signi®cant because the enzyme is susceptible to inactivation by various side reactions of the treatment process (Buchanan and Nicell, 1997). While recent work has shown that enzyme inactivation can be reduced using chemical additives (Nakamoto and Machida, 1991), the problem of enzyme cost could be circumvented by using a less expensive source of enzyme. In 1991, the seed coat of the soybean was identi®ed as a rich source of a single peroxidase isoenzyme with a molecular weight of 37,000 Daltons (Gillikin and Graham, 1991). Since the seed coat of the soybean is a byproduct of the soybean food industry, soybean peroxidase (SBP) has the potential of being a cost eective alternative to HRP for wastewater treatment. Therefore, the objectives of this study were to: (1) characterize SBP in terms of its catalytic activity and susceptibility to inactivation; and (2) compare the
0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 0 7 - 3
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H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
performance of SBP with HRP to assess its potential application to the treatment of industrial wastewaters containing phenolic contaminants. 2. Methods Medium purity soybean peroxidase, with a nominal activity of 50 PPU/mg based on a purpurogallin assay, was purchased as a dry powder from Enzymol International, Columbus, OH. Phenolic compounds were at least 98% pure and were purchased from either Fluka, Ronkonkoma, NY or Aldrich, Milwaukee, WI. ACS grade 30% H2 O2 w/v was purchased from BDH Chemicals, Toronto, Canada. ACS grade potassium ferricyanide and 98% pure 4-aminoantipyrine (4-AAP) were purchased from Fisher Scienti®c, Fair Lawn, NJ and Aldrich, respectively. ACS grade conjugate acids and bases were obtained from BDH Chemicals and were used to prepare buers in accordance with the methods of Gomori (1955). Except for SBP solutions, all aqueous solutions were prepared using deionized water obtained from a D4741 Nanopure Ultrapure Water System (Barstead/Thermolyne, USA). Aqueous solutions of SBP were prepared using either deionized water (adjusted to an alkaline pH using concentrated NaOH) or pH buer. SBP powder and all aqueous solutions were stored at 4°C until the time of use. All spectrophotometric measurements were performed in a 1 cm cuvette using a Beckman DU-65 Spectrophotometer. Reaction temperatures were maintained using a constant temperature room and a Haake A81 Temperature Bath. Phenolic polymers generated during phenol removal reactions were separated from the reaction mixtures by centrifugation at 3000 g in a Centra-8 Centrifuge (International Equipment, USA). Reaction pH and temperature were measured using a Radiometer-Copenhagen Ion 83 Meter. SBP activity was measured by a colorimetric assay in which the 1 ml assay volume was composed of 500 ll of 20 mM phenol, 250 ll of 9.6 mM AAP, 100 ll of 2 mM hydrogen peroxide (H2 O2 ), 50±150 ll of sample, and 0± 100 ll of 0.1 M monobasic±dibasic phosphate buer (pH 7.4) prepared according to the method of Gomori (1955). All assay reactants were prepared in the phosphate buer. Prior to signi®cant substrate depletion, the rate of colour development at 510 nm is proportional to the concentration of active SBP. The rate of colour development was converted to enzyme activity in the cuvette using a conversion factor of 7100 Mÿ1 cmÿ1 based on hydrogen peroxide. One unit of activity (U) is de®ned as the number of micromoles of peroxide consumed in one min at pH 7.4 and 25°C. Based on standard activity measurements of nine aqueous solutions of SBP, the solid enzyme preparation used in this study had a speci®c activity of 12.3 1.1 U/
mg. A Reinheitzahl (RZ) number of 0.49 0.06 was calculated based on a ratio of absorbance measured at 280 nm and 403 nm. Assuming a Soret extinction coef®cient (Dunford and Stillman, 1976) of e403 nm 105 Mÿ1 cmÿ1 is valid for ferric SBP and assuming all Soret absorbance is attributable to active enzyme, 1 U/ml of peroxidase activity corresponds to 0.37 0.02 lM of SBP. The eect of assay pH and H2 O2 concentration on the catalytic activity of SBP was investigated by varying the pH and H2 O2 concentration in the activity assay and measuring the resulting colour generation rate at 510 nm. After colour generation, adjustment of the pH to 7.4 did not change the peak wavelength nor absorbance significantly, therefore the absorbance of the coloured product was not a signi®cant function of pH over the range examined. The stability of SBP was evaluated by incubating the enzyme at 25°C in various pH buers, at elevated temperatures in various pH buers, and at 25°C and pH 7.4 with various concentrations of H2 O2 . SBP activity in the incubation mixture was measured over time using the standard activity assay and aliquots obtained from the incubation mixture. As the incubation progressed, 1 ml aliquots were removed from the incubation mixture and quickly cooled using an ice bath to halt the inactivation process. Once cooled, aliquots were removed and allowed to warm to 25°C. Repeated activity measurements on the aliquot gave reproducible measurements. When addition of the aliquot to the assay aected the assay H2 O2 concentration and pH, the measured activity was adjusted to the expected activity under standard conditions using the experimentally determined dependence of peroxidase activity on assay H2 O2 concentration and pH. The method is described in detail in Nicell and Wright (1997). Three-hour stirred batch reactions involving 1 mM phenolic compound, 2 mM H2 O2 and various doses of SBP reacting at 25°C in various pH buers were used to evaluate the ability of SBP to catalyze phenol removal from wastewaters. In all batch reactions, concentrated aqueous SBP and phenol were added to 10 ml of pH buer. The reaction was initiated by the addition of concentrated H2 O2 . After 3 h, the precipitated products were removed by centrifugation at 3000 g for 30 min and the residual phenol in the supernatant was measured. A reaction time of 3 h was sucient to ensure that all reactions had gone to completion over the range of pHs studied. Reaction blanks were used at each reaction pH to con®rm that phenolic compound removal was solely a consequence of SBP activity. Phenolic compound concentrations were measured using a colorimetric assay consisting of 100 ll of phenol sample, 50 ll of 41.6 mM 4-AAP, 800 ll of 0.15 M sodium borate, and 50 ll of 166.8 mM potassium ferricyanide. When the extent of the assay reaction is
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
limited only by the concentration of the phenolic compound, the quantity of colour formed under alkaline conditions at 510 nm is proportional to the initial concentration of phenolic compound in the assay. The sodium borate in the assay maintained the mixture at a ®xed alkaline pH such that molar absorptivities measured for various phenolic compounds were relatively insensitive to sample pH. Since phenolic compounds have absorbance peaks in the UV range, the phenolic concentrations in a batch reactions were also estimated by measuring the UV absorbance peak of the solution and then calculating concentration using an appropriate extinction coecient. Molar extinction coecients were evaluated for each phenolic compound and for each pH. An end-point assay, based on the AAP/phenol system used in the activity assay, was used to determine the hydrogen peroxide concentration. The concentration of peroxidase in the microcuvette was approximately 100 mg/l, while those of the other reactants (except peroxide) were as cited above for the peroxidase assay. The extent of color development at 510 nm was proportional to hydrogen peroxide concentration, provided this concentration did not exceed 0.05 mM in the cuvette. After a 10-minute reaction time, the absorbance at 510 nm was converted to peroxide concentration using a calibration curve. 3. Results and discussion Fig. 1 illustrates the catalytic cycle of peroxidase and its postulated side-reactions. This ®gure was constructed based on numerous studies of horseradish peroxidase (Arnao et al., 1990; Hewson and Dunford, 1976; Nakajima and Yamazaki, 1980, 1987) and forms the basis of some of the following discussion.
71
Fig. 1. The catalytic cycle and side-reactions of peroxidase: EN is native peroxidase; Ei , Eii and Eiii are compounds I, II and III, respectively; E-670 is verdohaemoprotein, and Ei -H2 O2 is an intermediate enzyme±peroxide complex; ROH is a phenolic substrate and RO is a phenoxy radical.
maximum peroxide consumption rate observed between pH 5.7 and 7.0, and >10% between pH 3 and 9. Nicell et al. (1993) reported that HRP experienced peak activity at pH 7.4, with >90% of the maximum rate observed between pH 5.7 and 8.0, and >10% between pH 3.5 to 10.0. A comparison of these results shows that the activity of SBP appears to be slightly more sensitive to pH than HRP; however, both peroxidases retain signi®cant activity over a wide range of pH conditions frequently encountered in wastewater treatment situations. The rate at which peroxidase passes through the catalytic cycle is limited by the slowest reaction of the cycle. With sucient H2 O2 in the reaction mixture, the reduction of Compound I (Eii ) to ferric enzyme (EN ) is usually the limiting step (Dunford and Stillman, 1976). For example, reported apparent rate constants for the reduction of HRP Compound II by 4-methylphenol are greatest near pH 7.4 but fall to 10% of peak value near pH 2 and pH 9.5 (Hewson and Dunford, 1976). This conforms with the activity measurements of Nicell et al. (1993). By analogy, the pH dependence of SBP activity likely re¯ects the pH dependence of the
3.1. Activity of soybean peroxidase The activity of peroxidase measured in a phenol/4AAP colorimetric assay is a measure of how quickly the enzyme passes through the catalytic cycle consuming H2 O2 and phenolic substrate (ROH) and generating phenoxy radicals (RO ). The impact of pH on SBP activity was measured by varying the pH of the assay buer. The relative activities as a function of pH are presented in Fig. 2. Relative activity is expressed as the rate of hydrogen peroxide consumption at a particular pH normalized with respect to the highest rate. Experiments were performed using dierent buer species to achieve identical pH conditions. Fig. 2 indicates that activity was not in¯uenced by the buer species used to maintain pH. The results demonstrate that SBP was active over a wide range of reaction pH with a maximum peroxide consumption rate at pH 6.4, with >90% of the
Fig. 2. Relative activity of SBP as a function of reaction pH with 5 lg/ ml SBP in the assay mixture.
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H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 3. Rate of H2 O2 consumption in the activity assay as a function of H2 O2 and SBP concentration. Smooth curves are based on a ®t of the model of Nicell and Wright (1997).
apparent rate constants describing the reactions of SBP Compound II. The rate of colour generation at pH 7.4 and 25°C was investigated as a function of the assay H2 O2 concentration. The resulting reaction rates, expressed as peroxide consumption rates, are presented in Fig. 3 as a function of initial H2 O2 concentration for three concentrations of SBP. The maximum activity was consistently observed at 0.33 mM H2 O2 . At low H2 O2 concentrations, the peroxidase cycle is limited by the rate at which ferric peroxidase is oxidized by H2 O2 to Compound I. At higher H2 O2 concentrations, the peroxidase cycle is limited by the formation of inactive Compound III (Eiii ). This is not a permanently inactivated form of peroxidase since it can revert to the ferric state and reenter the catalytic cycle, as shown in Fig. 1; however, the return is very slow and any accumulation in this state represents a loss in catalytic eciency. Nicell and Wright (1997) developed a kinetic model of this assay and extracted kinetic constants to describe the rate at which peroxidase passes through its catalytic cycle and its susceptibility to Compound III formation. A comparison of the rate constants for HRP and SBP revealed that SBP is more than an order of magnitude slower than HRP during the oxidation of phenol and SBP is far more susceptible to inactivation through Compound III formation than HRP (Nicell and Wright, 1997).
Fig. 4. Stability of SBP incubated at 25°C in various pH buers (pH 2.0 HCl/KCl buer, pH 3.0 citrate/phosphate buer, pH 5.0 citrate/ phosphate buer, pH 7.0 phosphate buer, pH 9.0 borate buer, pH 10.7 carbonate buer).
measured and the activity corrected to an equivalent value at pH 7.4 using the results presented in Fig. 2. In acidic buer, SBP undergoes time dependent inactivation whose rate and extent depend on the pH of the incubation mixture. At pH 2 and 3, ®rst order inactivation led to essentially zero activity within two and four days, respectively. Inactivation at pH 5 was biphasic and did not result in complete inactivation. In comparison, SBP incubated in either neutral or alkaline buer was essentially stable for over 20 days of incubation. 3.2.2. Thermal inactivation The stability of SBP when it was incubated at approximately 80°C in various pH buers is presented in Fig. 5. The thermal inactivation depended on incuba-
3.2. Stability of soybean peroxidase 3.2.1. Eect of pH The stability of SBP incubated at 25°C as a function of pH is presented in Fig. 4. Since activity was measured using the standard activity recipe, the addition of an aliquot of incubation mixture to the assay caused a notable pH shift that in¯uenced the rate of colour formation. Accordingly, the pH of the assay mixture was
Fig. 5. Stability of SBP incubated at 80.3 0.4°C in various buers (pH 5.0 citrate/phosphate buer, pH 6.0 phosphate buer, pH 7.0 phosphate buer, pH 8.0 phosphate buer, pH 9.0 borate buer, pH 10.3 carbonate buer).
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 6. SBP decimal reduction values as a function of temperature and pH.
tion pH and could be modelled using the ®rst order equation: t
1 A t A0 eÿkt A0 10ÿD ; where A(t) is the activity at time t, A0 is the initial activity, k is the inactivation decay constant, and D is the inactivation decimal reduction value (D-value). Fig. 6 presents D-values for SBP thermal inactivation as a function of incubation temperature and pH. The Dvalue at 70°C and pH 6.0 could not be measured due to the SBP's extreme thermal stability under these conditions. Consistently, SBP was most resistant to thermal inactivation at approximately pH 6. Inactivation decay constants may be related to incubation temperature using the Arrhenius Law and an activation energy (De Cordt et al., 1992). D-values over a limited temperature range may be related to temperature using: TREF ÿT
2 D DREF 10 z where DREF is the decimal reduction value at temperature TREF and Z is the temperature change required to obtain a ten-fold increase or decrease in the decimal reduction value (De Cordt et al., 1992). Unlike inacti-
73
vation decay constants and D-values, the activation energies and Z-values obtained from the data in Fig. 6 tend to be independent of pH with average values of 234 13 kJ/mol and 10.2 0.6°C, respectively. The thermal inactivation of SBP observed in this work is compared to observations reported in the literature in Table 1. Using SBP which was puri®ed using dialysis, fractionation and chromatography, Sessa and Anderson (1981) and Chuang and Chen (1988) observed considerably faster thermal inactivation of SBP. Since enzyme thermal inactivation is in¯uenced by a multitude of factors such as ionic strength and buer species (De Cordt et al., 1992), calcium ion concentration (Enzymol International Inc., 1994), and polyhydric alcohol and sugar concentration (Asther and Meunier, 1990), differences between the rates of thermal inactivation reported in the literature and the rates observed in this work may be due to dierences in the constituents of the incubation mixture which are a result of the puri®cation techniques used to extract SBP from the original plant material. The Z-value observed in this work compares well with the value reported by Chuang and Chen (1988). A pH optimum for peroxidase thermal stability has been previously reported with Japanese radish peroxidase (Tamura and Morita, 1975) and pH independent Z-values have been observed with non-peroxidase enzymes (De Cordt et al., 1992). While a 3±4 min D-value has been observed with HRP incubated at 72°C and pH 7.4 (from the data of Nicell et al., 1993), SBP incubated under similar conditions in this work exhibited a D-value of several days thereby indicating a much greater stability at elevated temperatures. 3.2.3. Inactivation by hydrogen peroxide The stability of SBP incubated at 25°C in pH 7.4 buer with various concentrations of H2 O2 is presented in Fig. 7. Since the concentration of H2 O2 within the assay was increased by the addition of an aliquot from the incubation mixture, the measured rate of colour generation was corrected to standard conditions using the curves of Fig. 3 and the method described by Nicell
Table 1 Thermal inactivation parameters of SBP Enzyme source
pH and buer species
Temperature (°C)
Composite of puri®ed isoenzymes Puri®ed 37 000 Dalton isoenzyme
pH 5.5 (0.1 M citrate/ phosphate buer) pH 7.0 (0.2 M phosphate buer)
70
Enzymol medium purity SBP
pH 5.0 (0.1 M citrate/ phosphate buer) pH 7.0 (0.1 M phosphate buer)
D (min)
Z (°C)
Reference
60
ÿ
70 80 90 70.2
103 9.1 0.38 1450
8
Sessa and Anderson (1981) Chuang and Chen (1988)
70.2
4000
80.3 90.8
300 30
10.2 0.6
This work
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H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 7. Stability of 0.37 U/ml SBP at 25°C as a function of time and initial concentration of hydrogen peroxide in pH 7.4 phosphate buer. Smooth curlves are based on Eq. (3) and parameters summarized in Table 2.
and Wright (1997). This correction was applied assuming that the catalytic consumption of H2 O2 in the incubation mixture was negligible. However, it should be noted that after 80 minutes of incubation with initial H2 O2 concentrations of 0.02, 0.2, 2.0, and 20 mM, the concentration of H2 O2 in the incubation mixtures fell by 62, 50, 30 and 20% of its initial value, respectively. This reduction in H2 O2 may result in a worst-case activity overestimation of 6.6% and 1.4% after 80 min of incubation with initial H2 O2 concentrations of 20 mM and 2.0 mM, respectively. When peroxidase and H2 O2 are incubated together without a phenolic reducing agent present, H2 O2 acts to reduce the +5 oxidation state of the enzyme in either one or two electron steps (Nakajima and Yamazaki, 1987). Arnao et al. (1990) reported that H2 O2 also reacts with HRP resulting in an inactive form referred to as verdohemoprotein (see E-670 in Fig. 1). While the exact mechanism of verdohemoprotein formation is uncertain and may involve a sequence of reversible and irreversible reactions (Nakajima and Yamazaki, 1980), Arnao et al. (1990) proposed the scheme shown in Fig. 1 for its formation. SBP inactivation over time presented in Fig. 7 increased with H2 O2 concentration and was biphasic. The biphasic inactivation observed with SBP can be ex-
plained based on the relative magnitudes of kinetic constants published for HRP reacting with H2 O2 (Arnao et al., 1990). Since the reaction of ferric peroxidase with H2 O2 occurs quickly (Yamazaki and Nakajima, 1986), ferric SBP is expected to oxidize to Compound I virtually immediately following the addition of H2 O2 to the incubation mixture. Since the reduction of Compound I by H2 O2 occurs relatively slowly (Arnao et al., 1990), the rate of verdohemoprotein formation at this point can be expected to occur at its fastest rate assuming its formation originates at the Compound I state. As the incubation proceeds, the majority of SBP is transformed to Compound II and Compound III at the expense of Compound I and the rate of verdohemoprotein formation slows. However, spontaneous decomposition of Compound III to ferric SBP ensures that a residual level of Compound I is maintained. Thus, the ®rst phase of the biphasic inactivation re¯ects the transformation of active enzyme to Compound III at the expense of Compound I thereby slowing verdohemoprotein formation. The second phase re¯ects the loss of active enzyme to verdohemoprotein due to the presence of small quantities of Compound I. In addition, the gradual catalytic consumption of H2 O2 simultaneously acts to slow compound III and verdohemoprotein formation. Arnao et al. (1990) modeled the biphasic inactivation of HRP incubated with H2 O2 by deriving two eigenvalues from a ®rst-order linear dierential equation approximation of the reacting system. The two eigenvalues, k1 and k2 , were estimated from the inactivation data by ®tting the equation: A t A1 eÿk1 t A2 eÿk2 t
3
Arnao et al. (1990) used these eigenvalues to estimate kinetic constants for verdohemoprotein formation and Compound I reduction by H2 O2 . The curves drawn through the SBP inactivation data presented in Fig. 7 were obtained by ®tting Eq. (3) using regression and the method of Arnao et al. (1990). The parameters of the curve ®t of SBP data for comparison with similar data published for HRP are presented in Table 2. While Eq. (3) provided for an excellent ®t to the data in Fig. 7, the consumption of H2 O2 mentioned above is so signi®cant as to invalidate treating the reacting system as a steady-state system that can be
Table 2 Summary of parameters describing the time-dependant inactivation of SBP and HRP by H2 O2 without the presence of a phenolic substrate Initial [H2 O2 ] (mM) 0.02 0.2 2 20
SBP ± This work
HRP±Arnao et al. (1990) ÿ1
ÿ1
A1 (U/mL)
k1 (min )
A2 (U/mL)
k2 (min )
k1 (minÿ1 )
k2 (minÿ1 )
ÿ 0.160 0.176 0.136
ÿ 0.0324 0.0353 0.0512
0.361 0.235 0.198 0.173
0.00084 0.00082 0.00133 0.00632
ÿ 0.159 0.357 0.695
ÿ 0.0104 0.0314 0.0379
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
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modelled using ®rst-order linear dierential equations, as was done by Arnao et al. (1990). Therefore, the terms k1 and k2 cannot be treated as eigenvalues and kinetic constants cannot be evaluated. Due to the similarity between these experiments and those of Arnao et al. (1990), it is very likely that H2 O2 consumption was also signi®cant in their experiments. For this reason, a direct comparison of kinetic coecients of SBP and HRP cannot be made. However, a comparison of the parameters presented in Table 2 suggests that with the same initial H2 O2 concentration in the incubation mixture, inactivation observed over time will be greater with HRP than with SBP. The results imply that either the formation of verdohemoprotein occurs at a faster rate with HRP, or SBP tends to partition more as Compound II and/or Compound III thereby protecting the enzyme from permanent inactivation. The suggestion that SBP tends to partition more as Compound III is supported by the kinetic model of Nicell and Wright (1997) which quanti®es the inhibition of peroxidase by hydrogen peroxide. 3.3. Phenolic compound removal The removal of seven phenolic compounds from synthetic wastewaters was investigated as a function of SBP dose and reaction pH using 3-h stirred batch reactions. Upon initiation of the reaction using an aliquot of concentrated H2 O2 , reaction mixtures changed colour indicating the formation of phenolic polymers. With the exception of reactions conducted between pH 9 and 10, ¯oc formation was apparent within a few minutes of reaction initiation, with larger ¯ocs observed at neutral pH and smaller ¯ocs observed at acidic pH. Centrifugation of the reaction mixtures usually resulted in a clear supernatant overlying coloured precipitate. A slightly discoloured supernatant was noted following the treatment of phenol at alkaline pH and the treatment of 2,4-dichlorophenol between pH 3 and 7. Subsequent treatment of the supernatant using alum as a coagulant accomplished the removal of the residual colour associated with phenol treatment. However, pale red reaction products observed with 2,4-dichlorophenol resisted alum coagulation indicating the products were likely in dissolved rather than colloidal form. The residual quantity of phenol, measured following treatment with SBP and after centrifugation, as a function of reaction pH and SBP dose is presented in Fig. 8(a). Phenol removal occurred over a wide pH range of 2.0 to 10. Maximum removal of 99.7% was observed at pH 6.0 using 3.3 U/ml SBP. Greater than 99% removal was observed over a pH 5 to 9 range using 3.3 U/ml SBP. At lower SBP doses, less phenol removal was observed and the optimal pH for removal shifted from pH 6 to pH 9. The reason for the shift in the pH optimum is currently unknown. Phenol removals at pH
Fig. 8. Removal of phenol in batch reactions at 25°C as a function of pH and SBP concentration for solutions initially containing 1.0 mM phenol and 2.0 mM H2 O2 with residual phenol measured by (a) colorimetric and (b) UV absorbance methods.
3.0 were poor compared to those at pH 2.0. Fig. 2 demonstrates that SBP demonstrates activity over the full range of pH from 3 to 9; therefore, since the batch reactions were allowed to go to completion, the residual phenol concentrations of Fig. 8(b) are not a consequence of a slower reaction rate (i.e. a lower activity) but were due to permanent and complete inactivation of the enzyme by the end of the reaction. The residual UV absorbance of reaction supernatants obtained after centrifugation is presented in Fig. 8(b). When expressed in terms of residual UV absorbance, phenol removal occurred over a wide pH range and was optimal at pH 6.0 with a high dose of SBP and pH 7.0 with a smaller dose of SBP. Phenol removals based on the residual UV absorbance in Fig. 8(b) were consistently less than phenol removals measured using colorimetry as shown in Fig. 8(a). For reactions at pH 4±8, the supernatant was clear and the discrepancy between residual UV absorbance (Fig. 8(b)) and residual phenol (Fig. 8(a)) averaged 4 3%. At higher and lower pH values, the supernatant contained signi®cant quantities of reaction products (as indicated by a high residual UV absorbance) and the discrepancy was 12 2%,
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H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
35 23%, and 93 77% at pH 2.0, 9.0 and 9.2, respectively. It is concluded that for pH <4 and pH >8 there is a tendency to form signi®cantly greater quantities of soluble reaction products. While the UV absorbance of residual SBP may explain a fraction of the discrepancies observed between the UV absorbance and colorimetric phenol measurements, it is not the most signi®cant factor. Rather, the higher absorbance values obtained using the UV absorbance method can most likely be attributed to soluble and colloidal reaction products that resist removal by centrifugation. Zou and Taylor (1994) reported that 0.40 lM o,o0 -biphenol, 0.083 lM p-phenoxyphenol and 0.90 lM p,p0 -biphenol remained as soluble byproducts after treatment of 1.0 mM phenol at pH 7.4 using excess HRP. Yu et al. (1994) also observed o,o0 -biphenol, pphenoxyphenol and p,p0 -biphenol in the precipitate phase after phenol treatment with HRP at neutral pH. Thus it appears that a percentage of the original phenol may be transformed to soluble reaction products in equilibrium with a solid precipitate phase. Furthermore, while these soluble reaction products may undergo further enzyme catalyzed oxidation (Yu et al., 1994) and combine to form less soluble reaction products, the equilibrium with the solid phase will ensure that some residual levels of these products are maintained. Soluble aromatic reaction products at the concentrations observed by Zou and Taylor (1994) would only require UV molar extinction coecients an order of magnitude larger than the coecients for phenol in order to explain the discrepancies observed between the UV and colorimetric phenol measuring methods. The presence of residual byproducts was particularly notable in the case of 2,4-dichlorophenol removal, as is shown in Fig. 9. While colorimetry indicated that 0.35 U/ml of SBP catalytically transformed all 2,4-dichlorophenol over a pH range of 4 to 9.2, UV absorbance
measurements indicated the presence of signi®cant quantities of reaction products. The pale red colour of supernatants observed following 2,4-dichlorophenol treatment were a visual con®rmation of the presence of reaction products for reactions conducted between pH 3 and 7. Since an increase in SBP dose above 0.35 U/ml did not result in a decrease in UV absorbance (data not shown), the reaction products were not substrates of SBP. Maloney et al. (1986) studied the products generated during HRP catalyzed 2,4-dichlorophenol oxidation and identi®ed oligomers whose structure lacked a hydroxyl group. Since these products lacked a hydroxyl group, it is unlikely that they can partake in the peroxidase cycle and radical transfer would be required for their further transformation. The presence of soluble reaction products that resist removal poses a serious limitation to peroxidase catalyzed treatment of wastewaters, especially if those products are more toxic than their precursors (Aitken et al., 1994). Since these products appear unreactive towards peroxidase, coprecipitation with another phenolic compound (Nicell et al., 1993) or controlled radical transfer (Ryu et al., 1993) may be the only means of converting them to insoluble forms or preventing their formation. Further work in this area is required especially since the role of radical transfer has not been fully explored. In addition, the problem of characterizing the reaction products becomes even more complex when evaluating the treatment of real wastewaters. These results point to the need to quantify the soluble reaction products and to assess their potential toxicity if peroxidase is to be used for the treatment of wastewaters. Fig. 10 presents the removal of seven phenolic compounds as a function of SBP dose for batch reactions at neutral pH. These and other experiments conducted at pH 2 and 9 (data not shown) indicate that 99% removal or better can be achieved with all seven phenolic com-
Fig. 9. Residual 2-4-dichlorophenol in batch reactions at 25°C as a function of pH for solutions initially containing 1.0 mM 2-4-dichlorophenol, 2.0 mM H2 O2 and 0.35 U/ml SBP.
Fig. 10. Removal of seven phenolic compounds as a function of initial SBP activity for reactions conducted at pH 7.0 and 25°C and solutions initially containing 1.0 mM phenolic compound and 2.0 mM H2 O2 .
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 11. Removal of seven phenolic compounds in batch reactions at 25°C as a function of reaction pH for solutions initially containing 1.0 mM phenolic compound, 2.0 mM H2 O2 and 0.17 U/ml SBP.
pounds over a wide range of reaction pH given a sucient dose of SBP. Examples of the removal of seven phenolic compounds as a function of reaction pH for a limiting dose of SBP are presented in Fig. 11. With all seven substrates, signi®cant removal was observed over a wide pH range of 2 to 10. The degree of removal achieved by a ®xed dose of SBP depended on the phenolic compound being treated. When the treatment was limited by SBP dose, and therefore the catalytic lifetime of the enzyme, removal eciency observed over a pH 2.0±9.2 range generally followed the order of 2,4-dichlorophenol > 4-chlorophenol > 2-chlorophenol > 2-methylphenol > 3-methylphenol 3-chlorophenol > phenol. Figs. 8 and 11 demonstrate that SBP enzyme appears to be most eective in accomplishing treatment over a pH range of 6 to 9. Nicell et al. (1993) reported that HRP was most eective in the pH range of 7±9 for the same phenolic substrates. Nicell et al. (1993) proposed the parameter `catalytic turnovers' as a measure of the lifetime of the enzyme. Turnovers are de®ned as the ratio of the number of
77
molecules of phenolic substrate removed from solution per molecule of enzyme inactivated. The ratios of the number of turnovers of HRP to SBP as a function of pH and dierent substrates are summarized in Table 3. Over the majority of the pH conditions examined, the ratio is greater than unity, thereby indicating that each molecule of HRP is usually capable of removing more molecules of substrate than SBP before being permanently inactivated. Therefore, each molecule of HRP is usually used more eectively than SBP during the treatment of phenolic substrates over a wide range of pH. However, there are exceptions to this trend as is shown by the results for 2,4-dichlorophenol for pH 4±9, and for 2- and 4-chlorophenol at particular pH values. The apparent disadvantage associated with the less ecient use of SBP might be unimportant, however, due to the relatively low cost of SBP compared to HRP. With all seven phenolic compounds investigated, removals observed at pH 3 were notably less than removals observed at pH 2.0. A pH of 3 was maintained using a citrate/phosphate buer while a pH of 2 was maintained using an HCl/KCl buer. Since phosphate has been implicated in promoting HRP inactivation with incubation at low pH (Bovaird et al., 1982), phenol removal was investigated as a function of the anionic species present in solution. Fig. 12 presents phenol removal obtained after a three hour batch reaction incubated at various acidic pH values obtained using 0.1 M buers adjusted using concentrated HCl and NaOH to a desired pH. Phenol removal observed with reaction mixtures containing either phosphate (PO3ÿ 4 ) or or) was signi®cantly less than phenol thophosphite (PO3ÿ 3 removal observed with mixtures containing either acetate, citrate or chloride. With reactions at or near pH 2, precipitate was observed in mixtures containing either acetate, citrate or chloride but was not observed in mixtures containing either phosphate or orthophosphite. The results suggest that these ions promote inactivation of SBP during enzymatic removal of phenols and that the inactivation becomes more signi®cant as the reaction pH decreases. This is particulary important because buers containing phosphate are commonly used when evaluating the ability of HRP to catalyze
Table 3 Ratio of catalytic turnovers of HRP to SBP as a function of pH for 1 mM solutions of phenolic substrate, 2.0 mM H2 O2 and a limiting dose of enzyme at 0.17 U/mL Phenolic substrate Phenol 2-chlorophenol 3-chlorophenol 4-chlorophenol 2,4-dichlorophenol 2-methylphenol 3-methylphenol
Ratio of catalytic turnovers of HRP to SBP pH 4
pH 5
pH 6
pH 7
pH 8
pH 9.2
Avg. Std. Dev.
2.4 1.4 1.3 1.1 0.8 2.2 3.1
2.0 0.9 1.1 0.8 0.7 1.2 2.0
2.7 1.0 1.1 0.8 0.6 1.3 1.9
3.7 1.3 1.3 0.8 0.7 1.7 2.1
4.7 1.5 1.3 1.0 0.8 1.7 2.0
3.7 0.2 7.0 1.3 0.2 2.2 3.5
3.1 1.1 2.2 1.0 0.6 1.7 2.4
1.0 0.5 2.4 0.2 0.2 0.4 0.7
78
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
water treatment process. Signi®cant quantities of soluble products were observed at extremes of pH and the tendency for the formation of such products was dependent on the phenol being treated. Additional research is required to assess the toxicity of these products and to explore methods to reduce their concentration in the treated euents.
Acknowledgements
Fig. 12. Eect of pH buer species on phenol removal for reactions at 25°C with initial concentrations of 1.0 mM phenol, 2.0 mM H2 O2 and 1.48 U/ml SBP and a buer concentration of 0.1 M.
phenolic compound removal from solution under acidic conditions (Klibanov et al., 1980; Dec and Bollag, 1990). In future work, the use of acidic buers (pH < 5) containing phosphate or orthophosphite should be avoided in order to prevent inaccuracies associated with inactivation of the enzyme by these ionic species.
4. Conclusions SBP has demonstrated its ability to catalyze the polymerization and precipitation of phenolic compounds from water. It can act on a broad range of compounds and retains its catalytic ability over wide ranges of temperature and pH. Its catalytic activity was optimal at pH 6.4, with more than 90% of its maximum activity observed between pH 5.7 and 7.0, and more than 10% between pH 3 and 9. SBP activity was very stable at 25°C at neutral and alkaline pHs but experienced rapid inactivation below pH 3. It was most thermally stable at approximately pH 6. SBP underwent biphasic inactivation by hydrogen peroxide in the absence of a reductant substrate. SBP was most eective when used to treat phenolic solutions between pH 6 and 9. In comparison with HRP, the activity of SBP was only slightly more sensitive to pH, was signi®cantly more stable at elevated temperatures, and was less susceptible to permanent inactivation by hydrogen peroxide. However, SBP was catalytically slower than HRP and a larger molar quantity of SBP was usually required to remove a given quantity of phenolic substrate. Both SBP and HRP were susceptible to increased inactivation in the presence of phosphate or orthophosphite ions under acidic conditions. The formation of residual byproducts is a particular concern which may limit the application of this waste-
This work was funded by the Natural Sciences and Engineering Research Council of Canada and the ``Fonds pour la formation de chercheurs et l'aide a la recherche'' of Quebec. The technical assistance of Caroline Korn is gratefully acknowledged.
References Aitken, M.D., 1993. Waste treatment applications of enzymes: opportunities and obstacles. Chem. Eng. J. 52, B49±B58. Aitken, M.D., Massey, I.J., Chen, T., Heck, P.E., 1994. Characterization of reaction products from the enzyme catalyzed oxidation of phenolic pollutants. Wat. Res. 28, 1879±1889. Arnao, M.B., Acosta, M., del Rio, J.A., Garcia-Canovas, F., 1990. Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent. Biochim. Biophys. Acta 1038, 85±89. Asther, M., Meunier, J.C., 1990. Increased thermal stability of Bacillus licheniformis a-amylase in the presence of various additives. Enz. Microb. Tech. 12, 902±905. Bovaird, J.H., Ngo, T.T., Lenho, H.M., 1982. Optimizing the ophenylenediamine assay for horseradish peroxidase: Eects of phosphate and pH, substrate and enzyme concentrations, and stopping reagents. Clin. Chem. 28, 2423±2426. Buchanan, I.D., Nicell, J.A., 1997. Model development for horseradish peroxidase-catalyzed removal of aqueous phenol. Biotech. Bioeng. 54 (3), 251±261. Chuang, W., Chen, A.O., 1988. Puri®cation and characterization of vegetable soybean peroxidase I: Puri®cation and heat stability of vegetable soybean peroxidase isoenzymes. Shih P'in K'o Hsueh (Taipei) 15, 12±26 (in Chinese). Cooper, V.A., Nicell, J.A., 1996. Removal of phenols from a foundry wastewater using horseradish peroxidase. Wat. Res. 30 (4), 954± 964. De Cordt, S., Vanhoof, K., Hu, J., Maesmans, G., Hendrickx, M., Tobback, P., 1992. Thermostability of soluble and immobilized a-amylase from Bacillus licheniformis. Biotech. Bioeng. 40, 396± 402. Dec, J., Bollag, J.M., 1990. Detoxi®cation of substituted phenols by oxidoreductive enzymes through polymerization reactions. Arch. Environ. Contam. Toxicol. 19, 543±550. Dunford, H.B., Stillman, J.S., 1976. On the function and mechanism of action of peroxidases. Coord. Chem. Rev. 19, 187±251. Enzymol International Inc. 1994. Personal Communication. Gillikin, J.W., Graham, J.S., 1991. Puri®cation and developmental analysis of the major anionic peroxidase from the seed coat of Glycine max. Plant Physiol. 96, 214±220. Gomori, G., 1955. Preparation of buers for use in enzyme studies. In: Colowick, S.P., Kaplan, N.O., (Eds.), Methods in Enzymology, vol. I, New York, Academic Press, p. 138.
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79 Hewson, W.D., Dunford, H.B., 1976. Stoichiometry of the reaction between horseradish peroxidase and p-cresol. J. Biol. Chem. 251, 6043±6052. Klibanov, A.M., Alberti, B.N., Morris, E.D., Felshin, L.M., 1980. Enzymatic removal of toxic phenols and anilines from wastewaters. J. Appl. Biochem. 2, 414±421. Maloney, S.W., Manem, J., Mallevialle, J., Fiessinger, F., 1986. Transformation of trace organic compounds in drinking water by enzymatic oxidative coupling. Env. Sci. Tech. 20, 249±253. Nakajima, R., Yamazaki, I., 1987. The mechanism of oxyperoxidase formation from ferryl peroxidase and hydrogen peroxide. J. Biol. Chem. 262, 2576±2581. Nakajima, R., Yamazaki, I., 1980. The conversion of horseradish peroxidase C to a verdohemoprotein by a hydroperoxide derived enzymatically from indole-3-acetic acid and by m-nitroperoxybenzoic acid. J. Biol. Chem. 255, 2067±2071. Nakamoto, S., Machida, N., 1991. Phenol removal from aqueous solutions by peroxidase-catalyzed reaction using additives. Wat. Res. 26, 49±54. Nicell, J.A., Bewtra, J.K., Biswas, N., St. Pierre, C.C., 1993. Enzyme catalyzed polymerization and precipitation of aromatic compounds from aqueous solution. Can. J. Civ. Eng. 20, 725±735.
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Nicell, J.A., Wright, H., 1997. A model of peroxidase activity with inhibition by hydrogen peroxide. Enz. Microb. Tech. 21 (4), 302± 310. Ryu, K., McEldoon, J.P., Pokora, A.R., Cyrus, W., Dordick, J.S., 1993. Numerical and Monte Carlo simulations of phenolic polymerizations catalyzed by peroxidase. Biotech. Bioeng. 42, 807±814. Sessa, D.J., Anderson, R.L., 1981. Soybean peroxidases: puri®cation and some properties. J. Agric. Food Chem. 29, 960±965. Tamura, Y., Morita, Y., 1975. Thermal denaturation and regeneration of japanese-radish peroxidase. J. Biochem. 78, 561±571. Yamazaki, I., Nakajima, R., 1986. Physico-chemical comparison between horseradish peroxidases A and C. In: Greppin, H., Penel C., Gaspar, T., (Eds.), Molecular and Physiological Aspects of Plant Peroxidases, 3rd ed., University of Geneva, Switzerland, pp. 71±84. Yu, J., Taylor, K.E., Zou, H., Biswas, N., Bewtra, J.K., 1994. Phenol conversion and dimeric intermediates in horseradish peroxidasecatalyzed phenol removal from water. Env. Sci. Tech. 28, 2154± 2160. Zou, H., Taylor, K.E., 1994. Products of oxidative coupling of phenol by horseradish peroxidase. Chemosphere 28, 1807±1817.
Characterization of soybean peroxidase for the treatment of aqueous phenols Harold Wright, James A. Nicell* Department of Civil Engineering & Applied Mechanics, McGill University, 817 Sherbrooke Street West, Montreal PQ,Canada H3A 2K6 Received 24 July 1998; revised 12 January 1999; accepted 18 January 1999
Abstract The application of soybean peroxidase (SBP) to catalyze the polymerization and precipitation of aqueous phenols by hydrogen peroxide is potentially promising because this peroxidase is less expensive than horseradish peroxidase (HRP), which has been the focus of most wastewater research. SBP can act on a broad range of compounds and retains its catalytic ability over wide ranges of temperature and pH. Activity was optimal at pH 6.4, with signi®cant activity observed between pH 3 and 9. SBP was very stable at 25°C at neutral and alkaline conditions but experienced rapid inactivation below pH 3. SBP underwent biphasic inactivation by hydrogen peroxide in the absence of a reductant substrate. SBP was most eective when used to treat phenolic solutions between pH 6 and 9. In comparison with HRP, the activity of SBP was only slightly more sensitive to pH, was more stable at elevated temperatures, and was less susceptible to permanent inactivation by hydrogen peroxide. However, SBP was catalytically slower than HRP and a larger molar quantity of SBP was usually required to remove a given quantity of phenolic substrate. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Soybean peroxidase; Wastewater treatment; Inactivation; Stability; Temperature; pH
1. Introduction The catalytic removal of phenolic and other aromatic compounds from wastewaters using peroxidase enzyme and hydrogen peroxide has been the focus of extensive research since the initial work of Klibanov et al. (1980). Treatment involves the catalytic oxidation of the aromatic contaminants by hydrogen peroxide to form aromatic free radicals that subsequently combine to create high molecular weight products. These products precipitate from solution due to their low solubility. The method presents a possible alternative for the treatment of industrial wastewaters when conventional methods such as biological treatment, activated carbon and advanced oxidation may be ineective due to the nature of the wastewater stream (Aitken, 1993; Nicell et al., 1993). While a number of peroxidase enzymes obtained from plant, animal and microbial sources have been investigated for their ability to catalyze the removal of aromatic compounds from wastewaters, the majority of studies have focused on using horseradish peroxidase *
Corresponding author. E-mail: [email protected].
(HRP). HRP is commercially available in puri®ed form and has a proven ability to remove a variety of phenolic contaminants from aqueous solution over relatively wide ranges of pH and temperature (Nicell et al., 1993). However, one of the major challenges associated with peroxidase catalyzed phenol removal is the prohibitive cost of the enzyme (Cooper and Nicell, 1996). Enzyme costs are signi®cant because the enzyme is susceptible to inactivation by various side reactions of the treatment process (Buchanan and Nicell, 1997). While recent work has shown that enzyme inactivation can be reduced using chemical additives (Nakamoto and Machida, 1991), the problem of enzyme cost could be circumvented by using a less expensive source of enzyme. In 1991, the seed coat of the soybean was identi®ed as a rich source of a single peroxidase isoenzyme with a molecular weight of 37,000 Daltons (Gillikin and Graham, 1991). Since the seed coat of the soybean is a byproduct of the soybean food industry, soybean peroxidase (SBP) has the potential of being a cost eective alternative to HRP for wastewater treatment. Therefore, the objectives of this study were to: (1) characterize SBP in terms of its catalytic activity and susceptibility to inactivation; and (2) compare the
0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 0 7 - 3
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H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
performance of SBP with HRP to assess its potential application to the treatment of industrial wastewaters containing phenolic contaminants. 2. Methods Medium purity soybean peroxidase, with a nominal activity of 50 PPU/mg based on a purpurogallin assay, was purchased as a dry powder from Enzymol International, Columbus, OH. Phenolic compounds were at least 98% pure and were purchased from either Fluka, Ronkonkoma, NY or Aldrich, Milwaukee, WI. ACS grade 30% H2 O2 w/v was purchased from BDH Chemicals, Toronto, Canada. ACS grade potassium ferricyanide and 98% pure 4-aminoantipyrine (4-AAP) were purchased from Fisher Scienti®c, Fair Lawn, NJ and Aldrich, respectively. ACS grade conjugate acids and bases were obtained from BDH Chemicals and were used to prepare buers in accordance with the methods of Gomori (1955). Except for SBP solutions, all aqueous solutions were prepared using deionized water obtained from a D4741 Nanopure Ultrapure Water System (Barstead/Thermolyne, USA). Aqueous solutions of SBP were prepared using either deionized water (adjusted to an alkaline pH using concentrated NaOH) or pH buer. SBP powder and all aqueous solutions were stored at 4°C until the time of use. All spectrophotometric measurements were performed in a 1 cm cuvette using a Beckman DU-65 Spectrophotometer. Reaction temperatures were maintained using a constant temperature room and a Haake A81 Temperature Bath. Phenolic polymers generated during phenol removal reactions were separated from the reaction mixtures by centrifugation at 3000 g in a Centra-8 Centrifuge (International Equipment, USA). Reaction pH and temperature were measured using a Radiometer-Copenhagen Ion 83 Meter. SBP activity was measured by a colorimetric assay in which the 1 ml assay volume was composed of 500 ll of 20 mM phenol, 250 ll of 9.6 mM AAP, 100 ll of 2 mM hydrogen peroxide (H2 O2 ), 50±150 ll of sample, and 0± 100 ll of 0.1 M monobasic±dibasic phosphate buer (pH 7.4) prepared according to the method of Gomori (1955). All assay reactants were prepared in the phosphate buer. Prior to signi®cant substrate depletion, the rate of colour development at 510 nm is proportional to the concentration of active SBP. The rate of colour development was converted to enzyme activity in the cuvette using a conversion factor of 7100 Mÿ1 cmÿ1 based on hydrogen peroxide. One unit of activity (U) is de®ned as the number of micromoles of peroxide consumed in one min at pH 7.4 and 25°C. Based on standard activity measurements of nine aqueous solutions of SBP, the solid enzyme preparation used in this study had a speci®c activity of 12.3 1.1 U/
mg. A Reinheitzahl (RZ) number of 0.49 0.06 was calculated based on a ratio of absorbance measured at 280 nm and 403 nm. Assuming a Soret extinction coef®cient (Dunford and Stillman, 1976) of e403 nm 105 Mÿ1 cmÿ1 is valid for ferric SBP and assuming all Soret absorbance is attributable to active enzyme, 1 U/ml of peroxidase activity corresponds to 0.37 0.02 lM of SBP. The eect of assay pH and H2 O2 concentration on the catalytic activity of SBP was investigated by varying the pH and H2 O2 concentration in the activity assay and measuring the resulting colour generation rate at 510 nm. After colour generation, adjustment of the pH to 7.4 did not change the peak wavelength nor absorbance significantly, therefore the absorbance of the coloured product was not a signi®cant function of pH over the range examined. The stability of SBP was evaluated by incubating the enzyme at 25°C in various pH buers, at elevated temperatures in various pH buers, and at 25°C and pH 7.4 with various concentrations of H2 O2 . SBP activity in the incubation mixture was measured over time using the standard activity assay and aliquots obtained from the incubation mixture. As the incubation progressed, 1 ml aliquots were removed from the incubation mixture and quickly cooled using an ice bath to halt the inactivation process. Once cooled, aliquots were removed and allowed to warm to 25°C. Repeated activity measurements on the aliquot gave reproducible measurements. When addition of the aliquot to the assay aected the assay H2 O2 concentration and pH, the measured activity was adjusted to the expected activity under standard conditions using the experimentally determined dependence of peroxidase activity on assay H2 O2 concentration and pH. The method is described in detail in Nicell and Wright (1997). Three-hour stirred batch reactions involving 1 mM phenolic compound, 2 mM H2 O2 and various doses of SBP reacting at 25°C in various pH buers were used to evaluate the ability of SBP to catalyze phenol removal from wastewaters. In all batch reactions, concentrated aqueous SBP and phenol were added to 10 ml of pH buer. The reaction was initiated by the addition of concentrated H2 O2 . After 3 h, the precipitated products were removed by centrifugation at 3000 g for 30 min and the residual phenol in the supernatant was measured. A reaction time of 3 h was sucient to ensure that all reactions had gone to completion over the range of pHs studied. Reaction blanks were used at each reaction pH to con®rm that phenolic compound removal was solely a consequence of SBP activity. Phenolic compound concentrations were measured using a colorimetric assay consisting of 100 ll of phenol sample, 50 ll of 41.6 mM 4-AAP, 800 ll of 0.15 M sodium borate, and 50 ll of 166.8 mM potassium ferricyanide. When the extent of the assay reaction is
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
limited only by the concentration of the phenolic compound, the quantity of colour formed under alkaline conditions at 510 nm is proportional to the initial concentration of phenolic compound in the assay. The sodium borate in the assay maintained the mixture at a ®xed alkaline pH such that molar absorptivities measured for various phenolic compounds were relatively insensitive to sample pH. Since phenolic compounds have absorbance peaks in the UV range, the phenolic concentrations in a batch reactions were also estimated by measuring the UV absorbance peak of the solution and then calculating concentration using an appropriate extinction coecient. Molar extinction coecients were evaluated for each phenolic compound and for each pH. An end-point assay, based on the AAP/phenol system used in the activity assay, was used to determine the hydrogen peroxide concentration. The concentration of peroxidase in the microcuvette was approximately 100 mg/l, while those of the other reactants (except peroxide) were as cited above for the peroxidase assay. The extent of color development at 510 nm was proportional to hydrogen peroxide concentration, provided this concentration did not exceed 0.05 mM in the cuvette. After a 10-minute reaction time, the absorbance at 510 nm was converted to peroxide concentration using a calibration curve. 3. Results and discussion Fig. 1 illustrates the catalytic cycle of peroxidase and its postulated side-reactions. This ®gure was constructed based on numerous studies of horseradish peroxidase (Arnao et al., 1990; Hewson and Dunford, 1976; Nakajima and Yamazaki, 1980, 1987) and forms the basis of some of the following discussion.
71
Fig. 1. The catalytic cycle and side-reactions of peroxidase: EN is native peroxidase; Ei , Eii and Eiii are compounds I, II and III, respectively; E-670 is verdohaemoprotein, and Ei -H2 O2 is an intermediate enzyme±peroxide complex; ROH is a phenolic substrate and RO is a phenoxy radical.
maximum peroxide consumption rate observed between pH 5.7 and 7.0, and >10% between pH 3 and 9. Nicell et al. (1993) reported that HRP experienced peak activity at pH 7.4, with >90% of the maximum rate observed between pH 5.7 and 8.0, and >10% between pH 3.5 to 10.0. A comparison of these results shows that the activity of SBP appears to be slightly more sensitive to pH than HRP; however, both peroxidases retain signi®cant activity over a wide range of pH conditions frequently encountered in wastewater treatment situations. The rate at which peroxidase passes through the catalytic cycle is limited by the slowest reaction of the cycle. With sucient H2 O2 in the reaction mixture, the reduction of Compound I (Eii ) to ferric enzyme (EN ) is usually the limiting step (Dunford and Stillman, 1976). For example, reported apparent rate constants for the reduction of HRP Compound II by 4-methylphenol are greatest near pH 7.4 but fall to 10% of peak value near pH 2 and pH 9.5 (Hewson and Dunford, 1976). This conforms with the activity measurements of Nicell et al. (1993). By analogy, the pH dependence of SBP activity likely re¯ects the pH dependence of the
3.1. Activity of soybean peroxidase The activity of peroxidase measured in a phenol/4AAP colorimetric assay is a measure of how quickly the enzyme passes through the catalytic cycle consuming H2 O2 and phenolic substrate (ROH) and generating phenoxy radicals (RO ). The impact of pH on SBP activity was measured by varying the pH of the assay buer. The relative activities as a function of pH are presented in Fig. 2. Relative activity is expressed as the rate of hydrogen peroxide consumption at a particular pH normalized with respect to the highest rate. Experiments were performed using dierent buer species to achieve identical pH conditions. Fig. 2 indicates that activity was not in¯uenced by the buer species used to maintain pH. The results demonstrate that SBP was active over a wide range of reaction pH with a maximum peroxide consumption rate at pH 6.4, with >90% of the
Fig. 2. Relative activity of SBP as a function of reaction pH with 5 lg/ ml SBP in the assay mixture.
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H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 3. Rate of H2 O2 consumption in the activity assay as a function of H2 O2 and SBP concentration. Smooth curves are based on a ®t of the model of Nicell and Wright (1997).
apparent rate constants describing the reactions of SBP Compound II. The rate of colour generation at pH 7.4 and 25°C was investigated as a function of the assay H2 O2 concentration. The resulting reaction rates, expressed as peroxide consumption rates, are presented in Fig. 3 as a function of initial H2 O2 concentration for three concentrations of SBP. The maximum activity was consistently observed at 0.33 mM H2 O2 . At low H2 O2 concentrations, the peroxidase cycle is limited by the rate at which ferric peroxidase is oxidized by H2 O2 to Compound I. At higher H2 O2 concentrations, the peroxidase cycle is limited by the formation of inactive Compound III (Eiii ). This is not a permanently inactivated form of peroxidase since it can revert to the ferric state and reenter the catalytic cycle, as shown in Fig. 1; however, the return is very slow and any accumulation in this state represents a loss in catalytic eciency. Nicell and Wright (1997) developed a kinetic model of this assay and extracted kinetic constants to describe the rate at which peroxidase passes through its catalytic cycle and its susceptibility to Compound III formation. A comparison of the rate constants for HRP and SBP revealed that SBP is more than an order of magnitude slower than HRP during the oxidation of phenol and SBP is far more susceptible to inactivation through Compound III formation than HRP (Nicell and Wright, 1997).
Fig. 4. Stability of SBP incubated at 25°C in various pH buers (pH 2.0 HCl/KCl buer, pH 3.0 citrate/phosphate buer, pH 5.0 citrate/ phosphate buer, pH 7.0 phosphate buer, pH 9.0 borate buer, pH 10.7 carbonate buer).
measured and the activity corrected to an equivalent value at pH 7.4 using the results presented in Fig. 2. In acidic buer, SBP undergoes time dependent inactivation whose rate and extent depend on the pH of the incubation mixture. At pH 2 and 3, ®rst order inactivation led to essentially zero activity within two and four days, respectively. Inactivation at pH 5 was biphasic and did not result in complete inactivation. In comparison, SBP incubated in either neutral or alkaline buer was essentially stable for over 20 days of incubation. 3.2.2. Thermal inactivation The stability of SBP when it was incubated at approximately 80°C in various pH buers is presented in Fig. 5. The thermal inactivation depended on incuba-
3.2. Stability of soybean peroxidase 3.2.1. Eect of pH The stability of SBP incubated at 25°C as a function of pH is presented in Fig. 4. Since activity was measured using the standard activity recipe, the addition of an aliquot of incubation mixture to the assay caused a notable pH shift that in¯uenced the rate of colour formation. Accordingly, the pH of the assay mixture was
Fig. 5. Stability of SBP incubated at 80.3 0.4°C in various buers (pH 5.0 citrate/phosphate buer, pH 6.0 phosphate buer, pH 7.0 phosphate buer, pH 8.0 phosphate buer, pH 9.0 borate buer, pH 10.3 carbonate buer).
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 6. SBP decimal reduction values as a function of temperature and pH.
tion pH and could be modelled using the ®rst order equation: t
1 A t A0 eÿkt A0 10ÿD ; where A(t) is the activity at time t, A0 is the initial activity, k is the inactivation decay constant, and D is the inactivation decimal reduction value (D-value). Fig. 6 presents D-values for SBP thermal inactivation as a function of incubation temperature and pH. The Dvalue at 70°C and pH 6.0 could not be measured due to the SBP's extreme thermal stability under these conditions. Consistently, SBP was most resistant to thermal inactivation at approximately pH 6. Inactivation decay constants may be related to incubation temperature using the Arrhenius Law and an activation energy (De Cordt et al., 1992). D-values over a limited temperature range may be related to temperature using: TREF ÿT
2 D DREF 10 z where DREF is the decimal reduction value at temperature TREF and Z is the temperature change required to obtain a ten-fold increase or decrease in the decimal reduction value (De Cordt et al., 1992). Unlike inacti-
73
vation decay constants and D-values, the activation energies and Z-values obtained from the data in Fig. 6 tend to be independent of pH with average values of 234 13 kJ/mol and 10.2 0.6°C, respectively. The thermal inactivation of SBP observed in this work is compared to observations reported in the literature in Table 1. Using SBP which was puri®ed using dialysis, fractionation and chromatography, Sessa and Anderson (1981) and Chuang and Chen (1988) observed considerably faster thermal inactivation of SBP. Since enzyme thermal inactivation is in¯uenced by a multitude of factors such as ionic strength and buer species (De Cordt et al., 1992), calcium ion concentration (Enzymol International Inc., 1994), and polyhydric alcohol and sugar concentration (Asther and Meunier, 1990), differences between the rates of thermal inactivation reported in the literature and the rates observed in this work may be due to dierences in the constituents of the incubation mixture which are a result of the puri®cation techniques used to extract SBP from the original plant material. The Z-value observed in this work compares well with the value reported by Chuang and Chen (1988). A pH optimum for peroxidase thermal stability has been previously reported with Japanese radish peroxidase (Tamura and Morita, 1975) and pH independent Z-values have been observed with non-peroxidase enzymes (De Cordt et al., 1992). While a 3±4 min D-value has been observed with HRP incubated at 72°C and pH 7.4 (from the data of Nicell et al., 1993), SBP incubated under similar conditions in this work exhibited a D-value of several days thereby indicating a much greater stability at elevated temperatures. 3.2.3. Inactivation by hydrogen peroxide The stability of SBP incubated at 25°C in pH 7.4 buer with various concentrations of H2 O2 is presented in Fig. 7. Since the concentration of H2 O2 within the assay was increased by the addition of an aliquot from the incubation mixture, the measured rate of colour generation was corrected to standard conditions using the curves of Fig. 3 and the method described by Nicell
Table 1 Thermal inactivation parameters of SBP Enzyme source
pH and buer species
Temperature (°C)
Composite of puri®ed isoenzymes Puri®ed 37 000 Dalton isoenzyme
pH 5.5 (0.1 M citrate/ phosphate buer) pH 7.0 (0.2 M phosphate buer)
70
Enzymol medium purity SBP
pH 5.0 (0.1 M citrate/ phosphate buer) pH 7.0 (0.1 M phosphate buer)
D (min)
Z (°C)
Reference
60
ÿ
70 80 90 70.2
103 9.1 0.38 1450
8
Sessa and Anderson (1981) Chuang and Chen (1988)
70.2
4000
80.3 90.8
300 30
10.2 0.6
This work
74
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 7. Stability of 0.37 U/ml SBP at 25°C as a function of time and initial concentration of hydrogen peroxide in pH 7.4 phosphate buer. Smooth curlves are based on Eq. (3) and parameters summarized in Table 2.
and Wright (1997). This correction was applied assuming that the catalytic consumption of H2 O2 in the incubation mixture was negligible. However, it should be noted that after 80 minutes of incubation with initial H2 O2 concentrations of 0.02, 0.2, 2.0, and 20 mM, the concentration of H2 O2 in the incubation mixtures fell by 62, 50, 30 and 20% of its initial value, respectively. This reduction in H2 O2 may result in a worst-case activity overestimation of 6.6% and 1.4% after 80 min of incubation with initial H2 O2 concentrations of 20 mM and 2.0 mM, respectively. When peroxidase and H2 O2 are incubated together without a phenolic reducing agent present, H2 O2 acts to reduce the +5 oxidation state of the enzyme in either one or two electron steps (Nakajima and Yamazaki, 1987). Arnao et al. (1990) reported that H2 O2 also reacts with HRP resulting in an inactive form referred to as verdohemoprotein (see E-670 in Fig. 1). While the exact mechanism of verdohemoprotein formation is uncertain and may involve a sequence of reversible and irreversible reactions (Nakajima and Yamazaki, 1980), Arnao et al. (1990) proposed the scheme shown in Fig. 1 for its formation. SBP inactivation over time presented in Fig. 7 increased with H2 O2 concentration and was biphasic. The biphasic inactivation observed with SBP can be ex-
plained based on the relative magnitudes of kinetic constants published for HRP reacting with H2 O2 (Arnao et al., 1990). Since the reaction of ferric peroxidase with H2 O2 occurs quickly (Yamazaki and Nakajima, 1986), ferric SBP is expected to oxidize to Compound I virtually immediately following the addition of H2 O2 to the incubation mixture. Since the reduction of Compound I by H2 O2 occurs relatively slowly (Arnao et al., 1990), the rate of verdohemoprotein formation at this point can be expected to occur at its fastest rate assuming its formation originates at the Compound I state. As the incubation proceeds, the majority of SBP is transformed to Compound II and Compound III at the expense of Compound I and the rate of verdohemoprotein formation slows. However, spontaneous decomposition of Compound III to ferric SBP ensures that a residual level of Compound I is maintained. Thus, the ®rst phase of the biphasic inactivation re¯ects the transformation of active enzyme to Compound III at the expense of Compound I thereby slowing verdohemoprotein formation. The second phase re¯ects the loss of active enzyme to verdohemoprotein due to the presence of small quantities of Compound I. In addition, the gradual catalytic consumption of H2 O2 simultaneously acts to slow compound III and verdohemoprotein formation. Arnao et al. (1990) modeled the biphasic inactivation of HRP incubated with H2 O2 by deriving two eigenvalues from a ®rst-order linear dierential equation approximation of the reacting system. The two eigenvalues, k1 and k2 , were estimated from the inactivation data by ®tting the equation: A t A1 eÿk1 t A2 eÿk2 t
3
Arnao et al. (1990) used these eigenvalues to estimate kinetic constants for verdohemoprotein formation and Compound I reduction by H2 O2 . The curves drawn through the SBP inactivation data presented in Fig. 7 were obtained by ®tting Eq. (3) using regression and the method of Arnao et al. (1990). The parameters of the curve ®t of SBP data for comparison with similar data published for HRP are presented in Table 2. While Eq. (3) provided for an excellent ®t to the data in Fig. 7, the consumption of H2 O2 mentioned above is so signi®cant as to invalidate treating the reacting system as a steady-state system that can be
Table 2 Summary of parameters describing the time-dependant inactivation of SBP and HRP by H2 O2 without the presence of a phenolic substrate Initial [H2 O2 ] (mM) 0.02 0.2 2 20
SBP ± This work
HRP±Arnao et al. (1990) ÿ1
ÿ1
A1 (U/mL)
k1 (min )
A2 (U/mL)
k2 (min )
k1 (minÿ1 )
k2 (minÿ1 )
ÿ 0.160 0.176 0.136
ÿ 0.0324 0.0353 0.0512
0.361 0.235 0.198 0.173
0.00084 0.00082 0.00133 0.00632
ÿ 0.159 0.357 0.695
ÿ 0.0104 0.0314 0.0379
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
75
modelled using ®rst-order linear dierential equations, as was done by Arnao et al. (1990). Therefore, the terms k1 and k2 cannot be treated as eigenvalues and kinetic constants cannot be evaluated. Due to the similarity between these experiments and those of Arnao et al. (1990), it is very likely that H2 O2 consumption was also signi®cant in their experiments. For this reason, a direct comparison of kinetic coecients of SBP and HRP cannot be made. However, a comparison of the parameters presented in Table 2 suggests that with the same initial H2 O2 concentration in the incubation mixture, inactivation observed over time will be greater with HRP than with SBP. The results imply that either the formation of verdohemoprotein occurs at a faster rate with HRP, or SBP tends to partition more as Compound II and/or Compound III thereby protecting the enzyme from permanent inactivation. The suggestion that SBP tends to partition more as Compound III is supported by the kinetic model of Nicell and Wright (1997) which quanti®es the inhibition of peroxidase by hydrogen peroxide. 3.3. Phenolic compound removal The removal of seven phenolic compounds from synthetic wastewaters was investigated as a function of SBP dose and reaction pH using 3-h stirred batch reactions. Upon initiation of the reaction using an aliquot of concentrated H2 O2 , reaction mixtures changed colour indicating the formation of phenolic polymers. With the exception of reactions conducted between pH 9 and 10, ¯oc formation was apparent within a few minutes of reaction initiation, with larger ¯ocs observed at neutral pH and smaller ¯ocs observed at acidic pH. Centrifugation of the reaction mixtures usually resulted in a clear supernatant overlying coloured precipitate. A slightly discoloured supernatant was noted following the treatment of phenol at alkaline pH and the treatment of 2,4-dichlorophenol between pH 3 and 7. Subsequent treatment of the supernatant using alum as a coagulant accomplished the removal of the residual colour associated with phenol treatment. However, pale red reaction products observed with 2,4-dichlorophenol resisted alum coagulation indicating the products were likely in dissolved rather than colloidal form. The residual quantity of phenol, measured following treatment with SBP and after centrifugation, as a function of reaction pH and SBP dose is presented in Fig. 8(a). Phenol removal occurred over a wide pH range of 2.0 to 10. Maximum removal of 99.7% was observed at pH 6.0 using 3.3 U/ml SBP. Greater than 99% removal was observed over a pH 5 to 9 range using 3.3 U/ml SBP. At lower SBP doses, less phenol removal was observed and the optimal pH for removal shifted from pH 6 to pH 9. The reason for the shift in the pH optimum is currently unknown. Phenol removals at pH
Fig. 8. Removal of phenol in batch reactions at 25°C as a function of pH and SBP concentration for solutions initially containing 1.0 mM phenol and 2.0 mM H2 O2 with residual phenol measured by (a) colorimetric and (b) UV absorbance methods.
3.0 were poor compared to those at pH 2.0. Fig. 2 demonstrates that SBP demonstrates activity over the full range of pH from 3 to 9; therefore, since the batch reactions were allowed to go to completion, the residual phenol concentrations of Fig. 8(b) are not a consequence of a slower reaction rate (i.e. a lower activity) but were due to permanent and complete inactivation of the enzyme by the end of the reaction. The residual UV absorbance of reaction supernatants obtained after centrifugation is presented in Fig. 8(b). When expressed in terms of residual UV absorbance, phenol removal occurred over a wide pH range and was optimal at pH 6.0 with a high dose of SBP and pH 7.0 with a smaller dose of SBP. Phenol removals based on the residual UV absorbance in Fig. 8(b) were consistently less than phenol removals measured using colorimetry as shown in Fig. 8(a). For reactions at pH 4±8, the supernatant was clear and the discrepancy between residual UV absorbance (Fig. 8(b)) and residual phenol (Fig. 8(a)) averaged 4 3%. At higher and lower pH values, the supernatant contained signi®cant quantities of reaction products (as indicated by a high residual UV absorbance) and the discrepancy was 12 2%,
76
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
35 23%, and 93 77% at pH 2.0, 9.0 and 9.2, respectively. It is concluded that for pH <4 and pH >8 there is a tendency to form signi®cantly greater quantities of soluble reaction products. While the UV absorbance of residual SBP may explain a fraction of the discrepancies observed between the UV absorbance and colorimetric phenol measurements, it is not the most signi®cant factor. Rather, the higher absorbance values obtained using the UV absorbance method can most likely be attributed to soluble and colloidal reaction products that resist removal by centrifugation. Zou and Taylor (1994) reported that 0.40 lM o,o0 -biphenol, 0.083 lM p-phenoxyphenol and 0.90 lM p,p0 -biphenol remained as soluble byproducts after treatment of 1.0 mM phenol at pH 7.4 using excess HRP. Yu et al. (1994) also observed o,o0 -biphenol, pphenoxyphenol and p,p0 -biphenol in the precipitate phase after phenol treatment with HRP at neutral pH. Thus it appears that a percentage of the original phenol may be transformed to soluble reaction products in equilibrium with a solid precipitate phase. Furthermore, while these soluble reaction products may undergo further enzyme catalyzed oxidation (Yu et al., 1994) and combine to form less soluble reaction products, the equilibrium with the solid phase will ensure that some residual levels of these products are maintained. Soluble aromatic reaction products at the concentrations observed by Zou and Taylor (1994) would only require UV molar extinction coecients an order of magnitude larger than the coecients for phenol in order to explain the discrepancies observed between the UV and colorimetric phenol measuring methods. The presence of residual byproducts was particularly notable in the case of 2,4-dichlorophenol removal, as is shown in Fig. 9. While colorimetry indicated that 0.35 U/ml of SBP catalytically transformed all 2,4-dichlorophenol over a pH range of 4 to 9.2, UV absorbance
measurements indicated the presence of signi®cant quantities of reaction products. The pale red colour of supernatants observed following 2,4-dichlorophenol treatment were a visual con®rmation of the presence of reaction products for reactions conducted between pH 3 and 7. Since an increase in SBP dose above 0.35 U/ml did not result in a decrease in UV absorbance (data not shown), the reaction products were not substrates of SBP. Maloney et al. (1986) studied the products generated during HRP catalyzed 2,4-dichlorophenol oxidation and identi®ed oligomers whose structure lacked a hydroxyl group. Since these products lacked a hydroxyl group, it is unlikely that they can partake in the peroxidase cycle and radical transfer would be required for their further transformation. The presence of soluble reaction products that resist removal poses a serious limitation to peroxidase catalyzed treatment of wastewaters, especially if those products are more toxic than their precursors (Aitken et al., 1994). Since these products appear unreactive towards peroxidase, coprecipitation with another phenolic compound (Nicell et al., 1993) or controlled radical transfer (Ryu et al., 1993) may be the only means of converting them to insoluble forms or preventing their formation. Further work in this area is required especially since the role of radical transfer has not been fully explored. In addition, the problem of characterizing the reaction products becomes even more complex when evaluating the treatment of real wastewaters. These results point to the need to quantify the soluble reaction products and to assess their potential toxicity if peroxidase is to be used for the treatment of wastewaters. Fig. 10 presents the removal of seven phenolic compounds as a function of SBP dose for batch reactions at neutral pH. These and other experiments conducted at pH 2 and 9 (data not shown) indicate that 99% removal or better can be achieved with all seven phenolic com-
Fig. 9. Residual 2-4-dichlorophenol in batch reactions at 25°C as a function of pH for solutions initially containing 1.0 mM 2-4-dichlorophenol, 2.0 mM H2 O2 and 0.35 U/ml SBP.
Fig. 10. Removal of seven phenolic compounds as a function of initial SBP activity for reactions conducted at pH 7.0 and 25°C and solutions initially containing 1.0 mM phenolic compound and 2.0 mM H2 O2 .
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
Fig. 11. Removal of seven phenolic compounds in batch reactions at 25°C as a function of reaction pH for solutions initially containing 1.0 mM phenolic compound, 2.0 mM H2 O2 and 0.17 U/ml SBP.
pounds over a wide range of reaction pH given a sucient dose of SBP. Examples of the removal of seven phenolic compounds as a function of reaction pH for a limiting dose of SBP are presented in Fig. 11. With all seven substrates, signi®cant removal was observed over a wide pH range of 2 to 10. The degree of removal achieved by a ®xed dose of SBP depended on the phenolic compound being treated. When the treatment was limited by SBP dose, and therefore the catalytic lifetime of the enzyme, removal eciency observed over a pH 2.0±9.2 range generally followed the order of 2,4-dichlorophenol > 4-chlorophenol > 2-chlorophenol > 2-methylphenol > 3-methylphenol 3-chlorophenol > phenol. Figs. 8 and 11 demonstrate that SBP enzyme appears to be most eective in accomplishing treatment over a pH range of 6 to 9. Nicell et al. (1993) reported that HRP was most eective in the pH range of 7±9 for the same phenolic substrates. Nicell et al. (1993) proposed the parameter `catalytic turnovers' as a measure of the lifetime of the enzyme. Turnovers are de®ned as the ratio of the number of
77
molecules of phenolic substrate removed from solution per molecule of enzyme inactivated. The ratios of the number of turnovers of HRP to SBP as a function of pH and dierent substrates are summarized in Table 3. Over the majority of the pH conditions examined, the ratio is greater than unity, thereby indicating that each molecule of HRP is usually capable of removing more molecules of substrate than SBP before being permanently inactivated. Therefore, each molecule of HRP is usually used more eectively than SBP during the treatment of phenolic substrates over a wide range of pH. However, there are exceptions to this trend as is shown by the results for 2,4-dichlorophenol for pH 4±9, and for 2- and 4-chlorophenol at particular pH values. The apparent disadvantage associated with the less ecient use of SBP might be unimportant, however, due to the relatively low cost of SBP compared to HRP. With all seven phenolic compounds investigated, removals observed at pH 3 were notably less than removals observed at pH 2.0. A pH of 3 was maintained using a citrate/phosphate buer while a pH of 2 was maintained using an HCl/KCl buer. Since phosphate has been implicated in promoting HRP inactivation with incubation at low pH (Bovaird et al., 1982), phenol removal was investigated as a function of the anionic species present in solution. Fig. 12 presents phenol removal obtained after a three hour batch reaction incubated at various acidic pH values obtained using 0.1 M buers adjusted using concentrated HCl and NaOH to a desired pH. Phenol removal observed with reaction mixtures containing either phosphate (PO3ÿ 4 ) or or) was signi®cantly less than phenol thophosphite (PO3ÿ 3 removal observed with mixtures containing either acetate, citrate or chloride. With reactions at or near pH 2, precipitate was observed in mixtures containing either acetate, citrate or chloride but was not observed in mixtures containing either phosphate or orthophosphite. The results suggest that these ions promote inactivation of SBP during enzymatic removal of phenols and that the inactivation becomes more signi®cant as the reaction pH decreases. This is particulary important because buers containing phosphate are commonly used when evaluating the ability of HRP to catalyze
Table 3 Ratio of catalytic turnovers of HRP to SBP as a function of pH for 1 mM solutions of phenolic substrate, 2.0 mM H2 O2 and a limiting dose of enzyme at 0.17 U/mL Phenolic substrate Phenol 2-chlorophenol 3-chlorophenol 4-chlorophenol 2,4-dichlorophenol 2-methylphenol 3-methylphenol
Ratio of catalytic turnovers of HRP to SBP pH 4
pH 5
pH 6
pH 7
pH 8
pH 9.2
Avg. Std. Dev.
2.4 1.4 1.3 1.1 0.8 2.2 3.1
2.0 0.9 1.1 0.8 0.7 1.2 2.0
2.7 1.0 1.1 0.8 0.6 1.3 1.9
3.7 1.3 1.3 0.8 0.7 1.7 2.1
4.7 1.5 1.3 1.0 0.8 1.7 2.0
3.7 0.2 7.0 1.3 0.2 2.2 3.5
3.1 1.1 2.2 1.0 0.6 1.7 2.4
1.0 0.5 2.4 0.2 0.2 0.4 0.7
78
H. Wright, J.A. Nicell / Bioresource Technology 70 (1999) 69±79
water treatment process. Signi®cant quantities of soluble products were observed at extremes of pH and the tendency for the formation of such products was dependent on the phenol being treated. Additional research is required to assess the toxicity of these products and to explore methods to reduce their concentration in the treated euents.
Acknowledgements
Fig. 12. Eect of pH buer species on phenol removal for reactions at 25°C with initial concentrations of 1.0 mM phenol, 2.0 mM H2 O2 and 1.48 U/ml SBP and a buer concentration of 0.1 M.
phenolic compound removal from solution under acidic conditions (Klibanov et al., 1980; Dec and Bollag, 1990). In future work, the use of acidic buers (pH < 5) containing phosphate or orthophosphite should be avoided in order to prevent inaccuracies associated with inactivation of the enzyme by these ionic species.
4. Conclusions SBP has demonstrated its ability to catalyze the polymerization and precipitation of phenolic compounds from water. It can act on a broad range of compounds and retains its catalytic ability over wide ranges of temperature and pH. Its catalytic activity was optimal at pH 6.4, with more than 90% of its maximum activity observed between pH 5.7 and 7.0, and more than 10% between pH 3 and 9. SBP activity was very stable at 25°C at neutral and alkaline pHs but experienced rapid inactivation below pH 3. It was most thermally stable at approximately pH 6. SBP underwent biphasic inactivation by hydrogen peroxide in the absence of a reductant substrate. SBP was most eective when used to treat phenolic solutions between pH 6 and 9. In comparison with HRP, the activity of SBP was only slightly more sensitive to pH, was signi®cantly more stable at elevated temperatures, and was less susceptible to permanent inactivation by hydrogen peroxide. However, SBP was catalytically slower than HRP and a larger molar quantity of SBP was usually required to remove a given quantity of phenolic substrate. Both SBP and HRP were susceptible to increased inactivation in the presence of phosphate or orthophosphite ions under acidic conditions. The formation of residual byproducts is a particular concern which may limit the application of this waste-
This work was funded by the Natural Sciences and Engineering Research Council of Canada and the ``Fonds pour la formation de chercheurs et l'aide a la recherche'' of Quebec. The technical assistance of Caroline Korn is gratefully acknowledged.
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