Grant Information

This work was supported in part by NIEHS Superfund Basic Research and Training Grant ES04922.

BACKGROUND OF THE INVENTION

1. Field ofthe Invention. This invention describes methods to enhance the rate and extent of biodegradation of organic compounds by peroxidase enzymes such as those produced by white rot fungi. It has been discovered that metal ions can stabilize the enzymes to the effects of increasing temperature and increasing pH. In particular, this invention involves the use of calcium to enhance the rate and the extent of biotransformation and biodegradation reactions, such as the biodegradation of environmental pollutants, by white rot fungi or, more specifically, by peroxidase enzymes derived from white rot fungi.

2. The Relevant Technology.

It has long been known that enzymes secreted by white rot fungi can biotransform lignin and many synthetic chemicals. The enzymes effect oxidation and reduction reactions using various mediators resulting in generation of powerful free radical oxidizing and reducing agents which biotransform even degradation-resistant organic compounds. Efforts to apply this ability of white rot fungi-derived enzymes to the biotransformation and biodegradation of environmental pollutants are well-developed. See, e.g., the following U.S. Patents commonly owned by the assignee ofthe present application, the disclosures of which are herein incoφorated by reference; U.S. Patent No. 4,891,320,

U.S. Patent No. 5,459,065, U.S. Patent No. 5,389,356, and U.S. Patent No. 5,468,268.

These biologically derived oxidation and reduction reactions are often superior to other oxidation and reduction methods because of their lower cost and a greater ability to more carefully control the reaction conditions. Enzymes have the advantage of being able to overcome high reaction barriers without the input and/or generation of large amounts of energy such as heat. In addition, as long as the biological agent is kept alive by ensuring that the system has adequate quantities of nutrients (some or all of which may be supplied by the chemicals targeted for degradation) it will continue to produce adequate quantities ofthe oxidation or reduction agents. In this manner, the reaction is often self-sustaining so that no new reactants need to be added to complete the oxidation and/or reduction reactions.

The white rot fungi system can be used for effective bioremediation of environmental pollution sites including contaminated soil, water, air, wood, etc. It is known that the rate of the enzyme reactions can be increased by increasing the temperature. Unfortunately, it is also known that some ofthe critical enzymes are heat- sensitive and loss of activity due to thermal inactivation occurs at higher temperatures.

It is also known that some ofthe enzymes are extremely pH-sensitive and loss of activity occurs at higher pH conditions.

From the foregoing it should be understood that it would be a significant advance to provide methods for enhancing the rate and the extent ofthe biodegradation of organic chemicals by white rot fungi-derived enzymes. In particular, it would be advantageous to stabilize the enzymes to the effect of increasing heat and thereby permit continued activity at higher rate-enhancing temperatures. It would be a further advantage to stabilize the enzymes to the effect of increasing pH and thereby permit continued activity under non-optimal pH conditions. Such methods are disclosed and claimed herein.

BRIEF SUMMARY OF THE INVENTION In accordance with the invention as embodied and broadly described herein, the present invention is directed to methods to enhance the rate and extent of biodegradation of organic compounds by peroxidase enzymes such as those produced by lignin-degrading fungi and particularly by white rot fungi. It has been discovered that metal ions can stabilize the enzymes against adverse effects of increasing temperature and increasing pH. In particular, this invention involves the use of calcium to enhance the rate and the extent of biotransformation and biodegradation reactions, such as the biodegradation of environmental pollutants, by white rot fungi or, more specifically, by peroxidase enzymes derived from white rot fungi. In addition, other divalent metal cations, such as magnesium, zinc, and manganese, are also able to stabilize the enzymes. BRIEF DESCRIPTION OF THE DRAWINGS Figure IA is a graph illustrating the effect of thermal incubation at various temperatures on the activity of MnP over time. The MnP samples were incubated in sodium succinate, pH 5.5, at 37°C (•), 42.5°C (■), 45°C (A), and 47.5°C (♦).

Figure IB is an Arrhenius plot ofthe data shown in Figure 1 A where k represents the inactivation rate constant for the first phase of inactivation.

Figure 2 is a graph illustrating the effect of thermal incubation at various pH values on the activity of MnP over time. The MnP samples were incubated at 37° C in sodium phosphate at pH 5.5 (A), pH 6.0 (■), pH 6.5 (•), pH 6.75 (T), and pH 7.1 (♦).

Figure 3 is a graph illustrating the effect of thermal incubation of MnP on the activity and absorbance of heme in the enzyme. The MnP samples were incubated in sodium phosphate, pH 6.5, at 37 °C and the remaining activity (•) and heme absorbance (o) were quantitated as described in Materials and Methods The data corresponding to the second phase of inactivation was replotted (■) to compare this rate the rate of decrease of heme absorbance.

Figure 4 is a graph illustrating the effect of Ca ²⁺ and EGTA on the thermal inactivation of MnP. The MnP samples were incubated in sodium phosphate, pH 6.5, at 37 °C in the presence of 100 μM CaSO ₄ (■), 25 μM CaSO ₄ (A), no added compound (•), or l mM EGTA (Φ).

Figure 5 is a graph illustrating the effect of Ca ²⁺ on reactivation of thermally inactivated MnP. The MnP samples were incubated for 2.5 minutes at pH 7.1 and at 37°C prior to the addition of CaSO ₄ to final concentrations of 1.0 mM (•), 0.5 mM (■), and 0.1 mM (A) or without added compound (T). Figure 6 is a graph illustrating the effect ofthe time of addition of Ca ²⁺ during thermal incubation on the reactivation of MnP activity. The MnP samples were incubated at pH 7.1 and at 37°C prior to the addition of CaSO ₄ to a final concentration of 1.0 mM at 1.0 minute, 2.5 minutes, 5.0 minutes, and 10.0 minutes.

Figure 7 is a graph illustrating the amount of enzyme remaining with heme and the maximum amount of enzyme activity recovered by the addition of Ca ²⁺ following the various times of thermal incubation shown in Figure 6.

Figure 8 is a graph illustrating the effect of various divalent cations on the thermal inactivation of MnP over time. The enzyme mixture was incubated at 32 °C and contained 0.3 μMMnP, 20 mM sodium phosphate, pH 6.8, and 100 μM CaSO ₄ (■), MnSO ₄ (A), ZnSO ₄ (T), MgSO ₄ (♦), or no added cation (•).

Figure 9 is a graph illustrating the effect of calcium on the thermal inactivation of MnP by crosslinked polyacrylamide superabsorbent polymer and by crosslinked polyacrylate superabsorbent polymer. MnP (0.16 μM) was incubated in 20 mM phosphate, pH 6.75, at 37°C in the presence of 2.0 mg (per 5 mL) crosslinked polyacrylamide superabsorbent polymer in the presence (□) and absence (■) of 1.0 mM calcium and in the presence of 2.0 mg crosslinked polyacrylate superabsorbent polymer in the presence (Δ) and absence (A) of 1.0 mM calcium and in the absence of both polymer and calcium (•).

Figure 10 is a graph illustrating the effect of calcium, manganese, and oxalate on manganese-dependent peroxidase activity of Phanerochaete chrysosporium at 37°C.

Phanerochaete chrysosporium grown in the presence (■) and absence (•) of additional calcium, manganese, and oxalate was periodically determined by monitoring the ability to oxidize 2-methoxy phenol. Calcium (1 mM), manganese (1 mM), and oxalate (2 mM) were added to cultures on day 3 as indicated by the arrow. Data represent the average and standard deviations of triplicate cultures.

Figure 11 is a graph illustrating the effect of calcium, manganese, and oxalate on manganese-dependent peroxidase activity of Phanerochaete chrysosporium at 32° C. Manganese-dependent peroxidase activity in the extracellular fluid from cultures of Phanerochaete chrysosporium grown in the presence (■) and absence (•) of additional calcium, manganese, and oxalate was periodically determined by monitoring the ability to oxidize 2-methoxy phenol. Calcium (1 mM), manganese (1 mM), and oxalate (2 mM) were added to cultures on day 3 as indicated by the arrow. Data represent the average and standard deviations of triplicate cultures.

Figure 12 is a graph illustrating the effect of calcium, manganese, and oxalate on manganese-dependent peroxidase activity of Bjerkandera adusta at 32 °C. Manganese- dependent peroxidase activity in the extracellular fluid from cultures of Bjerkandera adusta grown in the presence (■) and absence (#) of additional calcium, manganese, and oxalate was periodically determined by monitoring the ability to oxidize 2-methoxy phenol. Calcium (1 mM), manganese (1 mM), and oxalate (2 mM) were added to cultures on day 6 as indicated by the arrow. Data represent the average and standard deviations of quadruplicate cultures.

Figure 13 A is a graph illustrating the effect of thermal incubation at various temperatures on the activity of LiP over time. LiP (3.0 μM) was incubated in 20 mM sodium acetate buffer, pH 5.7, at 58°C (•), 61 °C (A), 63.5° (T) and 67°C (♦). At given times, aliquots of the enzyme mixture were taken and assayed for the veratryl alcohol oxidase activity. The assay mixture contained 2 mM veratryl alcohol, 0.2 μM LiP H8, 100 μM H ₂ O ₂ in 50 mM sodium acetate buffer, pH 4.5, at 25 °C.

Figure 13B is an Arrhenius plot of the data shown in Figure 13 A where k represents the inactivation rate constant. Figure 14 is a graph illustrating the effect of thermal incubation at various pH values on the activity of LiP over time. Incubation mixtures contained 3 μM LiP H8 in 20 mM sodium acetate buffer at 60°C and pH 4.5 (•), pH 5.5 (■), pH 5.7 (A), and pH 6.0 (T). At the intervals indicated, aliquots were assayed for veratryl alcohol oxidase activity. The reaction mixture for this assay was the same as that described for Figure 13. Figure 15 is a graph illustrating the effect of Ca ²⁺ on the thermal inactivation of

buffer, pH 6.0, were incubated in the water bath at 57°C with no calcium (•), 0.1 mM calcium (■), or 0.2 mM calcium (A). At the given times, aliquots were assayed for veratryl alcohol oxidase activity as described for Figure 13.

Figure 16 is a graph illustrating the effect of Ca ²⁺ chelators on the thermal inactivation of LiP H8. Mixtures containing 3 μM LiP H8 in 20 mM sodium acetate buffer, pH 5.7, were incubated at 58°C , in the presence of no chelator (•), 0.2 mM

EGTA (■), or 0.2 mM oxalate (A). At the desired time intervals, the activity remaining was assayed as described in the legend to Figure 13.

Figure 17 is a graph illustrating the effect of calcium concentration on the recovery of thermally inactivated LiP H8. Inactivated enzyme was prepared by incubating enzyme mixture in 20 mM Tris-HCl buffer, pH 7.9, and 51 °C, until totally inactivated. The inactivated enzyme mixtures were incubated at 25 °C and pH 7.9 to recover enzyme activity in the presence of no calcium (•), 0.1 mM Ca ²⁺ (■), 0.5 mM Ca ²⁺ (A), 1.0 mM Ca ²⁺ (T). and 2.0 mM Ca ²⁺ (♦). At the indicated times, aliquots were taken to measure enzyme activity as described in the legend to Figure 13. The recovered enzyme activity was expressed as percentage of active enzyme.

Figure 18 is a graph illustrating the temperature dependence of recovery of LiP H8 enzyme activity. Inactivated enzyme was prepared with no Ca ²⁺ as described in the legend to Figure 5. The inactivated enzyme mixtures were then incubated at different temperatures for reconstitution in the presence of 1.0 mM Ca ²⁺ at 25 °C (♦), 4 °C (■), and 37°C (A). The recovered enzvme activity was assayed as described in the legend to Figure 13.

Figure 19 is a graph illustrating the effect of inactivation conditions on the reconstitution ofthe thermally inactivated LiP H8. Inactivated enzyme was prepared with no Ca ²⁺ , as described for Figure 17 except some enzyme was also inactivated in the presence of 1 mM EGTA. Ca ²⁺ (2 mM) was added to the enzyme inactivated with EGTA (o) and 1 mM Ca ²⁺ was used for the enzyme inactivated without EGTA (•). Reactivation was then conducted in 20 mM Tris buffer, pH 7.9. At the indicated times, aliquots were taken and assayed for enzyme activity as described for Figure 13. Figure 20 is a graph illustrating the effect of Ca ²⁺ on the mineralization of 4.0 mg of a crosslinked polyacrylate superabsorbent polymer by nutrient-nitrogen limited liquid cultures of P. chrysosporium. The Ca ²⁺ was added on day 6. The cultures received no additions (0) or had CaCl ₂ to a final concentration of 1.0 mM (•) and 2.0 mM (■). All cultures were supplemented with glucose (100 mg/bottle) on days 9, 18, and 27. Figure 21 is a graph illustrating the effect of Ca ²⁺ and Mn ²⁺ on the mineralization

limited cultures of P. chrysosporium. The Ca ²⁺ and Mn ²⁺ was added on day 6 to a final concentration of 0.5 mM (•), 1.0 mM (■) and 2.0 mM (A). To the other set of cultures, no additions (0) were made. All ofthe cultures received additional glucose supplements (100 mg/bottle) on days 9, 18, 27, 36, 45 and 54. Figure 22 is a graph illustrating the effect of calcium and manganese on the depolymerization of a polyacrylamide superabsorbent by Phanerochaete chrysosporium. Liquid cultures of Phanerochaete chrysosporium were grown in the presence of 4.0 mg (0.3 μCi) of polyacrylamide superabsorbent. Calcium and manganese were added to cultures to a final concentration of 1.0 mM on day 3 (•) or day 6 (■). No additional calcium or manganese was added to the third set of cultures (A). An additional glucose supplement (100 mg) was added to all cultures on day 9. Depolymerization was monitored by determining the percent of original radioactivity appearing in the water soluble fraction ofthe cultures at various time points. Radioactivity was quantitated by liquid scintillation spectroscopy. Figure 23 is a graph illustrating the effect of calcium, manganese, and oxalate on pentachlorophenol (PCP) mineralization by Phanerochaete chrysosporium at 37 °C. Cultures of Phanerochaete chrysosporium were prepared and mineralization of PCP in the presence (■) and absence (•) of additional calcium, manganese, and oxalate was monitored. Calcium (1 mM), manganese (1 mM), oxalate (2 mM), and PCP (100 ppm) were added to cultures on day 3 as indicated by the arrow. Mineralization was quantitated by determining the percent of original ^M C-PCP appearing as ^u CO ₂ at the indicated time points. Data represent the average and standard deviations of quintuplicate cultures.

Figure 24 is a graph illustrating the effect of calcium, manganese, and oxalate on pentachlorophenol (PCP) mineralization by Bjerkandera adusta at 32°C. Cultures of Bjerkandera adusta were prepared from a liquid inoculum using standard media and mineralization of PCP in the presence (■) and absence (•) of additional calcium, manganese, and oxalate was monitored. Calcium (1 mM), manganese (1 mM), oxalate (2 mM), and PCP (100 ppm) were added to cultures on day 6 as indicated by the arrow. Mineralization was quantitated by determining the percent of original ^U C-PCP appearing as ^,4 CO ₂ at the indicated time points. Data represent the average and standard deviations of quintuplicate cultures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS White rot fungi are known for their ability to degrade lignin to carbon dioxide. In addition to ugnin, the fungi are known to mineralize a variety of chemicals including such recalcitrant environmental pollutants as CC1 ₄ , DDT, TCDD, Lindane, and PCBs. The

degradation system which the fungus secretes during secondary metabolism. Lignin peroxidase ("LiP"), manganese peroxidase ("MnP"), H ₂ O ₂ , veratryl alcohol, and oxalate are secreted extracellularly under these conditions. LiP and MnP are thought to be the major enzymatic components ofthe lignin degradation system. The LiP isozymes can oxidize a broad range of substrates directly while the manganese peroxidases can only directly oxidize Mn ²⁺ . The kinetics ofthe oxidation of Mn ²⁺ by the MnP enzymes have been well characterized. Enzymatically produced Mn ³⁺ is believed to be essential to the degradation system of white rot fungi due to its role as a diffusible, one-electron oxidant. It is known that the rate ofthe enzyme reactions can be increased by increasing the temperature. Unfortunately, it is also known that some of the critical enzymes are heat-sensitive and loss of activity due to thermal inactivation occurs at higher temperatures. It is also known that some ofthe enzymes are extremely pH-sensitive and loss of activity occurs at higher pH conditions. Due to the importance of the fungal peroxidases to the biodegradation system of white rot fungi, it is crucial to understand the thermal stability properties of these enzymes. In studies with the representative white rot fungus, Phanerochaete chrysosporium, the predominant LiP isozyme was found to be extremely stable to temperatures up to 40°C and in the pH range of 4.0-6.5. The only study known to Applicants which investigated the stability of MnP involved a crude preparation ofthe enzyme which was unstable at 4°C. It will be appreciated that methods for stabilizing the enzymes will enhance the biodegradation reactions.

A. MANGANESE PEROXIDASE STUDIES

The experimental data below demonstrates the loss of activity of a pure form of a manganese peroxidase isozyme under higher temperature and pH conditions. It has been discovered, however, that some metals play an important role in stabilizing the enzyme and that calcium can be utilized to reactivate manganese peroxidase enzyme that has been thermally inactivated. Thus, the addition of cations to a system utilizing white rot fungi or white rot fungi-derived enzymes to biodegrade chemicals will permit activity of MnP enzymes to be maintained under temperature conditions exceeding 37 °C and at pH values exceeding 4.5. Experimental Proςedures

I. Materials and Methods

Chemicals: Hydrogen peroxide, succinic acid and ethyleneglycol-bis-(β- aminoethyl ether) N,N,N',N'-tetraacetic acid ("EGTA") were purchased from Sigma Chemical Co. (St. Louis, MO). Manganese sulfate and calcium sulfate were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sodium oxalate, sodium succinate and

All solutions were prepared using purified water (Barnstead NANOpure II system; specific resistance 18.0 Mohπvcm ^"1 ). All buffers were passed through a column of Chelex 100 (Bio-Rad, Richmond, CA).

Enzyme Production and Purification- Manganese peroxidase isozyme H4 was purified from the extracellular fluid of cultures of Phanerochaete chrysosporium as previously described in Tuisel, H. et al, Arch. Biochem. Biophys, 279:158-66 (1990). For purposes of disclosure, this article is incoφorated by specific reference. The purity and identity ofthe enzyme preparation were confirmed by analytical isoelectric focusing. The identification of the isozyme was further verified by N-terminal amino acid sequencing. The enzyme concentration was quantified by the extinction coefficient, which is 127 mM"

'cm ^'1 at 406 nm.

Thermal Stability Studies: Enzyme incubation mixtures consisted of 0.16 μM MnP and 20 mM sodium succinate, pH 5.5, or 20 mM sodium phosphate buffer of varied pH. The enzyme mixtures were incubated in a water bath ofthe appropriate temperature At given times, aliquots of the incubation mixture were removed, brought to room temperature, and assayed for enzyme activity. The assay reaction mixtures contained 100 mM sodium succinate, pH 4.5, 400 μM Mn ²⁺ , 1.0 mM oxalate, 100 μM H ₂ O ₂ and the incubation mixture aliquot which was diluted 5.2 times such that the final concentration of MnP was 30 nM. The concentration of the sodium succinate buffer was 25 times greater than the final concentration of sodium phosphate buffer in the assay reaction mixture to ensure that the pH remained at 4.5. The activity of MnP was determined by measuring the formation ofthe Mn ³⁺ -oxalate complex which absorbs at 270 nm and has an extinction coefficient of 5500 M ^' 'cm ^'1 . The turnover number for MnP, prior to any inactivation, was 143 s ^"1 . The amount of MnP which contained heme was determined spectrophotometrically at 406 nm. The wavelength of maximum absoφtion did not change as the enzyme was inactivated. In the reactivation studies, the enzyme mixture (0.16 μM MnP H4, 20 mM Tris buffer) was incubated in a water bath for the indicated amount of time to inactivate the enzyme, the activity was assayed, and a concentrated CaSO ₄ solution was added to the enzyme mixture. The activity ofthe enzyme was then assayed immediately, correcting for dilution, and the mixture was placed in a constant temperature water bath at 37°C. Aliquots of the mixture were then removed at the appropriate times and assayed. The presence of CaSO ₄ and EGTA in the incubation mixtures, at the concentrations used in these experiments, had no effect on the activity assays for MnP. All data points represent the average and standard deviation of assays from at least three samples.

Determination of Calcium Concentration in MnP The amount of calcium in the MnP samples was determined using Inductively Coupled Plasma (ICP) Emission spectroscopy by the Utah State University Analytical Laboratory using a Thermo Jarrell Ash ICAP-9000 (Franklin, MA) The detection limit for calcium ion was 0 15 mg/L The samples which were analyzed had calcium concentrations greater than ten times the detection limit The samples of inactivated enzyme were prepared by incubating MnP with 5 mM EGTA at pH 7 1, 37° C, for 10 minutes The remaining activity ofthe enzyme was less than 5% The EGTA was then removed from the enzyme through serial concentration with a Centricon-10 concentrator (Amicon, Ine , Beverly, MA) Control samples which contained no MnP contained no detectable calcium

II. Results

The inactivation of MnP over time due to the incubation ofthe enzyme at pH 5.5 and various temperatures is shown in Figure 1 A The remaining activity of MnP is plotted on a log scale shown in Figure IB. The plot was biphasic, indicative of a two-step process, with a fast initial phase and a slower second phase At this pH, MnP was relatively stable at 37 °C but, upon exposure to higher temperatures, the enzyme was rapidly inactivated At 46 5° C, the half-life for the inactivation of MnP was 3 minutes

Rate constants for the inactivation of MnP at each temperature were determined from the data in Figure 1 A for the first phase ofthe plot, which accounted for most ofthe inactivation The rate constants were then plotted on a log scale versus the inverse of temperature (Arrhenius plot) as shown in Figure IB A linear relationship was observed over this temperature range and the slope of this plot was utilized to determine the activation energy for this step of the inactivation process The activation energy was calculated to be 275 kJ/mol which is within the range observed for the inactivation of most proteins.

The effect of incubating MnP at 37 °C, and various pH values, on enzyme activity is shown in Figure 2 Due to the range of pH in this experiment, sodium phosphate buffer was used The use of phosphate, however, did not affect the thermal inactivation of MnP The inactivation of MnP at various temperatures was also characterized in sodium phosphate and the activation energy was determined to be 280 kJ/mol The remaining activity was plotted on a log scale and, once again, biphasic kinetics were observed for the inactivation process As the pH of the incubation mixture increased, the rate of inactivation increased dramatically At pH 7 1, the enzyme was almost completely inactivated within 5 minutes The enzyme was less susceptible to thermal inactivation at greater concentrations of MnP (data not shown)

It is known that the heme is incoφorated into the folded structure of the MnP enzyme and that heme is readily lost in the presence of urea which disrupts the noncovalent interaction in MnP. Therefore, the absorbance due to heme could be monitored as an indicator of unfolding ofthe MnP enzyme. The effect of temperature on the activity of MnP and on the Soret absoφtion band of the heme in MnP at 406 nm were simultaneously monitored. It was thus possible to correlate the loss of activity with the loss of overall protein structure of MnP. The effect of incubating MnP at 37 °C and pH of 6.5 on enzyme activity and the absorbance of heme is shown in Figure 3. There was a decrease in the heme absorbance of MnP over time, however, the loss of enzyme activity occurred at a much greater rate during the first phase ofthe plot. This indicated that the first phase of inactivation of MnP was not due to heme loss but rather to a small change in the enzyme which specifically affected the activity of MnP. As indicated in Figure 3, however, the rate ofthe second, slower phase of enzyme inactivation corresponded to the rate of heme loss from MnP. The effect of Ca ²⁺ and EGTA, an efficient chelator of Ca ²⁺ , on the thermal inactivation of MnP is shown in Figure 4. In the presence of only 25 μM Ca ² \ the inactivation of MnP was almost completely inhibited. In contrast, EGTA increased the rate of MnP inactivation. As shown in Figure 4, in the presence of 100 μM Ca ²⁺ , MnP retained greater than 90% of its activity and heme absorbance following incubation at 37°C , pH 6.5, for 80 minutes. Therefore, the addition of calcium ions prevented the loss of heme from MnP and prevented enzyme inactivation.

Atomic absoφtion spectroscopy was utilized to determine the amount of Ca ²⁺ present in the active, untreated MnP and in the heat-inactivated MnP. As shown in Table I, below, Ca ²⁺ is indeed lost during the inactivation process. There were 4 mol Ca ² 7mol MnP as it was isolated but only 1 mol Ca ² 7mol MnP following thermal inactivation. To ensure that the initial concentration of Ca ²⁺ was representative of intrinsic Ca ²⁺ and not loosely bound exogenous Ca ²⁺ , the MnP sample was washed with EGTA at 4°C. This procedure did not change the concentration of Ca ²⁺ in, or the activity of, the MnP.

TABLE J

Effect of Thermal Inactivation on the Amount of Ca ²⁺ Present in MnP

* Concentrations of Ca ²⁺ determined by ICP Emission spectroscopy. The values represent the average and standard deviation of at least three samples. b The MnP sample was inactivated as described in Materials and Methods.

The activity of thermally inactivated MnP could be recovered upon the addition of Ca ²⁺ . The enzyme mixtures were incubated for 2.5 minutes at 37°C and a pH of 7.1 prior to the addition of Ca ²⁺ . The activity ofthe MnP samples were recovered upon the addition of Ca ²⁺ . As shown in Figure 5, increasing the concentration of Ca ²⁺ up to a final concentration of 1.0 mM, increased the amount of enzyme activity recovered. The reactivation process was time-dependent and the maximum amount of activity was recovered when the incubation mixture, with added Ca ²⁺ , was thermally incubated. In addition, the reactivated enzyme was stable to dialysis.

As shown in Figure 6, the amount of MnP activity which could be recovered decreased as the time of thermal incubation increased. As shown in Figure 7, the amount of activity recovered at each time point, following the addition of Ca ²⁺ , was correlated to the amount of enzyme remaining which contained heme. The addition of hemin, in addition to Ca ²⁺ , did not increase the amount of activity recovered from thermally inactivated MnP (data not shown). It has further been demonstrated that, in addition to calcium, other divalent metal cations also inhibited thermal inactivation of MnP. As shown in Figure 8, enzyme mixtures incubated at 32°C containing 0.3 μM MnP, 20 mM sodium phosphate, pH 6.8, and 100 μM of either CaSO ₄ , MnSO ₄ , ZnSO ₄ , or MgSO ₄ each demonstrated slower rates of MnP inactivation than enzyme mixtures having no added cation. Figure 9 illustrates the effect of calcium on the thermal inactivation of MnP by crosslinked polyacrylamide superabsorbent polymer and by crosslinked polyacrylate superabsorbent polymer. Minimal MnP inactivation occurred in mixtures containing either polymer and added calcium while more MnP was inactivated in the presence of crosslinked polyacrylamide or polyacrylate superabsorbent polymers in the absence of added calcium than even in the control mixture which contained neither polymer or calcium. In other experiments, the addition of calcium, manganese, and oxalate has been shown to increase manganese- dependent peroxidase activity in cultures of P. chrysosporium at 37°C (Figure 10) and at 32 °C (Figure 11) as well as in cultures of B. adusta at 32 °C (Figure 12). B. LIGNIN PEROXIDASE STUDIES The experimental data below demonstrates the loss of activity of a pure form of a lignin peroxidase ("LiP") isozyme under higher temperature and pH conditions. It has

and that calcium can be utilized to reactivate LiP enzyme that has been thermally inactivated.

Experimental Procedures I. Materials and Methods Chemicals: Hydrogen peroxide, succinic acid and ethyleneglycol-bis-(β- aminoethyl ether) N,N,N',N'-tetraacetic acid ("EGTA") were purchased from Sigma Chemical Co. (St. Louis, MO). Veratryl alcohol, calcium chloride and sodium acetate were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sodium oxalate, acetic acid, tris(hydroxymethyl)aminomethane (Tris), and hydrochloric acid were purchased from Mallinckrodt (Paris, KY). All buffers were prepared using purified water (Barnstead

NANOpure II system; specific resistance 18.0 Mohπvcm ^"1 ) and passed through a column of Chelex 100 (Bio-Rad, Richmond, CA).

Enzvme Production and Purification: Lignin peroxidase was produced and purified from the extracellular fluid of cultures of Phanerochaete chrysosporium as previously described in Tuisel, H. et al, Arch. Biochem. Biophys, 279: 158-66 (1990). For puφoses of disclosure, this article is incoφorated by specific reference. Lignin peroxidase isozyme H8 was further identified by analytical isoelectric focusing. The enzyme concentration was quantified by the extinction coefficient, which is 169 mM ^'1 cm ^'1 at 408 nm. Thermal Stability Studies: Enzyme incubation mixtures consisted of 3.0 μM LiP and 20 mM sodium acetate buffer, pH 5.7 placed in a plastic vial (to avoid the presence of boron in the enzyme which occurs when enzyme is incubated in borosilicate glass). The enzyme mixtures were incubated in a water bath ofthe appropriate temperature. At given time intervals, aliquots of the incubation mixture were removed, brought to room temperature, and assayed for the veratryl alcohol oxidase activity. The assay reaction mixtures contained 50 mM sodium acetate, pH 4.5, 2 mM veratryl alcohol, 100 μM H ₂ O ₂ , and an enzyme concentration of 0.2 μM. The final buffer concentration of the assay mixture (50 mM) was 37 times greater than the diluted buffer concentration of the incubation mixture to make sure that the final pH was 4.5 The reaction was initiated by adding H ₂ O ₂ and veratryl aldehyde formation was monitored at 310 nm using an extinction coefficient of 9300 M ^" 'cm ^'1 . The activity remaining was expressed as the percentage of the activity remaining at the indicated time. In the incubation studies involving EGTA and oxalate, the percentage ofthe activity remaining was compared to the activity of enzyme in the presence of EGTA and oxalate before inactivation. Reactivation studies: A 3.0 μM enzyme in 20 mM Tris-HCl buffer was incubated

remaining was assayed immediately after inactivation. Upon addition of Ca ²⁺ , the inactivated enzyme was studied under various conditions to determine reactivation of activity. The recovered enzyme activity was assayed as described above.

Determination of Calcium Concentration in LiP: The amount of calcium in the LiP was determined using Inductively Coupled Plasma (ICP) Emission spectroscopy by the

Utah State University Analytical Laboratory using a Thermo Jarrell Ash ICAP-9000 (Franklin, MA). The detection limit for calcium ion was 0.15 mg/L. The samples which were analyzed had calcium concentrations greater than ten times the detection limit. The samples of inactivated enzyme and reconstituted enzymes were washed with EGTA to remove free Ca ²⁺ by serial concentrations with a Centricon-10 concentrator (Amicon, Inc.,

Beverly, MA). Control samples which contained no LiP contained no detectable calcium. II. Results

The temperature-dependent inactivation of LiP H8 over time due to the incubation ofthe enzyme at pH 5.7 and various temperatures is shown in Figure 13 A. The remaining activity of LiP is plotted on a log scale shown in Figure 13B. The plot is a straight line, indicating a first-order inactivation mechanism. At pH 5.7, the enzyme was quite stable at 58 °C, as shown in Figure 13 A. The rate of enzyme inactivation was dramatically increased as the incubation temperature increased. At 67°C, the half-life for inactivation of LiP H8 was approximately 5 minutes. The thermal stability of LiP H8 could be improved by increasing enzyme concentration (data not shown). The rate constants for the inactivation of LiP H8 at each temperature were plotted on a log scale versus the inverse of temperature (Arrhenius plot) as shown in Figure 13B. The activation energy was calculated from this plot to be 467 kJ/mol.

The effect of pH on the thermal inactivation of LiP H8 is shown in Figure 14 where the remaining activity is plotted on a log scale. The plot is again indicative of a linear, first-order inactivation mechanism. The enzyme lost activity rapidly when the pH ofthe incubation mixture was increased. At pH 6.0, the enzyme was almost completely inactivated within 30 minutes.

Calcium prevented the thermal inactivation of LiP H8 and the protection was improved by increasing Ca ²⁺ concentration as shown in Figure 15. Addition of Mg ²⁺ ,

Mn ²⁺ , Co ²⁺ did not significantly prevent thermal inactivation of LiP H8. Interestingly, Zn ²⁺ , Cd ²⁺ , Tb ^3t , Ho ³⁺ increased the loss of enzyme activity (data not shown).

Thermal inactivation of enzyme was accelerated by the Ca ²⁺ -chelators, EGTA, and oxalate as shown in Figure 16. The rate of enzyme inactivation was higher in the presence of oxalate than in the presence of EGTA. This may be explained by the fact that oxalate

The effect of Ca ²⁺ on the recovery of thermally inactivated LiP H8 is shown in Figure 17. The extent ofthe recovered activity increased as both the Ca ^2* concentration and incubation time increased. The recovered enzyme was quite stable and did not lose activity following incubation for 3 days at 25 °C. Moreover, the enzyme did not lose activity after it was washed with EGTA at 4°C indicating that the Ca ²⁺ bound to the protein was intrinsic.

The most favorable temperature for enzyme reactivation was 25 °C as shown in Figure 18. Reactivation at 4°C was much slower than that at 25 °C. This may be due to low rate of protein motion and Ca ²⁺ association resulting in a lower rate of refolding. As the incubation temperature increases, the rate of Ca ²⁺ association may also increase. As the temperature increases further, however, the rate of Ca ²⁺ disassociation from the protein may also increase resulting in unfolding and inactivation ofthe enzyme The net increase of Ca ²⁺ association at 37°C was less than that at 25 °C thus resulting in a lower rate and lesser extent of reactivation compared to reconstitution at 25 °C. The enzyme incubated at 37°C also lost some activity after reaching a maximum activity. This may be because the rate of calcium ion disassociation overwhelmed the rate of association after equilibrium was reached.

The amount of activity which could be recovered in the presence of Ca ²⁺ was dependent on the conditions utilized for the thermal inactivation of LiP H8. As shown in Figure 19, reconstitution could reach up to 95% or original level when the inactivation was in the presence of EGTA suggesting that the thermal inactivation was due to the loss of calcium. To eliminate the possibility that thermal denaturing of the protein was contributing to the enzyme inactivation, the completely inactivated enzyme was further incubated at 51 °C for 40 min. in the presence of EGTA. In this case, enzyme activity still could be recovered up to 95% of original activity in the presence of extra Ca ²⁺ (data not shown) which further supports that thermal enzyme inactivation is the result of loss of calcium ions.

As seen in Table II, calcium (5 mol) was found in native LiP H8. To ensure that all initial Ca ²⁺ was intrinsic and not loosely bound exogenous Ca ²⁺ , the enzyme was washed with EGTA at 4°C This treatment neither changed the amount of Ca ²⁺ in the enzyme nor enzyme activity. The Ca ²⁺ concentration decreased to 1 mol Ca ² 7mol LiP as the enzyme was completely inactivated The amount of Ca ²⁺ in the enzyme increased to about 3 mol as the enzyme activity recovered to 64% of the original level when the enzyme was inactivated in the absence of EGTA, and to about 4 mol Ca ² 7mol LiP as the enzyme activity was recovered to 92% of its original level when the enzyme was thermally

TABLE II

Calcium Contents of Native, Inactive, and Recovered LiP

* The samples of native and inactivated LiP were prepared, including washing with EGTA, as described under Materials and Methods. For the recovered LiP-1, the enzyme was inactivated in the absence of EGTA and was reconstituted by adding 1 mM Ca ²⁺ at 25 °C for 3 hours. For the recovered LiP-2, the enzyme was inactivated in the presence of ImM EGTA and was reconstituted by adding 2 mM Ca ²⁺ at 25 °C for 20 hours.

^b Calcium concentration was determined by ICP emission spectroscopy. All data are the average and standard deviation of at least three analyses.

The heme absorbance decreased as the enzyme was inactivated (data not shown).

Additionally, the position of Soret band was red shifted approximately 2 nm in the inactivated enzyme. Changes in the a, β, and charge transfer band regions were observed (data not shown). The absoφtion band at 634 nm totally disappeared and the absoφtion at 502 nm in the native enzyme red shifted to 532 nm in the inactivated form. Upon reactivation in the presence of Ca ²⁺ , the Soret band returned to the original wavelength ofthe native enzyme. The absoφtion at 634 nm also reappeared. The data from the ICP emission spectroscopy indicated that iron concentration did not change during thermal inactivation and reactivation procedures. C. DISCUSSION Thermal stability studies demonstrated that MnP was extremely susceptible to inactivation upon exposure to temperatures of 45 °C or greater, or to pH 6.5 or greater. The kinetics of inactivation of MnP were biphasic suggesting a two-step process. The activation energy was determined to be 275 kJ/mol for the inactivation of MnP during the first phase (280 kJ/mol in phosphate buffer). This value is within the range, 105-544 kJ/mol, observed for the inactivation of most proteins.

The loss of heme from MnP was utilized as an indicator of overall protein

from MnP corresponded to the rate of inactivation during the second phase ofthe plot and was much less than the rate during the first phase. This indicated that the thermal inactivation during the first phase, which accounted for most ofthe inactivation, was due to a relatively minor change in the enzyme which directly affected MnP activity. Thus, the active site of MnP may be much more susceptible to the detrimental effects of heat than the overall protein structure.

The results described above demonstrated that the thermal inactivation of MnP was almost completely inhibited in the presence of Ca ²⁺ . Moreover, the addition of EGTA, a chelator of Ca ²⁺ which would enhance removal of Ca ²⁺ from MnP, caused acceleration ofthe thermal inactivation ofthe enzyme. Thermally inactivated MnP was shown to contain less Ca ²⁺ than the active MnP. The results of ICP analysis indicated that active MnP contained 4 mol Ca ² 7mol MnP and that, during inactivation, 3 mol of Ca ² 7mol of MnP were lost. These results suggest that there is Ca ²⁺ in MnP which is relatively labile and is required for enzyme activity. It was also demonstrated that loss of heme from the enzyme was prevented in the presence of higher concentrations of Ca ²⁺ .

It appears that the thermal inactivation of MnP involves two steps. First, Ca ²⁺ is lost from the enzyme which renders MnP catalytically inactive. The second step involves further structural loss, which occurs at a slower rate and results in the loss of heme from

MnP. Thermally inactivated MnP was reactivated upon the addition of Ca ²⁺ to the enzyme incubation mixture. In addition, the reactivated enzyme was stable to dialysis indicating that the Ca ²⁺ was intrinsic to the enzyme. This further supported the proposal that the first step was due to loss of Ca ²⁺ and that this process could be reversed. As the time of incubation prior to the addition of Ca ²⁺ increased, the amount of recovered activity decreased. The reactivation studies also revealed that the amount of activity which could be recovered upon the addition of Ca ²⁺ was correlated with the amount of MnP which still contained heme. Attempts to add heme to the enzyme were not successful.

The LiP studies showed that Ca ²⁺ also protected against the loss of enzyme activity and increased the thermal stability of LiP H8. It was found that LiP H8 was quite stable below 58 °C and at the physiological pH range ofthe fungus (pH 4.5). The activation energy was determined to be 467 kJ/mol for the inactivation of LiP H8 which is within the range, 105-544 kJ/mol, observed for the inactivation of most proteins.

In comparison to MnP, LiP was much more stable. LiP H8 could maintain 84% activity for up to 150 min at pH 5.7 and 58 °C, but the activity of MnP was nearly totally lost in 15 min at pH 5.5 and 47.5 °C. The activation energy of LiP H8 was higher than that of MnP. In addition, the inactivation kinetics of LiP H8 was first-order and the

cations could neither protect nor reactivate LiP H8. It may be that the Ca ²⁺ binding sites in LiP are different from those in MnP.

Addition of calcium to the active LiP H8 did not increase enzyme activity significantly. The presence of Ca ²⁺ , however, slowed the loss of enzyme activity while the addition of Ca ²⁺ chelators increased the inactivation rate. The active enzyme had 5 mol

Ca ² 7mol LiP but only 1 mol Ca ² 7mol LiP existed in the inactivated enzyme.

Both the rate and extent of recovery of enzyme activity upon the addition of calcium depended on the thermal inactivation and reconstitution conditions. Enzyme activity could be almost completely recovered in the presence of excess Ca ²⁺ when the enzyme was thermally incubated in the presence of EGTA. The extent of recovered activity was higher when LiP H8 was inactivated with EGTA than when inactivated without EGTA, indicating that thermal inactivation was related to the loss of Ca ²⁺ .

In addition to the amount of recovered enzyme activity, there is another difference in the rate of recovering thermally inactivated LiP and MnP. It took a few minutes for MnP to recover its maximum activity, but it took 4 hours for LiP to restore its maximum activity in the presence ofthe same amount of Ca ²⁺ at 37 °C. In the case of MnP, two- step, biphasic kinetics were observed for the loss of activity. The first, fast step appears to be related to the loss of Ca ²⁺ from the enzyme. The second, slow step appears to be due to the loss of protein structure and heme. It was found that Ca ²⁺ could reactivate the MnP activity only in the first step. In contrast, inactivation of LiP H8 was first-order and the loss of activity and heme absorbance were simultaneous (data not shown). When Ca ²⁺ was utilized to reactivate the thermally inactivated LiP, both the activity and heme absorbance were recovered.

From the foregoing, it should be understood that the addition of Ca ²⁺ ions to a biodegradation or bioremediation system utilizing white rot fungi or white rot fungi- derived enzymes, including recombinant DNA produced enzymes, will stabilize the peroxidase enzymes to higher temperatures and pH conditions to thereby permit activity of the peroxidases enzymes to be maintained under the higher temperature conditions which are known to enhance the overall rate and extent of biodegradation. As shown in Figure 20, the addition of calcium enhanced the mineralization of a crosslinked polyacrylate superabsorbent polymer in nutrient-nitrogen limited liquid cultures of Phanerochaete chrysosporium. Figure 21 illustrates the enhancement when both calcium and manganese are added. As shown in Figure 22, the addition of calcium and manganese enhanced the rate of biodegradation of a high molecular weight, highly cross-linked, superabsorbing polyacrylamide in a liquid culture of Phanerochaete chrysosporium. In

biodegradation of PCP by two white rot fungi, Phanerochaete chrysosporium, as shown in Figure 23, and Bjerkandera adusta, as shown in Figure 24.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency ofthe claims are to be embraced within their scope.

What is claimed is: