INTRODUCTION

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Lignin, a complex and heterogeneous aromatic biopolymer in woody and herbaceous plants, is one of the most abundant natural polymers on earth. White rot fungi are primarily responsible for initiating the depolymerization of lignin in wood (4, 7, 13). The extracellular lignolytic enzyme system of white rot fungi has been studied extensively in recent years. Lignin peroxidase, manganese peroxidase (MnP), and laccase are associated with the degradation of lignin. Several attempts to bleach hardwood kraft pulp by means of enzyme treatment have been reported. Arbeloa et al. (1) showed that treatment of unbleached kraft pulp with lignin peroxidase facilitated subsequent chemical bleaching. Bourbonnais and Paice (3) demonstrated that unbleached kraft pulp could be delignified with a laccase from Trametes versicolor in the presence of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate).

MnP is a heme-containing enzyme which was first isolated from the extracellular medium of lignolytic cultures of the white rot fungus Phanerochaete chrysosporium, and it is considered to be a key enzyme in lignolysis by white rot fungi. MnP requires H₂O₂ as a cosubstrate and catalyzes the oxidation of Mn²⁺ to Mn³⁺. Mn³⁺ complexed with an organic acid acts as a primary agent in lignolysis. Kondo et al. (16) reported that MnP could degrade residual lignin in kraft pulp. However, MnP is labile compared to other peroxidases, e.g., horseradish peroxidase, and its stability at elevated temperatures and H₂O₂ levels must be increased before it can be used for an application such as pulp bleaching.

The immobilization of a protein on a solid support can overcome these disadvantages (17, 21). In recent years, many support matrices and coupling chemistries have been developed and made commercially available for use for protein immobilization. The biochemical characteristics of MnP, i.e., a low lysine content and alkali lability, are not ideal for immobilization on a commercially available support.

Periodic mesoporous materials with uniform pore diameters of 10 to 300 Å have been synthesized (2, 12, 22). Because the pore diameters of these materials approximate those of enzyme molecules, their application as enzyme supports has been suggested. Members of our group previously reported (19) that horseradish peroxidase immobilized in mesoporous materials with suitable mesopore sizes had the best thermal stability and highest peak activity in an organic solvent.

Here, we report that MnP was successfully stabilized in a mesoporous material (FSM-16). When the mesopore size of FSM-16 was nearly the same as the diameter of the enzyme, the immobilized MnP had high stability. We suggest that a thermally discontinuous biobleaching system involving MnP immobilized in FSM-16 might be an important component in a new total chlorine-free (TCF) pulp-bleaching system.

	MATERIALS AND METHODS

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Fungal strains and culture conditions. Phanerochaete chrysosporium SC-26 (ATCC 64964) (14), a mutant of P. chrysosporium BKMF-1767, was used in all studies. P. chrysosporium was cultivated by a modification of the cultivation method of Gold et al. (8). Stationary cultures in 500-ml Erlenmeyer flasks containing 100 ml of medium (8) were inoculated with conidia and then incubated at 37°C for 3 days. The cultures were homogenized in a Waring blender for 20 s and then used to inoculate 2-liter flasks containing 1 liter of medium (20 g of glucose, 0.22 g of ammonium tartrate, 2 g of KH₂PO₄, 0.5 g of MgSO₄ · 7H₂O, 0.1 g of CaCl₂ · H₂O, 1.2 g of acetic acid, 0.4 g of NaOH, 11.7 ml of 6× trace elements [8], and 1 g of Tween 80). Flasks were incubated at 39°C with shaking at 120 rpm. Veratryl alcohol was added to the medium to a 3 mM concentration after 3 days of growth. From day 3, the flasks were purged with oxygen every day. The cultures were harvested after 6 days of growth.

MnP purification. Cultures were filtered through glass wool. Polyethylene glycol 4000 (average molecular weight, 3,000; Wako Pure Chem. Ind., Ltd., Osaka, Japan) was added to the resultant filtrate to give a 5% solution, and the pH was adjusted to 7.2 with NaOH. After the slime was filtered off, the filtrate was loaded onto a DEAE-Sepharose FF (Amersham-Pharmacia Biotech UK, Buckinghamshire, United Kingdom) column equilibrated with 10 mM sodium phosphate buffer (pH 7.2). The column was eluted sequentially with 20 mM phosphate buffer (pH 6.0), 20 mM succinate buffer (pH 5.5), and 50 mM succinate buffer (pH 4.5). The MnP fraction was eluted with 50 mM sodium succinate buffer (pH 4.5). After dialysis against 50 mM sodium succinate buffer (pH 4.0), the active fraction was loaded onto a Hiload 26/10 S Sepharose high-performance column (Amersham-Pharmacia Biotech UK) equilibrated with the same buffer. Proteins were eluted with a NaCl gradient (0 to 0.4 M). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and the preparation was stained with Coomassie blue. The purified MnP gave a single band on a sodium dodecyl sulfate-polyacrylamide gel and exhibited an RZ (A₄₀₅/A₂₈₀) value of 1.85.

MnP assays. The level of MnP activity was determined by monitoring the formation of the Mn³⁺-oxalate complex at 270 nm at 37°C. The reaction mixtures comprised 2 mM sodium oxalate, 0.5 mM MnSO₄, and 0.1 mM H₂O₂ in 25 mM sodium succinate buffer (pH 4.5). Assays were initiated by the addition of H₂O₂. One unit of MnP was defined as the amount of enzyme producing 1 µmol of the Mn³⁺-oxalate complex per min.

FSM-16 preparation. FSM-16 material with a pore diameter of 31 Å was prepared from kanemite using hexadecyltrimethylammonium chloride, as described by Inagaki et al. (12). FSM-16 materials with pore diameters of 69 and 89 Å were prepared from kanemite using hexadecyltrimethylammonium chloride and 1,3,5-tri-isopropylbenzene (TIPB) in the molar ratio of TIPB/surfactant = 3 and docosyltrimethyl ammonium chloride and TIPB in the molar ratio of TIPB/surfactant = 4, respectively. The other conditions were the same as for the synthesis of FSM-16 with the pore diameter of 31 Å (12).

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Immobilized MnP preparation. MnP was immobilized by adding 40 mg of FSM-16/d or silica gel 5D powder to an MnP solution (11 µmol ml¹) containing 1 mM CaCl₂ and 1 mM MnSO₄. The mixture was incubated at 12 rpm at 4°C for 16 h and then centrifuged at 15,000 × g for 10 min at 4°C, and the resultant precipitate was washed with deionized water and then stored at 4°C under dark conditions.

Thermal stability. A half-milligram of MnP immobilized in a support was suspended in 500 µl of 50 mM Na-succinate buffer (pH 4.5). The suspension was incubated at 60°C for 15, 30, or 60 min. After centrifugal separation for 5 min at 4°C, the precipitate was washed with deionized water, and then 500 µl of the MnP assay buffer (pH 4.5) was added to the precipitate, followed by vigorous mixing for 5 min at 37°C. After centrifugation at 20,000 × g for 5 min at 4°C, an aliquot of the supernatant was removed and the amount of Mn³⁺-oxalate complex was measured as the absorbance at 270 nm. The thermal stability of free MnP was determined as follows. Each MnP solution (2.5 µl) was added to 50 µl of 50 mM succinate buffer (pH 4.5), followed by incubation at 60°C for 15, 30, or 60 min. Forty microliters of the treated solution was used for the MnP assay. All data are the mean values for at least four samples.

H₂O₂ dependency. A half-milligram of MnP immobilized in a support was suspended in 500 µl of the MnP assay buffer (pH 4.5) containing H₂O₂. The suspensions were mixed vigorously at 37°C for 5 min. After centrifugation at 4°C at 20,000 × g for 5 min, the supernatant was immediately subjected to measurement of the A₂₇₀. The H₂O₂ dependency of free MnP was determined by adding 1.25 µl of the MnP solution to 500 µl of the MnP assay buffer containing H₂O₂. All data are the mean values for at least four samples.

Operational stability of MnP immobilized in FSM-16. Five milligrams of MnP immobilized in FSM-16/70 (FSM/70-MnP) was packed into a column (0.5 by 7 cm). The reaction buffer containing 30 mM sodium malonate buffer (pH 4.5), 10 mM MnSO₄, 0.05% Tween 80, and 1 mM H₂O₂ was continuously loaded onto the column at 1.5 ml/min at 40°C. FSM-16/70 MnP activity was measured by monitoring the generation of the Mn³⁺-malonate complex at 270 nm. All data are the mean values for at least three samples.

Mn³⁺-malonate complex reactivity. One milligram of FSM/70-MnP was suspended in 1 ml of MnP assay buffer (pH 4.5) comprising 50 mM sodium malonate buffer (pH 4.5), 0.5 mM MnSO₄, and 0.1 mM H₂O₂. The suspension was vigorously mixed at 37°C for 5 min. After centrifugation (18,000 × g) for 5 minutes at 4°C, the supernatant was removed and incubated at 5, 37, or 60°C, and then the A₂₇₀ was measured. The data are the mean values for at least four samples.

Enzyme bleaching in a two-stage reactor system. Eighteen milligrams of FSM/70-MnP was packed into a column, and 0.5 g (as dry weight) of unbleached hardwood kraft pulp (brightness, 59%; kappa number, 17) was placed in a bleaching vessel (50 ml). Pulp bleaching was performed by continuously feeding 30 mM sodium malonate buffer (pH 4.5) containing 10 mM MnSO₄, 0.1 mM H₂O₂, and 0.05% Tween 80 into the immobilized MnP column at the flow rate of 6 ml/min. The reactant from the column was allowed to flow into the bleaching vessel with stirring at 150 rpm. The enzyme column and the bleaching vessel were both maintained at 39 or 70°C. A part of the pulp solution was removed from the bleaching vessel, and a pulp sheet was prepared in order to determine its brightness. MnP activity was measured by monitoring the amount of the Mn³⁺-malonate complex at 270 nm at the outlet of the enzyme column. All data are the mean values for at least two samples.

Multiple pulp bleaching through enzyme treatment and alkaline extraction. Enzymatic treatment in a two-stage reactor system was performed as described above. The enzyme-treated pulp was washed with deionized water twice and then suspended in a 2.5% NaOH solution for alkaline extraction. This slurry was incubated at 70°C for 5 min and then washed with deionized water to remove alkaline components. The enzyme treatment (55 min) and alkaline extraction (5 min) were repeated alternately for seven cycles. A part of the pulp solution was removed from the bleaching vessel at the indicated times, and a pulp sheet was prepared to determine its brightness. All data are the mean values for at least four samples.

Analysis of pulp properties. Pulp sheets were prepared with a Buchner funnel to determine their brightness (15). Pulp brightness was determined with a colorimeter (model CR-14; Minolta, Tokyo, Japan). Handsheets for strength measurement were prepared according to ISO standard method ISO 5269-1. The tearing resistance, tensile properties, and bursting strength were determined by the ISO 1974, ISO 1924-2, and ISO 2758 methods, respectively.

	RESULTS

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Thermal stability and H₂O₂ dependency of immobilized MnP. Free MnP (i.e., without immobilization) was completely inactivated within 15 min at 60°C, as was MnP immobilized in silica gel (average mesopore size, 50 to 300 Å) (Fig. 1). The thermal stability of MnP immobilized in FSM-16/30 and FSM-16/90 was slightly higher than that of free MnP, but these enzymes were almost completely inactivated within 30 min at 60°C. MnP immobilized in FSM-16/70 (FSM/70-MnP) had the highest stability, retaining about 50% of its initial activity after 30 min at 60°C and about 35% even after 60 min.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Thermal stability of immobilized MnP in various supports. , soluble MnP; , FSM-16/30; , FSM-16/70; , FSM-16/90; , silica gel 5D. All data are the mean values for at least four samples. The error associated with each point without an error bar is less than 10% of the value of the point.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   H2O2 dependency of immobilized MnP in various supports. , soluble MnP; , FSM-16/70; , silica gel 5D. All data are the mean values for at least four samples. The error associated with each point without an error bar is less than 10% of the value of the point.

Operational stability. The buffer solution containing 0.1 mM H₂O₂ was allowed to flow continuously, and FSM/70-MnP retained more than 80% of its initial activity even after 10 days of continuous reaction.

Mn³⁺-malonate complex reactivity. At 5°C, the Mn³⁺ complex was very stable, and almost all of the initial amount remained even after 60 min (Fig. 3). At 37°C, about 30% of the initial amount remained after 60 min and the enzyme reaction proceeded normally. At 60°C, the amount of the Mn³⁺-malonate complex decreased to less than 10% of the initial amount after 15 min, suggesting that the complex stability decreased as the temperature increased.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Residual activity of the Mn3+-chelate complex after incubation at 5°C (), 37°C (), and 60°C (). All data are the mean values for at least four samples. The error associated with each point without an error bar is less than 10% of the value of the point.

The two-stage reactor system. We designed a two-stage reactor system (Grabski et al. [9] also proposed such a system) (Fig. 4), in which the Mn³⁺ generation step on an enzyme column and the pulp-bleaching step were separated. In the first stage, the substrate solution comprising Mn²⁺, hydrogen peroxide, and an organic acid (chelating agent) was introduced into an enzyme column packed with FSM/70-MnP to generate the Mn³⁺-chelate complex. In the next stage, the Mn³⁺-chelate complex generated by the immobilized MnP was transferred to a bleaching vessel containing unbleached treated kraft pulp.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Schematic diagram of the TSRS involving MnP immobilized in FSM-16/70.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Thermal effects on pulp bleaching in the TSRS. (a) Amount of the Mn3+-chelate complex in the eluent from the enzyme column. This value reflected enzymatic activity. (b) Brightness of the pulp. The MnP reaction and the pulp-bleaching reaction were performed at 39°C (, ); the MnP reaction and the pulp-bleaching reaction were performed at 70°C (, ); the MnP reaction and the pulp-bleaching reaction were performed at 39 and 70°C, respectively (, ). All data are the mean values for at least two samples. The error associated with each point without an error bar is less than 10% of the value of the point.

Multiple pulp bleaching with MnP. Treatment with MnP by means of the two-stage reactor system was repeated in combination with alkaline extraction. The pulp brightness increased to about 88% when the enzyme treatment was repeated seven times (Fig. 6). When the pulp was treated on the column without MnP, the brightness increased to only about 71%.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Multiple bleaching of kraft pulp with MnP combined with alkaline extraction. The brightness of pulp treated with FSM/70-MnP (), brightness of pulp without FSM/70-MnP (), amount of the Mn3+-chelate complex with FSM-70/MnP (), and amount of the Mn3+ chelate complex without FSM-70/MnP () are shown. All data are the mean values for at least four samples. The error associated with each point without an error bar is less than 10% of the value of the point.

	DISCUSSION

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Grabski et al. (10) reported that MnP was sufficiently immobilized on the NH₂-Emphaze polymer (Pierce, Rockford, Ill.) and that the stability of MnP immobilized on the polymer was greater than that of free MnP but still too low for industrial application. MnP immobilized on FSM-16 had higher thermostability and was more tolerant to H₂O₂ than MnP immobilized on the NH₂-Emphaze polymer (9). The Emphaze-MnP column system also requires NaCl to prevent the adsorption of Mn³⁺-chelate to the support matrix, and the salt conditions must be controlled carefully, but the FSM/70-MnP column system does not require such an additive.

Enzymes can be immobilized on FSM-16 under milder conditions than for other immobilization methods, and the loss of activity during immobilization at 4°C might be negligible. MnP immobilized on FSM-16/70, whose pore size is nearly the same as the diameter of the enzyme, had the highest thermal stability, whereas silica gel, which has a wide range of pore sizes (50 to 300 Å), had no stabilizing effect with respect to temperature. Thus, the pore size and pore distribution of mesoporous materials are critical for the stabilization of enzymes. Immobilization of MnP on FSM/70 might help overcome problems with the industrial application of MnP. This immobilization method also should be applicable to other useful enzymes.

During lignolysis involving MnP, the Mn³⁺-chelate complex generated by MnP acts as an oxidant (18). Lignin, the most recalcitrant component of wood, can be oxidized at a considerable distance from an oxidative enzyme through the use of an enzymatically generated diffusible reactive intermediate, e.g., chelated Mn³⁺. The reactivity of the complex increased sufficiently with temperature for the Mn³⁺-chelate to react with lignin in pulp. By using a two-stage reactor system, the conditions for the oxidation of Mn²⁺ to Mn³⁺ by MnP (enzymatic reaction) and the subsequent oxidation of lignin in pulp by Mn³⁺ (chemical reaction) can be optimized independently.

We suggest that a two-stage reactor system involving MnP immobilized on FSM-16 is important for a new TCF pulp-bleaching system. However, with only MnP treatment, seven cycles of enzyme treatment were required to achieve a practical level of bleaching. It is essential to reduce the number of treatment cycles if greater bleaching ability is required. We are now examining additives such as linoleic acid, which plays an important role in the fungal lignolytic system (20), to increase bleaching ability. The combination of MnP treatment and other chemical bleaching methods, in which no chlorine-containing reagents are involved, should be another choice for practical TCF use.

Also, since Mn³⁺ generated by MnP is a nonspecific oxidant (6), it can attack other organic substrates in a similar manner (11, 21). Deguchi et al. (5) recently reported that nylon could be degraded by MnP. Therefore, a two-stage reactor system (TSRS) including MnP immobilized in FSM-16 might have additional uses for the remediation of toxic organopollutants.