Coal Depolymerising Activity and Haloperoxidase Activity of Mn Peroxidase from Fomes durissimus MTCC-1173

Mn peroxidase has been purified to homogeneity from the culture filtrate of a new fungal strain Fomes durissimus MTCC-1173 using concentration by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The molecular mass of the purified enzyme has been found to be 42.0 kDa using SDS-PAGE analysis. The K m values using MnSO4 and H2O2 as the variable substrates in 50 mM lactic acid-sodium lactate buffer pH 4.5 at 30°C were 59 μM and 32 μM, respectively. The catalytic rate constants using MnSO4 and H2O2 were 22.4 s−1 and 14.0 s−1, respectively, giving the values of k cat/K m 0.38 μM−1s−1 and 0.44 μM−1s−1, respectively. The pH and temperature optima of the Mn peroxidase were 4 and 26°C, respectively. The purified MnP depolymerises humic acid in presence of H2O2. The purified Mn peroxidase exhibits haloperoxidase activity at low pH.


Introduction
Manganese peroxidase, MnP [E.C. 1.11.1.13], is a hemecontaining enzyme [1]. It has been shown to be present in the culture filtrates of a number of fungal strains [2][3][4][5]. The catalytic cycle of Mn peroxidase resembles those of other heme peroxidases such as horseradish peroxidase [6] and lignin peroxidase [7,8] and includes the native ferric enzyme as well as the reactive intermediates compound I and compound II. The catalytic cycle can be shown as follows: MnP is a biotechnological important enzyme having wide application in degradation of lignin [11], biopulping and biobleaching in paper industries [12], removal of recalcitrant organic pollutants [13], and enzymatic polymerization [14]. Keeping these points in view, we have purified Mn peroxidase from the culture filtrate of Fomes durissimus MTCC-1173 and its enzymatic characteristics like K m , pH, and temperature optima have been determined. Depolymerisation of coal by the purified enzyme has been demonstrated using humic acid as a model of coal. MnP from F. durissimus also possesses haloperoxidase activity at low pH.

Fungal Strain and Its
Growth. The indigenous ligninolytic fungal strain F. durissimus MTCC-1173 was procured from MTCC Centre and Gene Bank, Institute of Microbial Technology, Chandigarh, India. The fungal strain was maintained on growth medium which consisted of "malt extract 20.0 g and agar 20.0 g in 1.0 L double distilled water." The pH of the medium was adjusted to 6.5 at temperature 25 • C.

Enzyme
Assay. The activity of Mn peroxidase was determined spectrophotometrically [16] by monitoring the absorbance change at λ = 240 nm due to the formation of Mn(III) lactate and using the molar extinction coefficient value of 65,00 M −1 cm −1 . The reaction solution 1 mL consisted of "50 μM MnSO 4 , 50 μM H 2 O 2 , and a suitable aliquot of the enzyme solution in 50 mM sodium lactate/lactic acid buffer pH 4.5 at 30 • C." One enzyme unit transformed 1 μmole of the substrate into the product under the specified assay condition. UV/VIS spectrophotometer Hitachi (Japan) Model U-2000 which was fitted with electronic temperature control unit was used for spectrophotometric measurements. The least count of the absorbance measurement was 0.001 absorbance unit. Each data point is an average of triplicate measurements with standard deviation less than 4%.

Purification of the Enzyme.
For the purification of Mn peroxidase, the fungal cultures were grown in sixty 100 mL sterilized culture flasks each containing 20 mL of the growth medium as described above. On the fifth day of inoculation of the fungal spores when Mn peroxidase activity reached maximum value, the cultures were pooled, mycelia were removed by filtration through four layers of cheese cloth, and culture filtrate 800 mL with 0.95 IU/mL activity was concentrated with Amicon Concentration Cell Model 8200 using PM10 ultrafiltration membrane with molecular weight. Cut-off value 10 kDa to 10 mL. The concentrated enzyme was dialysed against 1000 times excess of 10 mM sodium succinate buffer pH 4.5 overnight at 20 • C. The dialysed enzyme was loaded on a DEAE cellulose column size 1 cm × 22 cm which was preequilibrated with the same buffer. The adsorbed enzyme was washed with 50 mL of the same buffer and was eluted by applying NaCl gradient (0-400 mM; 100 mL + 100 mL = 200 mL). The 5 mL fractions were collected and analysed for Mn peroxidase activity using the method reported by Gold and Glenn [16] and for protein concentration using Lowry method [17]. The active fractions were combined and concentrated with the Amicon Concentration Cell Model 8200 and thereafter with Model-3 using ultrafiltration membrane PM10. The concentrated enzyme was stored at 4 • C and was used for further studies. The enzyme did not loose activity for two months under these conditions.

SDS-Polyacrylamide Gel Electrophoresis.
The homogeneity of the enzyme preparation was checked by SDS-PAGE analysis [18], and molecular mass was determined using the method of Weber and Osborn [19]. The separating gel was 12% acrylamide in 0.375 M Tris-HCl buffer pH 8.  [20]. The reaction solution 1 mL consisted of, "100 μM H 2 O 2 in 50 mM lactic acidsodium lactate buffer pH 4.5 at 30 • C and MnSO 4 was varied in the range 0.02 mM to 15 mM." 5 μg of the enzyme with specific activity 4 IU/mg was added. The same procedure was adopted for determination of K m value for H 2 O 2 except that H 2 O 2 concentration was varied in the range from 0.01 mM to 12 mM at the fixed 100 mM concentration of MnSO 4 . The K m value was calculated by the linear regression analysis of the data points of double reciprocal plots. The pH optimum of the purified enzyme was determined by measuring the steady-state velocity of the enzyme catalysed reaction in solutions of the above composition of varying pH in the range 2.0 to 5.0 using 50 mM lactic acid/sodium lactate buffer and plotting a graph of the steady-state velocity against pH of the reaction solutions. The temperature optimum was determined by measuring the steady-state velocity of the enzyme catalysed reaction in solutions of the above composition in the temperature range 15 to 35 • C and plotting the steady-state velocity versus temperature.

Effect of Chelators on MnP.
The effect of different Mn(III) chelator molecules "oxalate, lactate and malonate" on the activity of the enzyme was determined by measuring the activity of the enzyme at different concentrations of Mn(II) ions in presence of buffers of the chelating carboxylic acids with their sodium salts using the method reported in the literature [20]. The initial velocity of Mn(III) formation

Results and Discussion
The maximum activity of Mn peroxidase in the liquid culture growth medium of F. durissimus MTCC-1173 appeared on the 5th day after the incubation of fungal spores and the peak value of the activity was 0.95 IU/mL. The purification procedure of the enzyme is summarized in Table 1. It involved concentration of the culture filtrate by ultrafiltration and column chromatography on anion exchanger diethylaminoethyl (DEAE) cellulose. The enzyme bound to DEAE cellulose equilibrated with 50 mM succinic acid sodium succinate buffer pH 4.5 at 20 • C and was eluted by the linear gradient of NaCl in the range 150 mM to 230 mM in the above buffer. The active eluted enzyme 35 mL was 30-fold concentrated and analysed by SDS-PAGE for purity. The results of SDS-PAGE analysis are shown in Figure 1. Lane 1 contains protein molecular weight markers and lane 2 contains the purified enzyme. The presence of a single protein band in lane 2 clearly indicates that the purified enzyme is pure. The calculated relative molecular mass of the enzyme from the SDS-PAGE analysis was 42.0 kDa. Though Mn peroxidase has been purified from a number of fungal sources, namely, P. chrysosporium [19], Phanerochaete sordida [24], Nematoloma frowardii [25], Ceriporiopsis subvermispora [26], Aspergillus niger [27], Coriolus versicolor [28], Aspergillus terreus LD-1 [29], Pleurotus ostreatus [30], and Lentinula edodes [4], in most of the cases purification procedure is not so simple as in the case of the purification of the enzyme from the culture filtrate of F. durissimus MTCC-1173. Michaelis-Menten plots and double reciprocal plots using MnSO 4 and H 2 O 2 as the variable substrates are shown in Figures 2(a) and 2(b). The calculated K m values for Mn(II) and H 2 O 2 were 59 μM and 32 μM, respectively. Mn peroxidase of P. chrysosporium has been most extensively studied [21]. The reported K m values for the H4 isoenzyme of Mn peroxidase of P. chrysosporium using Mn(II) and H 2 O 2 as the substrates are 41 μM and 39 μM, respectively. Thus the K m values using Mn(II) and H 2 O 2 as the substrates for the Mn peroxidase of F. durissimus are in the same range as reported [21] for the Mn peroxidase of P. chrysosporium. The  Thus, the catalytic efficiency of the purified enzyme is lower than the catalytic efficiency of the Mn peroxidase isozyme H4 purified from P. chrysosporium.
The variation of the activity of the purified Mn peroxidase with the pH of the reaction solutions is shown in Figure 3(a). The calculated pH optimum is 4.0 which is lower than the pH optimum reported [21] for the Mn peroxidase of P. chrysosporium. The effect of temperature variation on the activity of the purified Mn peroxidase is shown in Figure 3(b) from which it follows that the temperature optimum of the enzyme is 26 • C which is near to the temperature optimum value of 28 • C for the Mn peroxidase of P. chrysosporium [21].      [7,21]. Our steady-state kinetic studies using Mn peroxidase from F. durissimus also support the conclusions of Kuan et al. [21].

Humic Acid Degradation.
The recording of UV/VIS spectra of the solution containing humic acid, H 2 O 2 , and the purified enzyme in lactic acid-sodium lactate buffer pH 4.5 at 30 • C at the intervals of 30 minutes indicated increase of absorbance at 360 nm as shown in Figure 5(a) and decrease of absorbance at 450 nm as shown in Figure 5(b) similar to the results of depolymerisation coal in aqueous medium by lignin peroxidase and H 2 O 2 reported by Wondrack et al. [31]. The decrease in absorbance at 450 has been attributed to the disappearance of brown colour of the coal, and the increase in absorbance at 360 has been attributed to the formation of yellowish colour fulvic acid like compound in coal depolymerization [31]. Our results with humic acid in presence of the purified MnP and H 2 O 2 have indicated the depolymerization of humic acid. The time course of humic acid depolymerization was studied by measuring absorbance increase at 360 nm in buffers of succinic acid-sodium succinate, lactic acid-sodium lactate, and malonic acid-sodium malonate. It has already been shown in Figure 4 that the Vmax of the enzyme catalysed reaction is dependent on the chelating ions of Mn(III) present in the buffer. The order was Vmax(malonate) > Vmax(lactate) > Vmax(succinate). The increase in the absorbance at 360 nm as shown in Figure 5(a) with time was in the same order as the Vmax of the enzyme catalysed reaction in different buffers. The decrease in absorbance at 450 nm as shown in Figure 5(b) also followed the same order.

Haloperoxidase Activity.
The haloperoxidase activity of the purified Mn peroxidase was tested by recording the UV/VIS spectrum of the reaction solution 1 mL containing "2.5 μg of the purified MnP, 20 mM KBr, 100 μM H 2 O 2 in 20 mM succinic acid-sodium succinate buffer pH 3.0 at 30 • C." The spectrum resembles the characteristic spectrum of tribromide complex Br 3 − with λmax at 266 nm as reported [23]. Figure 6(b) shows the spectrum of the solution of the same composition as mentioned above except that KI has been used in place of KBr and only 0.5 μg of the enzyme has been added. This spectrum also resembles the characteristic spectrum of triiodide with λmax at 285 nm and 353 nm as reported [23]. Thus the purified MnP liberated Br 2 and I 2 in presence of H 2 O 2 at pH 3.0. The pH dependence of the rate of oxidation of Br − to Br 2 and I − to I 2 has also been done, and the results shown in Figure 6(c) have indicated pH optima of 3.0 and 2.5, respectively, for the oxidation of Br − and I − . Free Br 2 which is not ecofriendly is used for many bromination reactions in organic chemistry. The enzyme with H 2 O 2 and KBr is a possible ecofriendly reagent for bromination reactions in organic synthesis. In conclusion this communication reports purification and characterization of Mn peroxidase from the culture filtrate of a new fungal strain F. durissimus using a simple procedure. The purified enzyme has similar properties with the MnP of P. chrysosporium, an extensively studied MnP. The enzyme depolymerises humic acid and shows haloperoxidase activity for the oxidation of Br − and I − at low pH.