報文鯉淵研報 3016-22（2020）

Characterization of Manganese Peroxidase fromPholiota aurevella
Hideyuki KOBAYASHI1
Food and Nutrition Department, Koibuchi College of Agriculture and Nutrition

1（Received 2 December 2019 / Accepted 8 January 2020）

Abstract: Manganese peroxidase from Pholiota aurevella was puriﬁed to homogeneity using ammonium sulfate precipitation followed by Sephacryl S-300 HR, DEAE-Sepharose, Con A Sepharose and Mono Q HR 5/5 column chromatographies. The puriﬁed enzyme had a molecular weight of 40 kDa and N-terminal amino acid sequence exhibited more than
30% homology with the other manganese peroxidases from white rot fungi. The enzyme showed optimum pH and temperature at 5.5 and 45℃, respectively, indicating the enzyme showed higher optimum temperature than the other manganese peroxidases. It showed similar speciﬁcity against phenolic substrates and the presence of Mn2+ enhanced the enzyme
activity greatly while Fe2+ completely inhibited the enzyme. Salt tolerance of the enzyme will make itself to be a potent
candidate for the bioremediation of organic pollutants.
Key words: Pholiota aurevella, manganese peroxidase, characterization, substrate speciﬁcity

pollution abatement is well documented8,10). Most of theIIntroduction

reported works on them are related to Phanerochaetechrysosporium11), but some white rot fungi containing

Lignins occupy a unique place among the abun-the edible mushrooms are also exploited for enzyme

dant renewable aromatic polymers and occur as ligno-production. Earlier work on the screening of different

cellulosic composites in the woody materials. Lignin

edible mushrooms for enzyme production suggested

degradation is affected by fungi especially the white rotthat Pholiota aurevella is one of the potent MnP produc-basidyomycetes . Oxidative enzymes that commonlyers12). In this paper, the production of lignin degrading

occurred in lignin degradation are lignin peroxidase

enzymes from P. aurevella, and the puriﬁcation and(LiP, EC 1.11.1.14), manganese peroxidase (MnP, EC

characterization of MnP which is the major enzyme pro-

1.11.1.13), and laccase (Lac, EC 1.10.3.2)

duced from the above fungus are described.

1). Due to2~5)

their broad speciﬁcities, these enzyme systems ﬁnd theirapplications in the xenobiotic degradation and also inIIMaterials and methods

other biotechnological applications6~9). MnPs have been
puriﬁed and characterized from white rot fungi5,10). They

1. Microorganism and culture conditions

are mostly measophilic enzymes in their thermal stabili-Pholiota aurevella (IFO No. 30265), obtained fromties. As in the case of other lignin degrading enzymes,

the Institute for Fermentation, Osaka was grown on aMnP also ﬁnds applications in different ﬁelds especiallypotato dextrose broth (24.0 g/l), yeast extract (5 g/l), pH

in pollution abatement, biopulping and biobleaching,

5.5. The fungus was cultured on a rotary shaker at 100

lignin bioconversion via xenobiotic degradation andrpm and 25℃. After cultivation, the supernatant wasbiotransformations of value added products such as bio-separated by ﬁltration using ADVANTEC No.2 ﬁlter

degradable polymers. Application of these enzymes on

and was used as crude enzyme solution.

1

Koibuchi 5965, Mito, Ibaraki 319-0323, Japnan

16

Kobayashi：Characterization of Manganese Peroxidase from Pholiota aurevella

2. Estimation of biomass and soluble protein
Fungal biomass was estimated by dry weight andprotein content was estimated by DC protein assay kit

The enzyme solution was also exposed at pH 5.5
and above incubation temperatures for 30 minutes andthe residual activity was measured.

(BioRad Laboratories Inc) and also by measuring theabsorbance at 280 nm assuming that 1mg/ml protein is

6. Substrate specificity of MnP

1.0.

Several phenol derivatives such as 2,6-dimethoxy phenol (DMP 468nm: ε = 49,600 M-1cm-1), ABTS (2,2 -azino-

3. Enzyme assaysdi-[3-ethyl benzothiazoline-6-sulphonic acid] 420nm :ε= 36,000 M-1cm-1), guaiacol (436 nm : ε=6,400 M-1cm-1),

Manganese peroxidase (MnP) activity was determethoxyphenol (DMP). The reaction mixture contained

catechol (450nm: ε = 2,211 M-1cm-1), pyrogallol(450nm: ε= 4,400 M-1cm-1) were used at 0.4mM con-

0.4 mM DMP, 0.1 mM MnSO4, 0.1 mM H2O2, in 50 mM

centration in the reaction mixture.

mined13) at 468 nm by measuring the oxidation of 2,6-di-sodium acetate buffer, pH 5.5, and enzyme solution.
The enzyme reaction was carried out at 30℃. One unit

7. Kinetic parameters of MnP in the presence andof MnP activity was deﬁned as the amount of enzyme

absence of Mn2+

which oxidizes 1 μmole of DMP in 1 min.

The effect of substrate concentration on the enzy-

Laccase (Lac) activity was measured by monitor-

matic activity in the presence and absence of Mn2+ wasing the oxidation of ABTS (2,2 -azino-di-[3-diethylben-investigated. In the set of experiments, DMP at lower

zothiazoline-6-sulphonic acid], Boehringer) at 420 nm14)concentrations such as 0.025, 0.05, 0.1, 0.2 and 0.3mM,

dissolved in 50 mM sodium succinate buffer, pH 5.5.

and at higher concentrations such as 3.0, 3.5, 4.0, 4.5,

One unit of laccase activity was deﬁned as the amountand 5.0mM was used as the substrate.

of enzyme which oxidizes 1 μmol of ABTS in 1 min.

8. Effect of NaCl on MnP activity
4. Purification of manganese peroxidase

Enzyme solution was mixed with equal amount of

Puriﬁcation of MnP was carried out by ammonium

50mM sodium acetate buffer pH 5.5 containing NaCl

sulate precipitation followed by chromatographies on

concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0 M. Enzyme

Sephacryl S-300 HR, DEAE-Sepharose, Con A Sepha-solutions after treating with different concentrations ofrose and Mono Q HR 5/5 columns.

NaCl as given above were kept 4℃ overnight and residual activities of MnP were determined.

5. Effect of reaction pH and temperature on theMnP activity and stability

IIIResults and discussion

Buffers ranging from pH 3.0 to 8.0 were used withthe substrate concentration of 0.4mM DMP. The reac-1. Production of lignin degrading enzymes fromtion was carried out at 30℃ keeping the concentrations

Pholiota aurevellaof other reagents constant.

Pholiota aurivella was cultured for 8 days andThe enzyme solution was incubated at the above

samples were taken at every 24 h for the analysis of pH

pHs and at 30℃ for 30 minutes and the residual activity

change, biomass production and enzyme levels. The re-was measured.

sults were shown in Fig. 1. P. aurevella produced two

The enzyme reaction was carried out at different

lignin-degrading enzymes (MnP and Lac) at high level

temperatures ranging from 20 to 70℃. The bufferedunder the experimental conditions indicating that thissubstrate with enzyme (pH 5.5) and Mn2+ was preincu-fungus is a potent producer of these enzymes. White rot

bated at the required temperature and then measured thefungi especially the edible mushrooms prefer to secrete

absorbance change after the addition of H2O2 at the ap-MnP and laccase, but not lignin peroxidase (LiP)15,16).

propriate wavelength.

In addition, the enzyme production pattern suggests that17鯉研報

第 30 号
pHLaccase

2020Mycelium

25141220101581064502123456789pH, Mucelium (mg/ml)

MnP activity, Laccase activity (unit/ml)

MnP

淵0Time (d)Fig. 1. Growth and enzyme production of Pholiota aurivella
During the cultivation of P. aurevella, pH, enzymatic activities and mycelium(dry weight) were determined.

Table 1. Purification of manganese peroxidase from Pholiota aurevella

Total activity(unit)

Total protein(mg)Speciﬁc activity(unit/mg)

Yield(%)

Puriﬁcation(-fold)

9,622

154.0

63

100

16,596

47.5

139

68.5

2.2

5,569

5.9

944

57.8

15.1

4,564
3,081
1,953

2.9
1.6
1.0

1,577
1,879
1,987

47.4
32.0
20.3

25.7
30.0
31.8

Culture supernatantAmmonium sulfate
fractionation
Sephacryl
S-300
DEAE Sepharose
Con A Sepharose
Mono Q

When the concentrated protein from ammonium sulfate precipitation was loaded onto Sephacryl S-300 column which was equilibrated with
0.05M sodium acetate buffer containing 0.5M NaCl, pH 5.5, a single activity peak was observed.
The active fractions from the above were dialyzed against 50mM sodium acetate buffer, pH 5.5, and were loaded onto the DEAE-Sepharosecolumn which was equilibrated with the same buffer. The adsorbed protein was eluted with decreasing pH gradient from pH 5.5 (50mM sodium
acetate buffer) to pH 3.0 (0.1M acetic acid) with the combination of increasing NaCl gradient from 0 to 0.5 M.
The active fractions from this column were pooled, dialyzed against sodium acetate buffer, pH 5.5 containing 0.5M NaCl, 1mM CaCl2, andMnCl2, and MgCl2, and concentrated by Ultraﬁltration on YM10 membrane (Toyo Roshi) and then again loaded onto the Con A Sepharose column. After washing with the same buffer, the MnP activity was eluted with 0.2M α-methyl-D-glucoside.
The active fractions from afﬁnity column were pooled, dialyzed against 50mM sodium acetate buffer, pH 5.5, and then loaded onto Mono Q HR
5/5 column. The active fraction was eluted with the NaCl gradient from 0 to 0.5 M.

there can be the presence of two MnPs, major one ex-overall yield was 20.3 % with a puriﬁcation fold of 31.8.

pressed in the stage of the growth (3d) and the minor one
expressed in the later stage (7d).

3. Characterization of MnP
The puriﬁed MnP was subjected to SDS-PAGE and2. Purification of MnP

the results were given in Fig. 2. The protein migrated as

Puriﬁcation of MnP from the crude enzyme prepa-a single band and the molecular weight was estimated toration was carried out using different steps including

be 40 kDa. The molecular weights of MnPs are reported

afﬁnity chromatography as shown in Table 1. The pu-to be ranging from 40 to 50 kDa17).

riﬁcation step using Con A as a ligand is effective forThis protein was transferred to PVDF (polyvini-the puriﬁcation of the enzyme, indicating the enzyme

lidene diﬂuoride) membrane and its N-terminal amino

contains oligosaccharide chains in the molecule. The

acid sequence was determined (Fig. 3). The homology

18

Kobayashi：Characterization of Manganese Peroxidase from Pholiota aurevellasearch revealed that it had more than 30% homology
with the previously reported MnPs derived from white
rot fungi such as Pleurotus eryingii, Pleurotus ostreatus,
Ganoderma applantum, Trametes versicolor, etc.

Three Cys residues are conserved among all enzymes and these Cys residues are thought to be important because of the relation to the activity and/or themaintenance of enzyme structure. These three Cys residues are conserved at the same position of the primary
sequence of the protein belonging to the same protein
family.

4. Effect of pH and temperature on activity and stability of MnPMnP was most active at pH 5.5 and this enzyme isstable for 30 min around its optimum pH. The optimum
temperature of MnP is 45℃ and the enzyme was stable
up to 40℃. MnPs are susceptible to thermal inactivation
due to their relatively low afﬁnity to Ca2+ ions, which
are required for its stability and activity18). The optimum
Fig. 2.	SDS-PAGE of purified MnP from Pholiota aurivella
The puriﬁed protein was subjected to SDS-PAGE.
The gel concentration was 12.5 %(w/v) and theprotein markers were ranging from 10 to 100 kDa.
After electrophoresis, the gel was stained with 0.25%(w/v) CBB R250 and destained with a mixture ofmethanol/acetic acid/distilled water (3:1:6, v/v).

temperature of the enzyme is higher than those of MnPs
from white rot fungi (22℃-40℃)19) but heat stability isalmost the same as those of MnPs 20).

5. Substrate specificity of MnPDifferent phenolic substrates such as DMP, ABTS,Pholiota aurivella

VT AN KTXT-NAVPLFDXVDFLQ

Pleurotus eryngii

VT AT QTTA-NEA

ALFPILDDIQ

Pleurotus ostreatus

VT AT QTTA-NEA

ALFPILEDIQ

Ganoderma lucidum

AT AN KTTA-NDAVWFDVLDDIQ

Trametes versicolor

VA XD YNTATNAAQLFXVRDDIQ

Ceriporiopsis subvermispora

VT SD -TVVPDSMDFIPLAQDLG

Phanerochaete chrysosporium

AT PD -TKVNNAA

AFIPLAQXLQDochomitus squalens

TV SD -TVVPDSVEFVPLAQALQ

Tylospora fibrillose

AT PD -TKVNNAA

AFIPFig. 3.	Comparison of N-Terminal amino acid sequences of MnPs from various origins
including Pholiota aurevella
The SDS-PAGE was conducted for MnP as given above. The protein in the gel was transferred to PDVF membrane by electroblotting using semi dry transfer cell equipment (BioRad Laboratories Inc) at 15 V for 2 h. The transferred proteins in the membrane werestained with 0.1 %(w/v) CBB R250 and destained with 50 %(v/v) methanol. The transferred protein was sequenced using a protein sequencer G1000A (Hewlett Packard Corp.)
along with standard PTH amino acids. Identical amino acids among all and majority ofenzymes are indicated as open and underlined characters, respectively.

19

鯉淵研報

第 30 号

2020

Fig. 4.	Substrate specificities of manganese peroxidase from Pholiota aurivellaDifferent phenol derivatives such as dimethoxy phenol (DMP 468 nm: ε= 49,600 M-1cm-1), ABTS (420nm : ε = 36,000 M-1cm-1), guaiacol (436
nm : ε =6,400 M-1cm-1), catechol (450 nm: ε = 2,200 M-1cm-1), pyrogallol (450 nm: ε = 4,400 M-1cm-1) were taken at 0.4 mM concentration in 50mM sodium acetate buffer, pH 5.5 and used as substrates.

R elative activity (% )1008060

40200None

M n2+

Co2+

Fe2 +Ni2+

M g2+

Cu2+K+--Catio n sFig. 5.	Effect of cations on the activities of manganese peroxidase from Pholiota
aurevellaInstead of MnCl2, different chlorides of metals like CoCl2, FeCl2, NiCl2,MgCl2, CuCl2, KCl were used at the concentration of 0.1mM and DMP wasused as substrate.

pyrogallol, guaiacol, and catechol were used for inves-creased (11.79 mM) whereas in the presence of 0.1 mMtigation of the substrate speciﬁcity of MnP. The results

Mn2+, Km value decreased signiﬁcantly (0.104 mM).

were given in Fig. 4. The enzyme showed its maximum

Similar results are reported in the case of MnP fromafﬁnity towards ABTS followed by guaiacol, DMP, py-Phanerochaete crassa WD169417) and P. chrysospolium

rogallol, and catechol as in the case of MnPs from otherindicating MnP from P. aurivella is similar to the en-

white rot fungi11).

zymes from these basidiomycetes in terms of catalytic

In the absence of Mn , the Km value of DMP in2+

20

properties.

Kobayashi：Characterization of Manganese Peroxidase from Pholiota aurevella

Table 2. Effect of various organic acids on manganeseperoxidase activity from Pholiota aurevella

Organic acidoxalatesuccinatemaleatemalonate
citrateglycolatelactatemalategluconatetartrate
phosphateacetateRelative activity (%)
7428
791
3354891003749
174

Sodium salts of different organic acids listed above were used at
the concentration of 50mM at pH 4.5. The oxidation of DMP wasmonitored by the standard MnP assay conditions.

6. Effect of cations on MnP activity
When individual cations were added to the reaction
mixture, Mn2+ gave the maximum activity and the othercations such as Co2+, Ni2+, Mg2+, Cu2+, K+, showed lessthan 20% of the activity exhibited by Mn2+. In addition,
Fe2+ inhibited the enzyme almost completely. (Fig. 5)

7. Effect of organic acids on MnP activity
DMP oxidation in the different organic acids wasmonitored. The activity of the enzyme was enhanced by
the addition of organic acid chelators like malate, malonate, lactate and oxalate that stabilize the Mn3+ complexof the organic acids. At 50mM concentration, malate
showed the maximum activity followed by malonate,lactate, oxalate, glycolate, and tartate as shown in Table
2.
When the oxidation of DMP monitored with different chelating organic acids by MnP, according to the stability of chelator-Mn3+ complex, various enhancementof the activities were observed. Earlier reports suggested that oxalate, which was secreted by the fungus, andother organic acid chelators, such as malonate, stabilize
the enzyme-generated Mn3+ 21,22) and these Mn3+-chelatecomplexes moved away from the enzyme to oxidize
phenolic substrates, including lignin, lignin model compounds and aromatic pollutants.

8. Influence of NaCl on MnP activity
The residual activities of the enzyme treated withdifferent concentrations of NaCl showed 100% activities with 0.1M NaCl, followed by other concentrations
from 0.2M to 0.5M which retained more than 80%activities. This results indicate that the MnP from thisfungus is a salt tolerant and it is an advantage for the application of the enzyme for xenobiotic degradation evenin the sea water.

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