Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification

7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification

1/7

264

LIGNIN

t26l

also

stimulates

he

manganese

eroxidase

o

varying degfees,

epending

on

the substrate

used.2

Substrate

Specificity.

In the

presence

of

HrO2

the

enzyme

oxidizes

polymeric

and

other

dyes,

lignin

model compounds,

and various

phenols.2,3

Mn(III)

is

capable

of oxidizing

all of

the organic

substrates

which are

oxidized

by the

enzyme

system.

In the

absence

of

exogenous

HrO, the

enzyme

also

acts as

an

NAD(P)H

oxidase2'3

eneratin1HzOz. This

reaction can

be coupled

o

the

oxidation

of

ABTS

in the

absence

of exogenous

H2O2, suggesting

hat

the

manganese

eroxidase

may

play

a

role

in HrOrproduction

by

the

fungus

under

ligninolytic

conditions.2'3

t26)

Manganese

Peroxidase

of

Phanetochaete

dhry

so

sp

oriumi

Purifi cation

I

,ByANonzH

PtszczYftsrt,

Ror.Iero

L.

CnawFoRD,

and

VaN-Be

HuvNn

Two

types

of

extracellular

peroxidases

have been

discovered

in the

growth

medium

of

ligninolytic

cultures

of

white

rot

fungus

Phanerochaete

chrysosporium.2,3

ne

type

has been termed

ligninase 4

or

diarylpro-

pane

oxygenase,

and

appears

o

oxidize

lignin

and

many

lignin

model

compounds

by

extracting

an

electron

from an aromatic

nucleus, creating

an unstable

cation

radical species

which

undergoes

numerous degradative

transformations leading to substratedecomposition.6A secondtype of

peroxidase

excreted

by

P.

chrysosporium

has been

called

manganese

er-

oxidase,

as

it

oxidizes

Mn(II)

to

Mn(III).7'8

This

enzyme

also

shows

oxi-

dase

activity,

producing

hydrogen

peroxide

by oxidation

of

reduced sub-

strates

like

NAD(P)H,

glutathione

(GSH),

dithiothreitol

(DTE),

and

rThis

work

was

performed

at the

University

of

Minnesota,

Gray

Freshwater

Biological

Institute,

Navarre,

Minnesota

55392

under

National

Science

Foundation

Grant

PCM-8318151.

2

M.

Tien and

T. K. Kirk, Scimce

221' 66t

(1983).

3

M.

Kuwahara,

J.

K. Glenn,

M.

A. Morgan,

and

M. H. Gold,

FEBS

Lett.169,247

(1984).

a

M.

Tien and

T.

K. Kirk,

Proc.

Natl. Acad.

Sci. U.S.A.81,2280

(1984)-

5

J.

K. Glenn

and

M.

H.

Gold,

Arch.

Biochem.

Biophys.

A2,329

(1985).

5

P. J.

Kersten,

M.

Tien, B.

Kalyanaraman,

and

T. K. Kirk, J-

Biol. Chem.zffi,2609

(1985).

7

A.Paszczyfiski,

V. B. Huynh,

and

R. Crawford,

FEMS Microbiol.

Lett.29,37

(1985).

8

J.

K.

Glenn

and

M. H. Gold,

Arch.

Biochem.

Biophys.

U2,329

(1985).

Copynght

@ 1988

by

Academic Press, nc.

All rigbts of reproduction

in

any

form reserved.

ETHODS

IN ENZYMOLOGY.

VOL. 16I


2/7

126l

MANGANESE

ERoxrDAsE

oF ,P.

chrysosporium

265

dihydroxymaleic

acid.,,e

Both

Mn(III)

and hydrogen

peroxide

produced

by

the enzyme

may

be

involved

in

lignin

biodegradation.e

Here

we

outline

a

method

for

purification

of the

manganese

eroxidase

of P. chrysosporium.

Enzyme

Production

Phanerochaete

hrysosporium

we

employed

strain

BKM-l

767,

ATCC

24725)

was

maintained

and

grown

as

described

n

previous

work.7

Growth

was

n

a defined

medium

at

pH

4.5

on

glucose

n

the

presence

of

growth-

limiting

amounts

of nitrogen

to

ensure

hat

the

fungus

produced

tJtgnin-

olytic

system.r'

one liter

of medium

was

ptep*d

by

mixing

100

ml

of

lOx concentratedbasal medium (r-asparagine,

g;

NttoNor,

0.5

KH2PO4

2

g;

MgSOa

7H2O,0.5

g;

CaClr.2H2O,0.l-g;

hiamin,-0.001

mineral

elixer,

l0

ml;

distilled

water

to I

liter;

filter

steritirea;,

ioo

mt

0.1

M

sodium

dimethylsuccinate

(pH

4.5,

filter

sterilized),

lgii

ml

of

a

chrysosporium

conidiospore

suspension Aeso-

0.5),

and

600

ml

of

auto_

claved distilled

water.

The

mineral

elixer

used

n

the

basal

medium

con-

tained

(in

g/liter

distilled

water):

nitrilotriacetic

acid,

1.5;

MgSoa,

3.0;

MnSOo,0.5;

NaCl,

1.0;

FeSOa,0.l ;

CaClr,0.l ;

CoCl2,

O.i;

ZnSOo,0.l ' ;

cusoa, 0.01;AIK(so4)2,0.01;H3Bo3,0.01;andNaMooc,0.0r.

Fungal

mycelium

was

grown

attached

o

the roughslsd

interior

* ils

of

a 20-liier

polyethylene

carboy

containing

I

liter

of medium

and

rotated

at

about

0.2

rpm.

Every

second

day

the

carboy

was

filled

with

pure

oxygen

by

passing

Or

(filter

sterilized)

through

a tube

passed

hrough

the

carboy's

screw-top

cap.

Two

additional

tubes

passed

through

the

cap:

an

oxygen

outlet

(equipped

with

a bacteriological

filter)

and

a sampling

port.

al

*tt .

porrs

were

clamped

closed

during

incubations,

which

were

done

at

40

oni

day

prior to harvesting the culture fluid for isolation of enzymes, veratryl

dcohol

(0.4

mM)

and

rween

80

(20

mg/iter)

were

added

o

the

medium

lo

increase

nzyme

production.lr

Alternatively,

investigators

may

use

the

above

medium

in

agitated

arbmerged ultures

f

Tween

80

(0.10/o),

ween

20

(0.05%),

or

3-[(3-chola-

midopropyl)dimethylammoniol-l-propane

sulfonate (0.05v0)

s-added

to

rcome

the

previously

known

necessity

o

grow

ligninolytic

p.

chryso_

rium

in

stationary

culture.rr

Medium

(600-

l00b

ml)

is

placed

n

a

-liter Erlenmeyerflask, inoculated with conidia, and shakeout t50-200

,

A.Paszczyriski,

.

B.

Huynh,

and R.

crawford,

Arch.

Biochem.

Biophys.244,750(r9g6).

w.

J.

connors,

L.

F.

Lorenz,

and

J.

G. zeikus,

Arch.

Mi*obiol.

rr7,

.

Kirk,

F.

Shultz,

277

1978).

g;

g;

of

P.

A. Jiger,

S.

croan,

and r.

K. Kirk,

Appl.

Environ.

Microbiol.

so,1274(19g5).


3/7

266

LIGNIN

1261

rpm

at

40".

Development

of

ligninolytic

activity

is

idiophasic,ro'r1

s in

siationary

cultures.

Addition

of

veratryl

alcohol

(0.4

mM)

still

enhances

enzymeyields.

Assay

Methods

for

Manganese

Manganese

peroxidase

may

be

assayed

using

a

variety

of

aromatic

substrates,

particularly

those

that

are employed

for assays

of common

peroxidases uch

as

horseradish

peroxidase.

Reaction

mixtures'

however,

must

be

supplemented

with

Mn(II)

ions.

Table

I lists some

useable

sub-

stratesatongwith their extinction coefficientsat wavelengths o be moni-

tored

during

the

peroxidatic reaction.T

Reagents

0.5

M sodium

tartrate

buffer,

PH

5.0

t

mM substrate

n

water

(water-soluble

compounds)

or

5090

aqueous

M,N-dimethylformamide

(water-insoluble

compounds)

C.

I

mM

MnSOa

D.

I

mMIJrO,

E.

Enzyme

solution

containing

about

0.2U

ml-r

Assay

Procedure

One

milliliter

of

assay

solution

contains

about

0.02

U

of

peroxidase,

0.1

M sodium

tartrate

(pH

5.0),

0.1

mM substrate

(Table

I), 0.1

mM

TABLE

I

Ass.lvSunsrnarBsFoRMe'NcLNsse ERoxrplsB

A.

B.

Substrate

Wavelength

(nm)

E

(1,1-r

cm-t)

1.

TMPD'

2.

Vanillylacetone'

3.

2,6-Dimethoxyphenol

4. Syringic

acid

5.

Guaiacol

6. Curcumin

7. Syringaldazine

8. Coniferyl

alcohol

9. o-Dianisidine

G2HCI)

610

336

568

260

465

430

525

263

460

I1,600

18,300

110,000r

8,050

12,lN

23,100

65,000

13,400

29,4N

"

N,N,N,N-Tetramethyl-

,4-phenylenediamine

-2HCl).

b

4-(4-Hydroxy-3-methoxyphenyl 3-buten-2-one.

"

Estimated

for a

nonhomogeneous,

polymeric

product;

disappearance

of

absorbance

(removal

of

substrate)

is monitored

for substrates

2,

4,

and 6

while

increases

in

absorbance

product

formation)

are

monitored

for substrates

1,

3,

5,

7, 8, and

9.


4/7

126l

MANGANESE

PEROXTDASE

F P.

chrysosporium

267

Hror,

and 0.1 mM

MnSoa.

The

solution is

contained

n

a

1.5-ml

quartz

cuvette

of

l-cm

path

length.

The

spectrophotometer

s

set

at

the wave-

length appropriate to the assaysubstratechosen(Table I). As a standard

assay,

vanillylacetone

was

used

as

substrate

during

purification

of

the

manganese

peroxidase.

Decrease

n

absorbance

at

336 nm

was

observed

(e

:

18,300)

and

used

to calculate

enzyme

activity.

Assays

are

performed

at

room

temperature

about

22 ).

One

unit

(U)

of

peroxidase

oxidizes

I

prmol

of

substrate/min,

and

units may

be

calculated

based

upon

U mg-t

of

protein

ml-r

of

enzyme

solution.

Assays

hould

be

carried

out

in

duplicate,

and are

initiated

by addition

of HrOr.

The

reference

cuvette

does

not

receiveHrOr.

Assay

of

Manganese

Peroxidase

Using Mn(II)

as

Substrate

A very

convenient

assay

of

manganese

peroxidase

activity

involves

monitoring

the

enzyme's

oxidation

of

Mn(II)

to Mn(III).

This

assays

best

used

with

purified

preparations

of the

peroxidase,

as

contaminating

metals

such as

iron

and copper

inhibit

the reaction.

The reaction

catalyzed

s

as

follows: 2Mn2+ + H2O2 + /\413+ + H2O. The reaction mixture contains

enzyme, 0.1 M

sodium

tartrate

(pH

5.0), 0.1 mM

HrOr,

and

0.1 mM

MnSoo. The

product,

Mn(III), forms

a transiently

stable

complex with

tartanc acid,

showing

a characteristic

bsorbance

t

238

nm

(e:6500).

Reactions

are initiated

by

addition

of

HrOr,

and

the reference

cuvette

contains

(l-cm path

length)

all components

except Mn(II).

Increase

n

Args

o

is monitored

during

the first

5

-

30 sec

of

reaction.

one

unit

of

peroxidase

xidizes

pmol

of Mn(II)/min.

Assay

or

Ligninase

The

enzyme

purification

employed

here

will

allow

purification

of

the

principal

ligninase

of

P.

chrysosporium

simultaneously

with

the manga-

nese

peroxidase.

The

most

convenient

assay or

ligninase

s

a

spectropho-

tometric

assay hat

monitors

the

oxidation

of veratryl

alcohol

to veratryl

aldehyde.a

Purffication

Procedures

All

procedures

are

carried

out at 4 .

Culture fluids

of the fungus

are

separated rom

the mycelium

by

filtration

through

glass

wool.

The fresh

filtrate is

concentrated

each

I

liter

to 30 ml)

by

ultrafiltration

(e.g.,

using

a

stirred cell

ultrafiltration

device

equipped

with

an M,10,000

cut-offmem-


5/7

268

LIGNIN

t26l

lr)

T

z

E

.=

z

N

-i

a

m

40

z.v

Cq ,

o

L

o_

c ,

30

E

f

F 2 0

F

O

t n

g

t .w

a

(D

r20 r40

Froction

number

Frc.

l. Chromatography

of the crude extracellular

proteins

of

Phanerochaete

chrysospor-

ium on

fast-flow DEAE-Sepharose.

We

employed a

Pharmacia Kl5 column

packed

to a bed

height of l0 cm. Enzyme solution (200 ml; concentrated by ultrafiltration on an Amicon

PM10 membrane and

diluted

1: I with water)

was

applied to the column and

then the

column

was

washed with 100

ml HrO. Elution

conditions:

flow rate, 70 ml hrt; fraction

volume, 2.2 ml; the column

first

was washed with

50

ml of

HrO

and then

proteins

desorbed

with 20 mMsodium

tartrate

(pH

5.0) containing

0.4 MNaCI;

(A-A)

manganese

eroxidase

activity;

(O-O)

absorbance

t 406 nm;

(-)

absorbance

t 280 nm.

brane). Crude

culture

filtrate

usually

contains 6-8

pg

ml-r

of

protein,

as

determined by the Coomassie blue method.r2 After ultrafiltration, the

protein

concentration

is increased

to about

150

pg

ml-r.

Ultrafiltered

enzyme

(100

ml) is

diluted

1: I with

deionized

water

and appted

to a

column

(1.6

X l0

cm) containing

DEAE(fast

flow)-Sepharose

prewashed

with 200 ml of distilled

water). Proteins

absorb to the column

as a dark

12

M. M. Bradford,Anal.

Chem.72,248

(1976).

20


6/7

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q 6

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o

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MH

EF E

$

HEq?

E

E t

1 i

n

=

E

l5EF:

:

$g :e i

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Fi6

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E

gE

EE

Ii

Eg

isE

J

-i

uralo.rd

Or.uZ

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tr)

/

(

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)

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7/7

270

LIGNIN t26l

TABLE

II

PunrrlcnrtoN

oF M.lNclNnss

PsRoxloesr

(eNo

LtcNNess)

rnou

rnr

GnowrH

MEptuv

op Phanerochaete

hrysosporium

U

ml-r

Total

units

RZ

(P)

,

u

mrt

protein

Yield

(%)

Purifi-

cation

Stepo

Volume

(ml)

;

2

2.5

189

t26

9

6

150

r20

16 8

79 95

80

60

26

12

42

47

60

8

100

15 3r.7

6.3

0.02

0.24

r.7

1.5

2.8

3 .6

11 .1

0.06

0.04

1.5

1 .2

16

12

12

1.6

3000

100

5

s

(P)

5 (L)

I

2

3

4

o

I, Crude

filtrate;

2, ultrafiltrate

(10,000-Da

cut-off);

3,

DEAE-sepharose;

4,PBE-94

chromatofo

cusing

column

chromatography

and

ultrafiltration.

,

P,

Manganese

peroxidase;

L,ligninase;

both

enzymes

will

store

(freeze

dried

in sealed

ampoules)

a

-20

with

a halfJife

of about

I

year.

Both enzymes

contain

appreciable

amounts

of carbohydrates

'

RZ:A*n^lArsoo

.

yellow band. The loaded column is washedwith 50 ml of water, and the

protein

eluted

using

20

mM

sodium

tartrate

containing0.4

M NaCl.

The

manganese

eroxidase

peak

from the

DEAE column

also contains

ignin-

ase

activity

(Fig.

1).

Peak

fractions

are

combined

and

desalted/concen-

trated

by

ultrafiltration.

This

preparation

may be

freezedried and

stored

at

-20

up

to

I

year

without appreciable

oss of activity.

Freeze-drieden-

zyme

is dissolved

in 10

ml of 0.025

M imidazole

buffer

(pH

7.4) and

applied

to a

column

(35

X

0.9 cm)

of

Polybuffer

Exchanger

(PBE-94,

Pharmacia) that had beenpreequilibrated with the samebuffer (seeFig. 2).

The column

is

then

eluted

with Polybuffer

74-HCl

(Pharmacia)

diluted

I : 8,

at

pH

4.0, and

containing

a linear

gradient

of

NaCl

(0-0.2

M).

About

l0

fractions

containing

the

highest activities

of

manganese

peroxidase

and

ligninase

are

pooled

separately

and

concentrated

by

ultrafiltration

to

a

final

volume

of

about

5

ml.

The concentrated

solutions

are

diluted

to 30

ml

with

deionized

water and

reconcentrated,

repeating

this operation

three

times

to

remove

dissolved

salts

and buffer.

Both enzyme

preparations

should show a singleband of protein upon polyacrylamide gel electropho-

resis

n

the

presence

of SDS.

After the

final ultrafiltration,

both enzyme

preparations

may be

freezedried

for

long-term storage

n

sealed

ampoules.

A summary

of

the above

purification

scheme

s shown

n Table

II.

Documents

Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification