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
7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification
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).
7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification
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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.
7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification
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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-
7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification
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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
7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification
6/7
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7/23/2019 Methods Enzymol_161 - Manganese Peroxidase of Phanetochaete Dhrysosporium Purification
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.