Solubilization of Leonardite by an Extracellular

Fraction from Coriolus versicolor

John W. Pyne Jr., Dorothy L. Stewart, James Fredrickson and
Bary W. Wilson
Appl. Environ. Microbiol. 1987, 53(12):2844.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1987,

0099-2240/87/122844-05$02.00/0
Copyright 1987, American Society for Microbiology

p.

Vol. 53, No. 12

2844-2848

Solubilization of Leonardite by an Extracellular Fraction from

Coriolus versicolor
JOHN W. PYNE, JR.,* DOROTHY L. STEWART, JAMES FREDRICKSON, AND BARY W. WILSON

Battelle, Pacific Northwest Laboratories, Richland, Washington 99352

Received 3 June 1987/Accepted 10 September 1987

Microbial biosolubilization, as evidenced by the formation

of discrete product droplets from low-rank coal by Coriolus
versicolor and Poria monticola, was first reported by Cohen
and Gabriele (2) and has since been confirmed and extended
to include Candida sp. strain ML113, Penicillium waksmanii, Sporothrix sp., Aspergillus sp. (9), Streptomyces sp.
(8), Bjerkandera adusta, Poria placenta, and Fomes annosus (10). The term biosolubilization has been coined to
emphasize the metabolic origin of the soluble product. In
nature, many fungi use complex plant organic molecules as
sources of carbon and energy, and some fungi are prominent
lignin degraders, which may account for their ability to
degrade low-rank coals.
The material used by Cohen and Gabriele (2) in their initial
work was present as an overburden to a lignite deposit and
was considered a leonardite (4). The product formed by the
action of C. versicolor on leonardite is a water-soluble
material that is dark brown or black. This material is very
anionic and weakly acidic and contains both aromatic and
aliphatic protons in about a 1:1 proportion. Approximately
85% of the product has a molecular mass greater than 50,000
daltons at pHs 3 and 6.4. At pH 10.4, 40% of the solute has
a molecular mass greater than 50,000 daltons, and about 45%
has a molecular mass between 10,000 and 50,000 daltons.
Metal ions are important elements in the structure of
low-rank coals (5), and the cell-free biosolubilization of coal
may be due only to chelation. A second possible role of the
cell extract is as a surfactant. A protein fraction from
Pseudomonas fluorescens lowers surface tension and increases coal solubility (R. M. Fakoussa, Ph.D. thesis,
Friedrich-Wilhelms University, Bonn, Federal Republic of
Germany, 1981). The results presented here show that the
biosolubilizing activity of C. versicolor is due neither to
chelation nor to surfactant characteristics of the cell extract,
but rather to another mechanism such as enzymatic bond
cleavage. A laccaselike activity may be involved in
*

biosolubilization by C. versicolor (11). This article presents

further evidence supporting the participation of laccaselike
activity in biosolubilization of leonardite by an extracellular
fraction.

MATERIALS AND METHODS

Materials. C. versicolor ATCC 12679 and product derived
from fungal action on leonardite were gifts of Martin Cohen
of the University of Hartford. Leonardite was obtained from
American Colloid Co., Skokie, Ill. DEAE-cellulose (MatrexCellufine AM), GC-200 Matrex-Cellufine for gel filtration,
and ultrafiltration membranes were obtained from Amicon
Corp., Lexington, Mass. Piricularia oryzae laccase was
from Sigma Chemical Co., St. Louis, Mo. Proteinase K (20
U/mg) was from Sigma. Bovine pancreas trypsin (2,916
U/mg) and three-times-crystallized porcine stomach mucosal
pepsin (2,700 U/mg) were obtained from CalbiochemBehring, La Jolla, Calif. Buffers and other inorganic and
organic salts were of reagent-grade quality or better.
Determination of leonardite-biosolubilizing activity. An in
vitro assay was used to measure leonardite biosolubilization.
Leonardite was ground to pass a 28-mesh sieve, and 10-mg
samples were placed in separate culture tubes, which were
loosely capped and autoclaved at 121C for 15 min.
Autoclaved leonardite samples were cooled and used immediately or stored at 5C until needed. The total volume of the
assay mixture was 1 ml, and the mnixture contained 0.1 mmol
of sodium phosphate at pH 5.2. The assay mixture was
incubated at the desired temperature with manual mixing
every 3 min. Mixing was kept gentle to keep particles
suspended but off the test tube walls. After incubation, the
mixture was briefly centrifuged at 3,000 x g in a benchtop
clinical centrifuge for 30 s to separate the coal from the
liquid. An aliquot of the supernatant was diluted in water,
and its A290 was measured spectrophotometrically. The
absorbances of a blank lacking enzyme and an enzyme blank
were subtracted from the absorbance of the reaction mix-

Corresponding author.
2844

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Coriolus versicolor has previously been shown to degrade leonardite, an oxidized form of lignite. An
extracellular fraction containing protein purified from a C. versicolor culture solubilized leonardite in vitro.
Expression of the activity did not require the presence of leonardite and appeared during idiophase. During
ion-exchange and gel filtration column chromatography, leonardite-biosolubilizing activity eluted with
syringaldazine oxidase activity and with protein, as measured by A280 and the biuret protein assay.
Syringaldazine is a substrate of the polyphenol oxidase formed by C. versicolor. Comparison of leonarditebiosolubilizing activity with the effects of chelators and surface-active agents on leonardite showed that
biosolubilization was not due to either surfactant or chelating ability. Heat treatment of the preparation at 60C
for 30 min significantly reduced both syringaldazine oxidase and leonardite-biosolubilizing activities. Cyanide,
azide, and thioglycolate, which are known inhibitors of syringaldazine oxidase activity of C. versicolor, also
inhibited leonardite biosolubilization. From these data, we conclude that the purified protein fraction from C.
versicolor contains a syringaldazine oxidase activity that participates in leonardite biosolubilization by
enzymatic action.

VOL. 53, 1987

2.7

1.8

0.9

EXTRACELLULAR SOLUBILIZATION OF LEONARDITE

..

....

purification scheme followed that of Bollag and Leonowicz

(1). Their purification system yielded a protein preparation
that was homogeneous by cetylpyridinium bromide-polyacrylamide electrophoresis. The syringaldazine oxidasecontaining fraction was frozen at -15C until used; it retained 100% of its activity after several months of storage at
that temperature.

._'
OC.200
250
300

350
Wavelength (nm)

400

450

phosphate, pH 5.2; B, 0.1 M sodium citrate, pH 5.2; C, 0.1 M MES,

pH 5.2; and D, 8 ,ug of fungally produced product per ml.
was the value reported for
the action of the active fraction. At pH 5.2, the 0.1 M
phosphate buffer caused very little solubilization of leonardite.
Preparation of crude fraction biosolubilizing leonardite. C.
versicolor was grown in a Chemap CF-20 fermentor at 25C
supplied with 4 liters of filtered air per min at atmospheric
pressure and with 15 liters of growth medium. The growth
medium used was that reported by Fahraeus and Reinhammar (3). A standard two-stage Rushton turbine (six-blade)
agitator system was used at a speed of 400 rpm. Inocula for
the fermentor were three 50-ml Sabouraud maltose broth
cultures of C. versicolor grown for 7 to 10 days at 23C with
no agitation (2). Hyphal mats were transferred to a 500-ml
Erlenmeyer flask with a ground-glass fitting containing 200
ml of distilled water and 100 ml of 3-mm-diameter glass
beads and were shaken vigorously. Mycelial fragments were
then transferred to the fermentor. After 3 days of growth, 0.3
ml of 2,5-xylidine was added to increase the levels of
extracellular laccase activity (3). The production of extracellular enzymes was monitored by measuring syringaldazine
oxidase activity in the culture broth with 7.5 ,uM syringaldazine in a 0.1 M citrate-phosphate buffer (pH 5.2) (1) or a
0.1 M phosphate buffer (pH 5.2) (6). Protein levels were
determined by the biuret method (7).
At 7 days after inoculation, the fermentation broth was
separated from the C. versicolor cell mass by filtration
through three layers of cheesecloth. The resultant fluid was
then filtered through an ultrafiltration membrane with a
molecular weight cutoff of 100,000 (H5P100-43; Amicon).
The high-molecular-weight material in the extracellular fluid
was concentrated with an ultrafiltration membrane with a
molecular weight cutoff of 10,000 (H5P10-43; Amicon). The
resulting retentate (1 liter) was filtered by dialysis with three
300-mi portions of deionized water while being continually
passed through the membrane to maintain a constant volume
of 1 liter. The retentate was clarified by centrifugation at
6,000 x g for 10 min. The resulting supernatant was applied
to a DEAE-cellulose column (2 by 30 cm) that had been
equilibrated with 0.01 M sodium phosphate buffer (pH 7.0).
The column was first washed with 100 ml of 0.01 M sodium
phosphate buffer (pH 7.0), and then adsorbed protein was
eluted from the column with a linear gradient of 200 ml of
0.01 M sodium phosphate (pH 7.0) and 200 ml of 0.5 M
sodium chloride-0.01 M sodium phosphate (pH 7.0). This

RESULTS AND DISCUSSION

Leonardite, an oxidized form of lignite (11), was the
substrate for the biosolubilizing activity of an extracellular
fraction obtained from C. versicolor. The cell-free reaction
was further characterized to develop a dependable in vitro
assay for studying the optimization and mechanisms of the
activity of the cell extract. Figure 1 shows the UV-visible
spectrum of the biosolubilization product obtained by the
action of C. versicolor cultures grown in the presence of
leonardite. Also shown in Fig. 1 are the spectra of buffers
commonly used to assay laccase activity, including 0.1 M
MES (morpholineethanesulfonic acid) (5), 0.1 M citrate, and
0.1 M phosphate (1). From these spectra, it is clear that a
wavelength of 290 nm or above would be useful for the
assay. At wavelengths -290 nm, considerable product
absorbance is evident, while background absorbance due to
buffer is minimal. Table 1 shows A350 of leachates from
representative coals with phosphate, citrate, and acetate
buffers. The phosphate buffer was chosen for routine use
because of its low solubilizing effect on leonardite and its low
absorbance in the spectral region where the product absorbs
strongly.
The enzyme preparation used was a fraction obtained
from DEAE-cellulose by the procedure of Bollag and
Leonowicz (1) for the isolation of laccases. The enzyme
preparation had a barely discernible blue color which, we
presumed, was due to the presence of laccase. Figure 2
shows the time course of the biosolubilizing activity on
leonardite at 23C. Various concentrations of the extracellular fraction were used, and the biosolubilizing activity was
not linear with respect to concentration (Fig. 2, inset). On
the basis of these experiments, an assay period of 20 min was
used in succeeding experiments. The stability of the product
absorbance was constant for more than 2 h after the remaining leonardite particles were filtered off.
The degree of leonardite solubilization by several surfactants was determined. The surfactants tested included
sodium dodecyl sulfate, Tween 80 (polyoxyethylenesorbitan
monooleate), and cetylpyridinium chloride, each in 0.1 M
sodium phosphate buffer (pH 5.2). None of the surfactants
tested solubilized leonardite significantly. Therefore, biosolubilization is probably not due to surfactant ability. When
TABLE 1. Coal leaching by syringaldazine oxidase assay buffersa
A350 of filtrate from:

Buffer and control

0.1 M sodium
phosphate
0.1 M sodium citrate
0.1 M sodium acetate
Water

Leonardite

Texas
lignite

Texas

Beulah
eah
zap

Beulah
standard
no33
no.

3.9

0.65

0.071

0.069

14.5b
3.48

0.84
0.047
0.28

1.455
0.037
0.163

2.74
0.036
0.051

2.62

" A 400-mg sample of coal was added to 4 ml of buffer or water. The mixture
was incubated for 2 days. Coal was removed by filtration, and the optical
density of the filtrate was measured at 350 nm.
b Estimated by diluting an aliquot of the solution in water.

Downloaded from http://aem.asm.org/ on February 14, 2012 by guest

FIG. 1. Absorption spectra of reaction buffers and reaction product. The UV-visible spectra of laccase assay buffers are compared
with the spectrum of the biosolubilization product. A, 0.1 M sodium

ture. This corrected absorbance

2845

2846

APPL. ENVIRON. MICROBIOL.

PYNE ET AL.

Time (min)

FIG. 2. Initial rates of leonardite biosolubilization at different

enzyme concentrations. Each point represents a value for a separate
assay from which the values for blanks were subtracted. Incubation
temperature was 23C. The ordinate shows the optical density of the
reaction mixture estimated from a diluted sample taken from the
reaction mixture. The initial reaction rates are plotted in the inset,
which shows the nonlinearity of the rates with protein concentrations (in milligrams per milliliter) as follows: 0, 0.06; A, 0.12; *,
0.18.

'._
0.

< I

.)

c
0 l

.Z

.5

=m: "IE

_ g

SCC.;.,
cn <
4D
Ij

WO E
C

co

C.

0
.
-

Co

;J

Citrate Concentration (mM)

FIG. 3. Effects of citrate on in vitro biosolubilization of

leonardite and syringaldazine oxidation activity. Each point represents the difference between the optical density of the reaction
mixture with purified extracellular fraction and that of a blank
without extracellular fraction. The incubation time was 40 min. The
amount of protein used was 0.1 mg of the crude preparation
containing biosolubilizing activity.

suggests that the reaction is not due just to chelation,

because chelation by citrate and the extracellular fraction
should be merely additive. These results are consistent with
the ability of syringaldazine oxidase or a similar protein to
solubilize leonardite. At low citrate concentrations, chelation by citrate and the action of the preparation are additive.
At higher citrate concentrations, direct inhibition by citrate
lowers net biosolubilizing activity.
The above-mentioned results differentiated leonarditebiosolubilizing activity from chelation or detergent action
and suggested that the activity may be enzymatic in nature.
To further test this hypothesis, the effect of heat treatment
on syringaldazine oxidase activity and leonardite biosolubilization was determined. Boiling the enzyme solution
for 10 min destroyed both activities. Loss of activity after
boiling occurs with most enzymes. Another characteristic of
proteins is that heat denaturation has a rather sharp transition compared with denaturation by other types of catalysts.
This type of transition does occur with the preparation
containing leonardite-biosolubilizing activity; activity was
partially destroyed by heating a sample of the purified
preparation (0.7 mg of protein per ml) for 30 min at 60C.
Under these conditions, 81% of the original leonarditebiosolubilizing activity and 29% of the syringaldazine oxidase activity remained. At and below 53C, there was no loss
in syringaldazine oxidase or leonardite-biosolubilizing activity after 30 min of treatment. The difference in the heat
labilities at 53 and 60C of the preparation containing
biosolubilizing activity further suggests that the activity is
enzymatic.
Another way to determine whether the leonardite-biosolubilizing activity is enzymatic is to test the lability of
activity in the presence of proteases. The purified preparation was treated with trypsin at 1 mg/ml, pepsin at 1 mg/ml,
or proteinase K at 0.1 mg/ml in the presence of 0.2% sodium
dodecyl sulfate for 3 days at room temperature and pH 5.2.
This treatment caused no loss of syringaldazine oxidase
activity or leonardite-biosolubilizing activity. However, a
commercial preparation of laccase from P. oryzae was also
not degraded by trypsin or pepsin under the conditions
described above. The resistance of laccase and C. versicolor
syringaldazine oxidase activities to proteases is probably

Downloaded from http://aem.asm.org/ on February 14, 2012 by guest

present in the laccase assay, surfactants inhibited the activity of C. versicolor culture preparations. On a molar basis,
cetylpyridinium chloride was the most potent inhibitor of
syringaldazine oxidase activity of all the surfactants tested.
Sodium dodecyl sulfate at 35 mM inhibited syringaldazine
oxidase activity by 83%, 1% Tween 80 inhibited activity by
56%, and 1 mM cetylpyridinium chloride inhibited activity
by 45%. With 1 mM cetylpyridinium chloride, biosolubilization of leonardite was inhibited by 83%. These results
suggest that biosolubilization is enzymatic, since proteins
are often denatured or inhibited by surfactants. Other catalysts, such as metal ions or low-molecular-weight organic
compounds, would probably not be affected by surfactants.
Low-rank coals may be held together, in part, by salt
bridging by multivalent metal ions (5). Control experiments
were conducted to show that biosolubilization is not due to
the chelating ability of the extracellular fraction. Several
chelators significantly solubilized leonardite in solutions
containing 0.1 M sodium phosphate buffer (pH 5.2). After
incubation for 20 min at 23C with gentle agitation, A290
values were 0.12 for 1 mM citrate, 0.42 for 1 mM
nitrilotriacetate, 0.81 for EDTA, and 0.30 for 1 mM tartrate.
These chelators affected the C. versicolor syringaldazine
oxidase activity to various degrees. At a concentration of 1
mM, chelators either had no effect or stimulated syringaldazine oxidase activity slightly. Nitrilotriacetate (1 mM)
added to the assay mixture yielded 112% of the control
activity; EDTA, tartrate, and citrate added to the assay
mixtures yielded 109, 103, and 99%, respectively, of the
control activity.
The effects of various concentrations of citrate on
leonardite biosolubilization, with or without the extracellular
fraction, are shown in Fig. 3. Citrate effectively solubilized
leonardite at concentrations greater than 0.3 mM and inhibited syringaldazine oxidase activity at concentrations greater
than 1 mM. Interestingly, citrate stimulated the solubilization of leonardite by the extracellular fraction at concentrations between 0.1 and 0.3 mM. This stimulation strongly

VOL. 53, 1987

EXTRACELLULAR SOLUBILIZATION OF LEONARDITE

1 .4

U.4
V.

1.2

_ 1.75-

U2-

1.t

0.3

x
1'

I 1.0

z
0.2 o

E
E

0.8

0.1 E
E

co

i?

o~~~~~~~~~r
n
:o 0.4

0.2

-.7
-

s,

10

20

30

40

m
N

50

.Z
._

_0.50 .'
'D

0,

_0.25 cnc
._

I
0

C
0

_ 0.75
0

_ 1.00 E

.4

._
0

\
I

_ 1.25 E

CD

60

70

co
J

FIG. 4. Chromatography of leonardite-biosolubilizing activity on

DEAE-cellulose. Crude preparation was diluted 1:10 with water and
then applied to a DEAE-cellulose column (1.5 by 28 cm; MatrexCellufine AM; Amicon) previously equilibrated with 0.01 M sodium
phosphate (pH 7.0). Syringaldazine oxidase and leonardite-biosolubilizing activities were estimated as described in Materials and
Methods. The elution profile of protein was determined spectrophotometrically by its A280. Protein was eluted with a gradient of 0 to 0.3
M sodium chloride, and 3 ml of eluate was collected. Salt concentration was determined by measuring ionic strength.

because these enzymes are glycoproteins and are adapted

for function in an extracellular milieu containing proteases.
High syringaldazine oxidase activity in the purified cellfree preparation suggested that this activity may be related
to leonardite-biosolubilizing activity. The purified preparation containing leonardite-biosolubilizing activity was rechromatographed on DEAE-cellulose. A gradient of 100 ml
of 0.01 M sodium phosphate (pH 7.0) and 100 ml of 0.3 M
sodium chloride-0.01 M sodium phosphate (pH 7.0) was
used to elute adsorbed proteins from the column. Leonardite-biosolubilizing activity eluted with syringaldazine
oxidase activity. A280 paralleled the elution patterns of
syringaldazine oxidase and leonardite-biosolubilizing activities. The coelution of syringaldazine oxidase and leonarditebiosolubilizing activities, along with protein, is strong evidence that syringaldazine oxidase activity is responsible for
leonardite-biosolubilizing activity (Fig. 4).
The concentration of protein in the peak fraction from the
DEAE-cellulose column was estimated to be 0.2 mg/ml. By
using the concentration of protein calculated from the biuret
assay and the molecular mass for syringaldazine oxidase of
64,000 daltons (1), the concentration of protein in the peak
fractions (Fig. 4, fraction 41) was determined to be approximately 7 F.M. To produce the color equivalent to that given
by the fractions containing biosolubilizing activity, the concentration of chelator should be on the order of 0.1 M, or
more than 10,000 times greater than the protein concentration. The concentration of protein in the active fractions was
too low for the protein to be an effective metal ion chelator.
Each peptide chain would have to chelate at least 5,000
divalent ions, which carry at least 100 times more negative
charges than the positively charged side chains in the
amount of protein present (3).
A second chromatography procedure supplied additional
information about the relationship between leonarditebiosolubilizing activity and syringaldazine oxidase activity.
The purified protein preparation (2.8 g of protein) was

precipitated by adding solid ammonium sulfate to saturation.

The precipitate was collected by centrifugation at 8,000 x g,
dissolved in 0.5 ml of 0.05 M ammonium bicarbonate, and
applied to the gel filtration column (GC-200 Matrex-Cellufine
[Amicon] equilibrated with 0.05 M ammonium bicarbonate).
Fractions (1.5 ml) were collected and assayed for syringaldazine oxidase activity (Fig. 5). Syringaldazine oxidase
activity could be readily detected, but leonardite-biosolubilizing activity could not be detected in the columneluted fractions. The inability to detect leonardite biosolubilization was expected because of the diluting effect of
gel permeation chromatography. Fractions from the column
with A280 were combined in three pools, lyophilized, and
dissolved in a small volume of 0.01 M ammonium bicarbonate. Only the first of the three pools of fractions (Fig. 5)
contained significant leonardite-biosolubilizing and syringaldazine oxidase activities. The coelution of syringaldazine
oxidase and leonardite-biosolubilizing activities from the gel
filtration column is additional evidence that these two activities result from the same protein. The elution position of the
activities corresponds to a protein having a molecular mass
of at least 50,000 daltons.
Cyanide and azide are effective inhibitors of laccase
activity (1), and both inhibit cell-free biosolubilization of
leonardite. Cyanide at 1 mM inhibits C. versicolor syringaldazine oxidase activity by 40% and leonardite-biosolubilizing activity by 10%, whereas azide at 0.01 mM inhibits
syringaldazine oxidase activity by 70% and biosolubilizing
activity by 30%. Syringaldazine and leonardite, the substrates used in the assays, are different enough chemically
that the difference in inhibition is expected. There may be a
difference between the rate-limiting steps of the two reactions or, more likely, there may be two or more partially
rate-limiting steps in the catalytic reaction sequence which
are affected differently by the inhibitors. The effects of
cyanide and azide on the leonardite-biosolubilizing reaction
are further evidence that the reaction is catalyzed by enzymes.

3 Pooled Fractions

0.7

7.0

lb

0.6

I I

6.0

5.0

5.0En

D 0.5

II

al4
v
c

0.4

4.0 X

I_

x
0

3.0

o 0.3

._

to

0.2

2.0 X

9~

C
1.0un

0.1
c
--

10

15

.I

20
25 30
Fraction Number

35

40

45

50

FIG. 5. Gel permeation chromatography of crude preparation

containing leonardite-biosolubilizing activity. The crude preparation
containing leonardite-biosolubilizing activity (2.8 mg of protein) was
first precipitated by saturating the solution with ammonium sulfate.
The precipitate was collected by centrifugation at 3,000 x g. The
precipitate was suspended in 0.5 ml of 0.05 M ammonium bicarbonate, and this solution was applied to a gel filtration column (1 by 40
cm; GC-200 Matrex-Cellufine; Amicon). Fractions (1.5 ml) were
collected at 2-min intervals. Syringaldazine oxidation (0) and protein A280 (
) were determined for individual fractions.

Downloaded from http://aem.asm.org/ on February 14, 2012 by guest

Fraction Number

2847

2848

PYNE ET AL.

This project was

Research Institute.

ACKNOWLEDGMENT
funded by a contract with the Electric Power

LITERATURE CITED
1. Bollag, J.-M., and A. Leonowicz. 1984. Comparative studies of
extra-cellular fungal laccases. Appl. Environ. Microbiol. 48:
849-854.
2. Cohen, M. S., and P. D. Gabriele. 1982. Degradation of coal by
the fungi Polyporus versicolor and Poria monticola. Appl.

Environ. Microbiol. 44:23-27.

3. Fahraeus, G., and B. Reinhammar. 1967. Large scale production
and purification of laccase from cultures of the fungus Polyporus
versicolor and some properties of laccase A. Acta Chem. Scand.
21:2367-2378.
4. Fowkes, W. W., and C. M. Frost. 1960. Leonardite: a lignite
byproduct. Bureau of Mines, U.S. Department of Interior
report no. 5611. Government Printing Office, Washington, D.C.
5. Knudson, D. L., and S. A. Farnum. 1985. Subbituminous coal
characterization, p. 1.1-1.26. In Proceedings of the Tenth
Annual EPRI Contractors' Conference on Clean Liquid and
Solid Fuels, April 23-25, 1985, Palo Alto, Calif. Electric Power
Research Institute, Palo Alto, Calif.
6. Leonowicz, A., and K. Grzywnowicz. 1981. Quantitative estimation of laccase forms in some white-rot fungi using syringaldazine as a substrate. Enzyme Microb. Technol. 3:55-58.
7. Robinson, H. W., and C. G. Hogden. 1940. The biuret reaction
in the determination of serum proteins. II. Measurements made
by a Duboscq colorimeter compared with values obtained by the
Kjeldahl procedure. J. Biol. Chem. 135:727-731.
8. Strandberg, G. W., and S. N. Lewis. 1987. The solubilization of
coal by an extracellular product from Streptomyces setonii
75Vi2. J. Ind. Microbiol. 1:371-375.
9. Ward, H. B. 1985. Lignite-degrading fungi isolated from a
weathered outcrop system. System. Appl. Microbiol. 6:236238.
10. Wilson, B. W., E. J. Lewis, D. L. Stewart, and S. M. Li. 1985.
Microbial conversion of coal, p. 31.1-31.8. In Proceedings of
the Tenth Annual EPRI Contractors' Conference on Clean
Liquid and Solid Fuels, April 23-25, 1985, Palo Alto, Calif.
Electric Power Research Institute, Palo Alto, Calif.
11. Wilson, B. W., E. J. Lewis, D. L. Stewart, S. M. Li, R. M. Bean,
E. K. Chess, J. Pyne, M. Cohen, and H. Aronson. 1986.
Microbial processing of fuels, p. IV88-IV98. In Proceedings of
the Department of Energy Coal Liquefaction Contractors'
Meeting, Pittsburgh, Pa., November 19-21, 1985. NTIS, Springfield, Va.

Downloaded from http://aem.asm.org/ on February 14, 2012 by guest

Several lines of evidence demonstrate that leonardite

biosolubilization is catalyzed by a C. versicolor syringaldazine oxidase. Both syringaldazine oxidase and biosolubilizing activities are reduced by preincubation at 60C but not
at 53C. A sharp decline in activity with a small increase in
temperature indicates an enzymatic activity. Both activities
were inhibited to various degrees by surfactants, azide, and
cyanide. The two activities coeluted during chromatography
on DEAE-cellulose and gel filtration columns. The accumulated evidence strongly suggests that the agent responsible
for leonardite biosolubilization is an enzyme and that the
enzyme has a syringaldazine oxidase activity.
The leonardite-biosolubilizing activity of C. versicolor can
possibly be used as the first step in a low-temperature,
low-pressure approach to coal beneficiation. The results
presented here show that biosolubilizing activity is associated with a specific enzymatic activity. Future work will
focus on enhancing the production of the enzyme activity by
both selection techniques and manipulation of fungal cultural
parameters. A second area of research will be the examination of the use of the extracellular material for solubilizing
lignites and coals.

APPL. ENVIRON. MICROBIOL.