APPLIED MICROBIOLOGY, Nov., 1966 Vol. 14, No.

6
Copyright ( 1966 American Society for Microbiology Printed in U.S.A.

Solubilization of Acid-Swollen Cellulose by an

Enzyme System from a Species of Alternariat
ROBERT M. LOGAN2 AND DONALD J. SIEHR
Department of Chemical Engineering and Department of Chemistry, University of Missouri at Rolla,
Rolla, Missouri
Received for publication 14 July 1966

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ABTRACT
LOGAN, ROBERT M. (University of Missouri, Rolla), AND DONALD J. SIEHR. Sol-
ubilization of acid-swollencellulose by anenzyme system from a species of Alternaria.
Appl. Microbiol. 14:1015-1018. 1966.-An unknown species of Alternaria, when
grown on a medium containing carboxymethylcellulose as a carbon source produced
a mixture of extracellular enzymes which solubilized acid-swollen cellulose. The prod-
uct of the hydrolysis was a 1:2 molar mixture of cellobiose and glucose. The or-
ganism apparently produced no cellobiase. It is suggested that the mixture of cell-
ulolytic enzymes contains at least two different enzymes which degrade cellulose in an
endwise manner.

The cell-free filtrates of Myrothecium verrucaria substances to test its cellulolytic activity. It grew well
(7) and Tricoderma viride (10) and Tricoderma in a salts medium containing filter paper as the sole

konigi (6) are among the most active sources of carbon source.
cellulolytic fungal enzymes studied so far. These The cultures of Tricoderma viride NRRL-2314 and
mixtures of enzymes have been extensively Myrothecium verrucaria NRRL-2003 were obtained
from the Northern Utilization Research and Develop-
studied, with the aim of determining the manner ment Division of the Agricultural Research Service,
in which they bring about the breakdown of Peoria, Ill.
cellulose. The Alternaria as a class, however, are Cultivation of the organism. The culture fluid used
not known to produce active cellulases, although in this study was obtained from fermentations cafried
a recent paper (9) described an exoenzyme from out in a 5-liter apparatus constructed previously (R.
A. tenuis which degrades cellulose. M. Logan, M. S. Thesis, University of Missouri,
We wish to report some observations we have Rolla). The apparatus was very similar to the one
made on a mixture of at least two enzymes found described by Ellsworth (1).
in the fluid culture in which a species of Alter- Temperature was controlled within 0.5 C, and
foaming was controlled by the addition of General
naria was grown. This enzyme mixture rapidly Electric Silicone Antifoam 10 as required.
degrades swollen cellulose to a nearly 1:2 molar The medium used for the cultivation of the organ-
ratio mixture of cellobiose and glucose, and ap- ism was that described by Reese and Mandels (10).
parently no extracellular cellobiase is formed by The carbon source was carboxymethylcellulose (CM
the organism. cation exchanger, fine mesh; Sigma Chemical Co.,
St. Louis, Mo.).
MATERIALS AND MErHODS In each run, a volume of 2.5 liters was used. A
Organisms. A species of Alternaria was isolated 500-ml separatory funnel was connected to a port in
during work with Sorangium cellulosum. It appeared, the top of the fermentor, and was filled with water
apparently as a contaminant, in attempts to grow the and sterilized with the fermentor for each run. This
myxobacterium on starch in shake-flask culture. The water was used to make up for evaporation losses
Alternaria grew in the starch medium, forming a occurring during the fermentation.
heavy white filamentous growth. The organism was The fermentor and substrate were sterilized in an-
isolated on 4% malt-agar and applied to cellulosic autoclave at 120 C for 40 min. The inoculum for the
fermentor was grown in shake flasks on 4% malt ex-
tTaken from the dissertation submitted by Robert tract.
M. Logan as partial fulfillment of the requirements Analytical. Two substrates were used for the assay
for the Ph.D. degree, The University of Missouri at of cellulase activity. "Swollen cellulose" was prepared
Rolla. by the method described by Reese and Mandels (10).
2Present address: Clinton Corn Processing Co., Whatman cellulose powder was also used. Cellulase-
Clinton, Iowa. 35 obtained as a gift from Rohm and Hass, Phila-
1015
1016 LOGAN AND SIEHR APPL. MICROBIOL.
delphia, Pa., was used as a standard for determining TABLE 2. Comparison of the cellulolytic activities
the uniformity of the swollen cellulose preparation p rof enzymes from the Aliernaria sp. and of cellu-
and as a comparison of cellulase activity. Total or- lase-35 upon swollen cellulose
ganic solids were determined by the method of Halli-
well (3, 4), reducing sugars by the nitrosalicylate Enzyme Protein Per cent Specific
method (11), and protein by a spectroscopic method concn hydrolysis activity
(8). Glucose was determined by incubating a pH 5.6 mg/ml
glucose solution and glucose oxidase for 4 to 8 hr at
30 C and determining the amount of reducing sugar Cellulase-35 ........ 2.40 18.8 7.84
left in the solution at the end of the incubation. Alternaria sp ........ 0.265 40.8 154
The qualitative analysis of the hydrolysis products
was done on ascending, single-directional paper chro-
matograms on 5 cm wide strips of Whatman no. 4 TABLE 3. Comparison of the increase in reducing
chromatographic paper. The solvent systems and sugar with increase in total organic solids in

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locating reagent were those described by Smith (11) assay hydrolysate
for the analysis of carbohydrates by paper chro-
matography. The solvent systems employed were Hydrolysis products (mg/ml) Amt (mg). of
isopropanol-water and isopropanol-pyridine-acetic Assay organic solids
per mg of
acid-water. The locating reagent was aniline-diphenyl- Reducng sugar Organic solids reducing sugar
amine.
A 50-ml sample of culture fluid was removed from 1 2.10 2.64 1.26
the fermentor. After centrifugation, the supernatant 2 2.10 2.83 1.35
fluid was filtered through a sintered-glass bacterial 3 1.06 1.44 1.36
filter to remove particles of mycelium. The filtrate was
diluted to one-half of its original volume, and the pH Avg 1.32
was adjusted to 5.5 with 1:4 phosphoric acid. A 4-ml
amount of the enzyme solution was added to 1 ml of
a swollen cellulose suspension containing 10 mg (dry
weight) of cellulose per ml of water, and the mixture mixture formed by the Alternaria is shown in
was incubated at 40 C for 2.5 hr. The incubation was Table 2. The activity of the Alternaria enzymes on
carried out in 50-ml Erlenmeyer flasks in a New Bruns- a protein basis is about 20 times that of cellulase-
wick model RW152 water bath. At the end of the 35. A similar comparison with the cellulases
incubation period the contents of the flasks were produced by Tricoderma viride NRRL-2314 and
transferred to centrifuge tubes and were centrifuged Myrothecium verrucaria NRRL-2003 showed
to remove the unreacted cellulose. The increase in that the Alternaria enzymes were two to four
reducing sugar over the diluted-enzyme blank was times more active.
determined. The per cent hydrolysis was then calcu-
lated as the milligrams of reducing sugar formed per Paper chromatographic separation of the
milliliter divided by the weight of cellulose per milli- hydrolysis products gave two spots which were
liter in the original assay solution. identified as glucose and cellobiose from com-
parisons of the RF values of standards and un-
RESULTS knowns run simultaneously. Also, it was found
The results from a typical fermentation are that, if either glucose or cellobiose was added to
shown in Table 1. a solution of the hydrolysis products and these
A comparison of the cellulase activity on solutions were chromatographed, only two spots
swoUlen cellulose of cellulase-35 and of the enzyme were visible on the paper chromatograms after
spraying with a solution of aniline-diphenyl
TABLE 1. Cellulase activity of cell-free mediuma amine. No polysaccharides of higher molecular
weight were observed on the chromatograms.
Age Proei
Sample Culture P (mtein
Substrate AsyPer cent The total organic solids present at the end of a
uure g5 Assay hydrol-
/ml)
~~~~~~~ysis 2.5-hr assay period was always greater than the
total reducing sugar (Table 3). The ratio of
hr mg/ml organic solids to reducing sugars was 1.32 on a
3 181 ND SC 3.9 11.9 weight basis. Based on the fact that ceUobiose
4.4 23.1 and glucose were the only products formed dur-
5.3 36.5 ing the hydrolysis of cellulose by the Alternaria
5.4 38.2 enzymes, the hydrolysis mixture contained 51.5%
5.8 37.0
4 205 ND PC 5.5 0.34 glucose and 48.5% cellobiose.
5 208 0.265 SC 5.5 40.8 To confirm the presence of glucose in the
hydrolysis mixture, a sample of a hydrolysis mix-
a SC = swollen cellulose; PC = Whatman cellu- ture was treated with glucose oxidase. At the
lose powder; ND = not determined. start of the incubation, the total reducing sugar
VOL. 14, 1966 ENZYME SYSTEM FROM ALTERNARIA
in the sample was 2.49 mg/ml, as determined by
the nitrosalicylate method. After 4 hr, 1.72 mg/ml
of reducing sugar remained. Thus, during the
incubation, 1.77 mg/ml of reducing sugar had
disappeared. This experiment substantiates the
observation that, on a weight basis, cellobiose and
glucose occur in a 1:1 ratio in the hydrolysis
mixture.
Because of the relatively large amount of
cellobiose formed upon hydrolysis, it was pos-
sible that there was no cellobiase present in the
Alternaria culture medium. To determine whether
cell-free extracts from Alternaria cultures con-
tained cellobiase, two separate culture media were
tested for their action on cellobiose.
In the first experiment, an enzyme solution was
applied to swollen cellulose in the usual manner.
After 2.5 hr of incubation, the hydrolysate was
separated from the insoluble cellulose by centrif-
ugation. A sample was taken and analyzed for
reducing sugar content. The remainder of the
hydrolysate was incubated for an additional 65
min. After the second incubation period, the
solution was again analyzed for reducing sugar
content to determine whether any increase had
occurred. The total amount of reducing sugar
present after the 1-hr incubation period was
1.70 mg/ml. After the additional incubation of
65 min, the reducing sugar concentration was
1.73 mg/ml. Since the two values were very
nearly the same, it was concluded that little or
no hydrolysis of cellobiose occurred during the
second incubation period.
In another test for the presence of cellobiase, an
enzyme solution was added to a solution of cello-
biose in two 50-ml Erlenmeyer flasks. The two
flasks were incubated at 40 C, one for 2.5 and the
other for 7.5 hr. The analysis of the solutions
after incubation showed a very small increase
in reducing sugar. It would appear that this
species of Alternaria produces very little if any
extracellular cellobiase.
DISCussIoN
The use of the fermentor rather than shake
flasks was preferred, since a fairly large volume
(50 ml) of culture media could be removed with-
out an appreciable change in the volume of
medium in the fermentor. This also eliminated
the variations in activity found when a series of
shake flasks was used.
Cell-free extracts from cultures of Alternaria
were found to rapidly hydrolyze swollen cellulose.
Little breakdown of filter paper was brought
about by these extracts. The hydrolysis products
formed from the action of Alternaria culture
broths on swollen cellulose were found to be
glucose and cellobiose in nearly a 1:1 weight
1017
ratio. Since the culture broths were also found to
be essentially free from cellobiase and polysac-
charide, it can be concluded that a minimum of
two enzymes attacking swollen cellulose are
present in the culture fluid of this species of
Alternaria.
The enzyme solution prepared from the Alter-
naria cultures differed from cellulase-35 and from
cellulase prepared from cultures of M. verrucaria.
Glucose was found to be the only hydrolysis
product when the latter two enzyme preparations
were applied to swollen cellulose under the con-
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ditions used.
Since a significant amount of cellobiose was
formed when enzyme solutions prepared from
cultures of Alternaria were used, the per cent
hydrolysis of the swollen cellulose, calculated on
the basis of the reducing sugar, was not a true
indication of the actual solubilization of the
substrate. The solubilization, as determined by
total organic solids analyses, was found to be
1.32 times greater than that obtained on the
basis of the reducing sugar. Consequently, when
a 40% hydrolysis of the swollen cellulose was
reported, the actual per cent solubilization was
nearly 53 %. With some of the more active Alter-
naria extracts, total solubilization of the swollen
cellulose substrate was approached in the 2.5-
hr incubation period. This finding was substan-
tiated by observations of the amount of in-
soluble cellulose remaining after incubation.
The hydrolyzing power of Alternaria culture
media was found to be about 20 times that of
cellulase-35 on a protein content basis. Since
only glucose was produced by the action of
cellulase-35 on swollen cellulose, but both glucose
and cellobiase were produced by Alternaria cul-
ture media, the solubilization power of the
Alternaria enzymes was still more efficient.
Since the product formed during the hydrolysis
of swollen cellulose is a 2:1 molar ratio of glu-
cose and cellobiose, and since no cellobiase
activity was found in cell-free extracts, apparently
there exist in the mixture of cellulolytic enzymes
produced by this Alternaria species two enzymes
which attack the cellulose in an end-wise manner.
It is possible that the failure to observe any cel-
lobiase activity might be due to the fact that the
cellobiase formed by this species of Alternaria is
strongly inhibited by glucose. However, the ob-
servation that there was very little hydrolysis of
cellobiose by the cell-free culture fluid would
tend to negate this possibility.
AcKNowLEDmENTrs
We are indebted to C. W. Hesseltine of the
Northern Utilization Research and Development Divi-
sion, U.S. Department of Agriculture, Peoria, Ill.,
1018 LOGAN A.NID SIEHR
who identified the organism as a member of the genus
Alternaria. No species name could be assigned to it.
The culture was lyophilized and stored as NRRL-13, 7.
646.
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