Inexpensive, Rapid Procedure for Bulk
Purification of Cellulase-Free
P-l,4-~-Xylanaseof High Specific Activity
Larry U. L. Tan,* Ernest K. C. Yu, Gerald W. Louis-Seize, and
John N. Saddler
Biotechnology and Chemistry Department, Forintek Canada Corp.,
800 Montreal Road, Ottawa, Canada
Accepted for publication July 7, 1986
A process has been developed for the bulk purification
of cellulase-free P-1,4-o-xylanase from the fungus Tri-
choderma harzianum E58. The process involved the pri-
mary step of ultrafiltering the culture filtrate via a 10,000-
molecular-weight cut-off membrane to separate the cel-
lulase (retentate) and xylanase (permeate) fractions. The
cellulase component was concentrated by 40- to 60-fold,
resulting in an enzyme complex that could effectively
hydrolyze high concentrations of cellulose and xylan to
glucose and xylose. The xylanase was concentrated and
solvent exchanged by adsorption to a cationic exchanger,
SP-ZetaPrep 250, followed by elution with a pH change
in the buffer to give a purified and concentrated xylanase
complex dissolved in a low-salt buffer. The resultant xy-
lanase system was pure by the criteria of sodium dodecyl
sulfate polyacrylamide electrophoresis, had a very high
specific activity of 2400 IUimg protein, was virtually free
of filter paper activity, and had a ratio of contaminating
filter paper activity of 2 x (0.009% endoglucanase
activity). Approximately 3.3 g protein, which contained
in excess of 7 x lo6 IU xylanase activity, was obtained
from 17 L original culture filtrate. The process scheme
was designed to facilitate scale-up to an industrial level
of production.
INTRODUCTION
Lignocellulose, the world’s largest renewable bio-
mass resource, is composed mainly of lignin, cellulose,
and hemicellulose, of which a large part of the latter
is xylan.’ Much of the current research and develop-
ment effort has been directed toward the utilization of
the cellulose fraction for liquid fuel production. How-
ever, if value-added products could be obtained from
the hemicellulose and lignin streams, the economics of
the process could be significantly improved.
For a number of years, our laboratory2 has been
working on a process where aspenwood chips are first
* To whom all correspondence should be addressed; present ad-
dress: Allelix, Inc., 6850 Goreway Dr., Mississauga, Ontario L4V
IP1. Canada.
Biotechnology a n d Bioengineering, Vol. XXX, Pp. 96-100 (1987)
0 1987 ..John Wilev 81S o n s , Inc.
steam exploded to enhance their subsequent enzymatic
hydrolysis to fermentable sugars. The hemicellulose,
lignin, and cellulose streams can then be separated by
selectively extracting the pretreated material by water
and dilute alkali. The cellulose and hemicellulose streams
are then, respectively, hydrolyzed by cellulases and
xylanases and fermented to ethanol and 2,3-butanediol
by the appropriate microorganisms.
Previously, we had shown that high levels of cel-
lulase and xylanase were efficiently produced by the
fungus Triclioderma harzianum E58.3 As the hemicel-
lulose- and cellulose-derived sugars are normally uti-
lized by different microorganisms for various products,
the hemicellulose and cellulose fractions are routinely
separated into different streams in our process.2 Ide-
ally, if an inexpensive process is available for the sep-
aration of the xylanase complex from the cellulase
complex, the two enzyme streams could be efficiently
utilized for the hydrolyses of the hemicellulose and
cellulose streams, respectively, while reducing the
overall cost of separate enzyme production steps.
A purified cellulase-free xylanase could also be used
for the removal of contaminating hemicellulose com-
ponents from high-grade cellulose pulps. Other workers4
have been partially successful at reducing the amount
of hemicellulose in aspen mechanical pulps using pu-
rified xylanase from Schizophyllum commune. Unfor-
tunately, the viscosity of the pulp was also reduced,
possibly because of the relatively high concentration
(-3%) of contaminating endoglucanase activity that
was present in their preparation. In addition, the cost
of producing the xylanase may be high since their pro-
cess depended on fractional precipitation using a vol-
ume ratio of 3: 1 ethanol-culture filtrate. An inexpen-
sive process that produces virtually cellulase-free
xylanase may be more amenable to this application.
Other potential uses of xylanasesS include processes
for the manufacture of liquid coffee, the adjustment of
CCC 0006-3592/87/’010096-05$04.00
wine characteristics, and the enhancement of astax-
anthin (3,3'-dihydroxy-4,4'-diketo-p-carotene) extrac-
tion. Hemicellulases (a group of enzymes that includes
the xylanases) may also be used in the food industry
for the clarification of fruit juices.6
MATERIALS AND METHODS
Culture Conditions
The fungus Trichoderma harzianum E58 was ob-
tained from the Forintek culture collection and grown
in a 30-L fermentor using 1% (w/v) Solka Floc B.W.
300 FC (Brown and Co.,Berlin, NH) as carbon source
as described e1sewhe1-e.~
Bulk Purification of Xylanase
Seventeen liters of a 4-day-old T . harzianum E58
culture was filtered through Whatman No. 1 paper to
obtain the crude culture filtrate. The culture filtrate was
ultrafiltered on a Pellicon apparatus (Millipore Ltd.)
fitted with 0.47 m2 polysulfone membrane with a mo-
lecular weight cut-off of 10,000 daltons. Ultrafiltration
was performed at approximately 6 and 45 L/h for the
filtration and recirculation rates, respectively, until ap-
proximately I-2% of the retentate remained. To obtain
maximum recovery of retained protein, the membrane
was flushed with 125 mL 50mM sodium citrate buffer,
pH 4.8. The cellulase complex, which was retained,
was concentrated by 40- to 60-fold, while 62% of the
original xylanase was detected in the filtrate. The ul-
trafiltrate containing the xylanase was diluted with an
equal volume of water, and the pH was adjusted to 4
with acetic acid. The xylanase was concentrated and
solvent exchanged by binding to a cationic exchanger,
SP-ZetaPrep 250 cartridge (7 cm diameter by 7 cm
height) (AMF Molecular Separations Division, Meri-
den, CT), and equilibrated with lOmM sodium acetate
buffer, pH 4, at a flow rate of 7.5 L/h. The cartridge
was washed with 2 L lOmM sodium acetate buffer, pH
4, and eluted with 2 L 50mM sodium phosphate, pH
8. Fractions of 250 mL were collected. The eluates
were immediately titrated with acetic acid to pH 5.
Alternative Methods of Xylanase Concentration
Ultrafiltration was performed with the Pellicon ap-
paratus fitted with 0.47 m2 of a cellulosic membrane
with a molecular weight cut-off of 1000 daltons. The
filtration and recirculation rates were 0.9 and 9 L/h,
respectively. Concentration by ammonium sulfate pre-
cipitation was carried out at 4°C using an ammonium
sulfate concentration of 20,40,60,and 80% saturation.
The mixtures were stirred for 30 min and vacuum fil-
tered via glass fiber disks. The disks containing the
precipitated xylanase were macerated in 5 mL 50mM
TAN ET AL.: PURIFICATION OF CELLULASE-FREE B-1.4-D-XYLANASE
sodium acetate, pH 4.8,stirred at 4°C for 30 min, and
centrifuged at 10,000g for 15 min. The supernatants
were assayed for enzyme recovery. Ethanol and ace-
tone precipitations were performed using final concen-
trations of 20,40,60,and 80% of the solvents prechilled
to -60°C. Following the addition of the solvents, the
mixtures were stirred at 4°C for 1 min, and the pre-
cipitates were processed identically to those of the
ammonium sulfate precipitates.
Assays
Xylanase and endoglucanase activities were assayed
in 50mM sodium citrate buffer, pH 4.8,at 50°C.One
milliliter of an appropriately diluted enzyme was added
to an equal volume of 1% (w/v) substrate and incubated
for 30 min. Reducing sugar was determined by the 3 5
dinitrosalicyclic acid method.* Oat spelt xylan and car-
boxymethylcellulose (Sigma Chemicals, St. Louis, MO)
were used as substrates for the xylanase and endog-
lucanase assays, respectively. Filter paper activity was
assayed by the method of Mandels et al.' Enzyme units
were expressed as micromoles of D-xylose or D-glucose
equivalents released per minute. Protein was deter-
mined by the method of Lowry et a1.I0 as modified by
Tan et al."
RESULTS AND DISCUSSION
As shown elsewhere,I2 the xylanase and cellulase
enzyme components present in the culture filtrates of
T. harzianum E58 can be efficiently and rapidly sep-
arated by Pellicon ultrafiltration using a polysulfone
membrane with a molecular weight cut-off of 10,000
*r
0 1 2 3 4 5
FRACTION N U M B E R
X Y L A N A S E (IUi PROTEIN (UG)
0 SPECIFIC ACTIVITY x (iU/rng)
Figure 1. Elution profile of SP-ZetaPrep 250 column charged with
6 x 106 IU xylanase activity (3.3 g protein). Fractions of 250 mL
were collected.
97
daltons. The ability of the xylanases to penetrate this
membrane was unique since their molecular sizes were
between 20,000 and 29,000 d a l t o n ~ . ’ The
~ ~ ’ cellulases
~
n
Z 25i
contained in the retentate were concentrated between
40- and 60-fold and were ideally suited for the hy-
drolysis of cellulose at high concentrations. The xy-
lanases, which were present in dilute solution in the
ultrafiltrate, were concentrated and solvent exchanged
by ion exchange chromatography using the cationic
SP-Zetaprep 250 cartridge. The Pellicon filtrate was
yi
v)
<
Z
4
2X
LL
0
k-
2ott.‘
0 5
15

10

diluted onefold prior to adsorption because the undi- 0

luted filtrate contained excessive salt concentrations,
which interfered with efficient binding. Elution of the
column-bound xylanases was carried out by a com-
bination of increased ionic strength and increased pH
in the buffer. The relatively low salt concentration and
the use of the nontoxic salt sodium phosphate in the
eluate avoided desalting of the enzyme when it was
used in conjunction with fermentative organisms. The
eluted xylanase had a very high specific activity of
approximately 2400 IU/mg protein and was concen-
trated in the second and third fractions (Fig. 1). A
summary of the enzyme recovery is shown in Table I.
A comparison of different aliquots of the enzyme
before and after passage through the ion exchange col-
umn showed that the percentage of xylanase that was
not bound increased linearly with the volume of en-
zyme applied, up to a ratio of 0.25% (Fig. 2). By taking
the difference, it was apparent that more than 99.7%
of the applied xylanase (3.3 g protein, 6,070,000 IU)
was bound. The recoveries of protein and xylanase
activity were 100% and 132%, respectively, if all the
fractions containing xylanase activity were combined.
The greater than 100% xylanase recovery may be due
to the elimination of enzyme inhibitors in the Pellicon
filtrate or to the reported problems associated with the
dinitrosalicylic acid reducing sugar assay. l4
Since the objectives of the ion exchange step were
to concentrate the xylanase, as well as to change the
solvent in which the enzymes were dissolved, a plot
n
w
LL
0 5 10 15 20 25 30
LITERS OF DILUTED PELLICON FILTRATE APPLIED
Figure 2. Relationship between percentage of initial xylanase ac-
tivity not bound and volume of xylanases applied to SP-ZetaPrep
250 column. Column was equilibrated with lOmM sodium acetate
buffer, pH 4. Xylanases applied to column were Pellicon filtrate
diluted with equal volume of water. The pH of enzyme solution was
adjusted to 4 with acetic acid prior to application.
corresponding 4 1-fold increase in concentration was
obtained while 127% of the original activity was re-
covered.
The isolated xylanase was essentially pure (Fig. 4)
and was composed largely of the 20,000-dalton xylan-
ases and small amounts of the 22,000-dalton xylanases.
Previously, we had shown1*J3that these were the pre-
dominant xylanases found in T . harzianum culture fil-
trates. These partially purified xylanase preparations
were shown to be compatible with the bacteria Kleb-
siella pneumoniae, and a simultaneous saccharification
z

\
of protein recovery vs. degree of concentration was W
made (Fig. 3). If fractions 2 and 3 were combined, a 0
k

3 I-fold increase in protein concentration was obtained IT

R
while approximately 96% of the original protein was
recovered. When the xylanase activity was assayed, a I-
\
Z
W
0
cc
W
n
Table I. Effect of various treatments on enzyme recoveries. 85C
’F
I I 1 I
Volume Xylanase FPase” Protein 4 / 1
15 25 35 45 55
(L) (106 IU) (IU) (B)
~ ~ ~ ~
NO OF FOLD CONCENTRATED
Culture filtrate 17.2 9.84 28600 33.3
Ultraretentate 0.3 2.78 21700 22.3 Figure 3. Relationship between percentage of protein recovered
Ultrafiltrate 17.0 6.07 6300 3.30 and degree of concentration relative to undiluted Pellicon filtrate.
SP-zeta 0.515 7.70 15.4 3.18 Various eluted fractions from the SP-ZetaPrep 250 column were
preparation pooled and total protein content was used in calculating protein
recovery. From left to right, points represent pooling of fractions
Filler paper activity. 1 4 , fractions 2 4 , fractions 2 and 3, and fraction 2 alone.

98 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 30, JULY 1987

 Figure 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of xylanase preparations. Lane 1
contained molecular weight standards corresponding to sizes of 94,000,67,000,43,000, 30,000,20,100, and
14,400 daltons. Lanes 2 and 3 contained 50 pg protein from Pellicon retentate and Pellicon filtrate, re-
spectively. Fifty micrograms of xylanase concentrated by ion exchange column was applied onto lane 4.
Lanes 5-7 contained 30, 20, and 5 pg purified xylanases with molecular weights of 20,000, 22,000, and
29,000 daltons, respectively.

and fermentation approach could be used to produce
2,3-butanediol from aspenwood hemicellulose. I s
The 10,000-dalton cut-off polysulfone membrane in
the ultrafiltration step was durable and was resistant
to hydrolysis by the cellulase enzymes. The same
membrane was used for over 3 years, during which
more than 20 runs were carried out without a significant
deterioration of the membrane being observed. Since
particulates were totally removed in the ultrafiltration
step, the ultrafiltrate containing the xylanase enzyme
was ideal for subsequent ion exchange treatment. As
a result, column clogging was not a problem, and the
same column was used for more than 10 runs without
change of properties.
The above-described procedure for the production
of cellulase-free xylanase has potentials for scale-up

Table 11. Comparison of methods for xylanase concentration.

Xylanase activity Processing Reagents needed per

recovered (%) rateb (Lih) liter processed' (g)

Ion exchange I27 2.7 Na acetate (0.15)

Na phosphate (0.36)
Acetic acid (4.4)
Ultrafiltration 93.7 0.8 Na dodecyl sulfate (0.12)
Na acetate (0.1)
Rotary ND 0.9 none
evaporation
Ammonium sulfate 70 ND ammonium sulfate (390)
precipitation
Ethanol 63 ND ethanol (3200)
precipitation
Acetone 55 ND acetone (3200)
precipitation

a ND = not determined.
Average rate including time needed for regeneration.
Reagents needed for regenerating apparatus included.

TAN ET AL.: PURIFICATION OF CELLULASE-FREE p-l.4-D-XYLANASE 99

studies. The ultrafiltration process has been proven to
be cost-effective on an industrial scale in comparison
to other processes such as evaporation, lyophilization,
and salt and solvent Scaling up of
the ion exchange step using the SP-ZetaPrep cartridges
has been claimed by the manufacturer (AMF Molec-
ular Separations Division, Meriden, CT) to be straight-
forward. Industrial-scale apparatus (multicartridge
systems) with a filtration rate of 720 Wh and a capacity
to process kilogram quantities of protein is already
available.
The efficiency of the ion exchange method for con-
centrating the xylanase in the ultrafiltrate was com-
pared to other methodsI9 that are frequently used in
industrial processes (Table 11). Salt or solvent precip-
itation was found to result in poor enzyme recoveries
of between 55 and 70%, probably because of the low
initial protein concentration. When the large quantities
of reagents needed for precipitation were taken into
consideration, these processes were concluded to be
economically nonviable. In addition, these processes
result in the need for wastewater treatment and/or re-
quire devices for the removal of toxic and explosive
vapors as well as explosion-proof motors and switches.19
These capital expenditures all add to the cost of en-
zyme production. Xylanases concentrated by rotary
evaporation were found to contain inhibitory sub-
stances that interfered with their utilization in com-
bination with K . pneumoniae during the simultaneous
hydrolysis and fermentation of various hemicellulose
fraction^.'^ The need to dialyze the enzyme to remove
these inhibitory materials would add another step to
the process. Concentration by ultrafiltration was pos-
sible using the 1000-dalton cut-off membrane; how-
ever, the processing rate was less than one-third that
of the ion exchange method. In addition, the necessity
to direct 90% of the flow into recirculation rather than
filtration, as recommended by the manufacturer for the
ultrafiltration process, ultimately resulted in higher
capital costs for high-output pumps and increased
pumping costs. l6 Such recirculation is not necessary
in the ion exchange method so that pumping costs can
be expected to be lower. The ion exchange method
was found to have the highest processing rate when
compared to the other concentration methods as well
as resulting in the greatest percentage of xylanase ac-
100 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 30, JULY 1987
tivity recovered. The quantities of chemicals needed
for column regeneration, enzyme elution, and pH ad-
justments were small when compared with those for
precipitation processes.
In conclusion, we have shown that a highly purified
xylanase system with high activity can be prepared in
large quantities using relatively simple procedures. We
are currently assessing the commercial opportunities
for applying this technology.
We wish to thank D. Turnbull and M. K.-H. Chan for ex-
cellent technical assistance and the Canadian Forestry Ser-
vice for funding this work.
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