[1] ISOLATION OF LIGNIN 3
[ 1] Isolation of Lignin
By JoI-IN R. OBST and T. KENT KIRK
General Considerations
Introduction
Significant gains have recently been made in understanding the bio-
chemistry of the microbial degradation of lignin.l Further advances will be
facilitated through studies using isolated lignins. This chapter presents
some of the most useful methods for lignin isolation.
Critique of Lignin Preparations. Probably the best known isolated lig-
nin is Klason lignin, which is obtained by treating wood with sulfuric acid.
The polysaccharides are hydrolyzed to water-soluble sugars, and the lignin
is recovered as an insoluble residue. Although this method for lignin
isolation has great utility as an analytical means of determining lignin
content (see chapter [12], this volume), the highly condensed and altered
Klason lignin is generally unsuited for either chemical characterization or
studies of biological modification and degradation. For such studies, what
is needed is an isolated lignin that is representative of the lignin in the
original lignocellulose (which is sometimes referred to as protolignin).
Because the methods for isolating lignin have been devised by wood
scientists, the procedures discussed here are those used to isolate lignin
from wood (see Table I). Often these methods will be suitable for other
lignocellulosics, but sometimes modifications will be required. In particu-
lar, it may be desirable to treat certain plant materials, such as forages and
immature woody tissues, to remove protein prior to lignin isolation. This
can be accomplished by treatment with proteases or by extraction with hot,
neutral detergent (see chapter [12], this volume).
The most useful lignin preparation is Bjrrkman lignin, also known as
Bjrrkman milled wood lignin or simply milled wood lignin (MWL). Milled
wood lignin is purified from the aqueous p-dioxane extract of finely milled
wood, which has been first extracted with organic solvents to remove
extraneous components. Although it has not been rigorously proved that
MWL is representative of protolignin, it is considered to be appropriate for
most chemical and biological studies. Milled wood lignin can be obtained
t T. Higuchi (ed.), "Biosynthesis and Biodegradation of Wood Components," Chap. 19-21.
Academic Press, San Diego, California, 1985.
METHODSIN ENZYMOLOGY,VOL. 161
4 LIGNIN [1]
TABLE I
LIONINISOLATIONMETHODS
Preparation Methodology Remarks
Milled wood lignin (MWL) Aqueous dioxane extraction Obtained m about 20%
of finely milled wood yield; considered to be
representative of the
original lignin
Milled wood enzyme lignin Residue left after polysac- Ninety five plus percentage
(MWEL) charidase hydrolysis of the yield, but contains
carbohydrates in finely 10-12% carbohydrate;
milled wood not completely soluble in
common lignin solvents
Cellulase enzyme lignin Solvent-soluble fraction of Similar to MWL
(CEL) MWEL
Brauns' native lignin Ethanol extract of ground Lower yield and lower
wood (fine sawdust-size molecular weight than
particles) MWL
Brown rot lignin Ethanol or aqueous dioxane Probably not severely
extract of brown-rotted altered, but some
wood demethylation of meth-
oxyls and oxidation of
side chains has occurred
Chemical lignins (kraft and Dissolution of lignin at high Not representative of the
sulfite) temperature and pressure original lignin; major by-
with chemicals products in pulp
production to make paper
Klason lignin Insoluble, condensed Not representative of the
residue left after original lignin; often used
hydrolysis of polysaccha- as a measure of lignin
rides with sulfuric acid content (see chapter [12],
this volume)
in 2 0 - 3 0 % yields, based on the total lignin. This m e t h o d requires either a
vibratory or rotary ball mill.
O n e lignin preparation which does not require a n y ball-milling equip-
m e n t is Brauns' lignin (sometimes referred to as Brauns' native lignin or
native lignin, which should not be confused with protolignin). T h e w o o d is
first extracted with cold water a n d then with ether to r e m o v e extraneous
components. Subsequent extraction o f s o m e o f the lignin with ethanol
followed by purification steps gives Brauns' native lignin. This lignin prep-
aration has fallen into disfavor a m o n g w o o d chemists, w h o consider its low
yield (about 80/0 based on the total lignin) and its low molecular weight to
be disadvantageous for investigations o f structure a n d reactivity. However,
the low-molecular-weight distribution o f Brauns" lignin m a y be beneficial
[ 1] ISOLATION OF LIGNIN 5
in some lignin biodegradation studies by providing greater accessibility of
the substrate and increased degradation rates. Basically, the structure of
Brauns' native lignin is similar to that of MWL except for its molecular
weight and associated properties. Brauns' native lignin may be used as the
first lignin substrate, and successful investigations can then move on to use
other more representative isolated lignins.
Ball-milled wood, prepared in the same manner as that used for MWL
extractions, may be treated with polysaccharidase enzymes to solubilize the
carbohydrate components. In this way, a lignin residue is produced which
contains nearly all of the lignin in the wood. This lignin, termed milled
wood enzyme lignin (MWEL), has not been severely modified by any
chemical treatment. Milled wood enzyme lignin is the most representative
of all the isolated lignins. Unfortunately, it contains a relatively high
residual carbohydrate content of 10-12%, a result of covalent linkages
between lignin and polysaccharide fragments. Also, due to its high molecu-
lar weight, it is not completely soluble in c o m m o n lignin solvents, such as
aqueous dioxane, acetic acid, dimethylformamide, and dimethyl sulfoxide.
This insolubility presents experimental difficulties in handling, purifying,
and analyzing MWELs.
Fractionation of MWEL, based on solubility in dioxane-water, is a
means of preparing a soluble lignin which can then be purified in the same
manner as MWL. This lignin was originally termed cellulase enzyme lignin
(CEL). It is thought to be more representative of protolignin than MWL
but has a lower yield than MWEL. Milled wood lignin is probably ade-
quate for most studies, and the additional steps in preparing CEL are
usually not justifiable.
There are numerous other lignin preparations, including hydrochloric
acid lignin, periodate lignin, cuoxam lignin, enzymatically liberated lignin,
alcohol-HC1 lignin, thioglycolic acid lignin, acetic acid lignin, dioxane-
HC1 lignin, phenol lignin, and hydrogenolysis lignin. 2 These preparations
usually are not adequate as substrates for biochemical studies of protolig-
nin. However, "enzymaticaUy liberated lignin," or brown rot lignin, may
be useful in certain circumstances. When wood is rotted by brown rot
fungi, the lignin is not substantially degraded while the carbohydrates are
removed. The rotted wood, largely lignin, is extracted with lignin solvents
and the lignin is purified; yields of over 20% of the original lignin are
obtained. Although brown rot fungi demethylate aromatic methoxyl
groups and cause a limited amount of oxidation, the lignin is not otherwise
severely damaged. The demethylation may even be considered advanta-
2 D. Fengel and G. Wegener, "Wood, Chemistry, Ultrastructure, Reactions," p. 50.
de Gruyter, Berlin, Federal Republic of Germany, 1983.
6 LIGNIN [I]
geous: subsequent methylation of phenolic hydroxyls using a ~ac label
would provide a substrate for monitoring lignin degradation by measuring
the evolution of labeled carbon dioxide or for studying demeth(ox)ylation.
Another class of lignins is produced by chemical pulping processes.
Most of the chemical pulp produced in the United States is by the kraft
process. Kraft lignin is highly modified; it is lower in molecular weight, has
a higher phenolic content and a lower methoxyl content, and has under-
gone extensive side-chain reactions.
Lignosulfonate, as implied by its name, is the sulfonated lignin re-
moved from wood by sulfite pulping. Lignosulfonates have higher molecu-
lar weight than kraft lignin but are not representative of protolignin.
Hydrolysis reactions have occurred, and sulfonation (to give a water-solu-
ble product) can be extensive.
Whereas kraft lignin and lignosulfonates are not suitable for studies
modeling the behavior of protolignin, they are important in their own right
as industrial by-products, and research is warranted on their biodegrada-
tion and bioconversion.
Finally, after a lignin has been isolated and purified, it is essential to
analyze it to be certain that it is not grossly contaminated. Often, determi-
nations, such as carbohydrate content, methoxyl content, and ultraviolet
absorption, are sufficient, but sometimes more detailed, analyses are re-
quired. An overview of quantitative lignin determinations is given in
chapter [12] in this volume.
Isolation of Lignin
Milled Wood Lignin (BjOrkman Milled Wood Lignin, BjOrkman Lignin)
The wood used for lignin isolation should be sapwood; heartwood often
contains polyphenolics which are difficult to remove and may even have
condensed with the lignin. Air-dried wood is milled in a Wiley mill to pass
40 mesh and extracted first with acetone:water (9: 1, v:v) by percolation
at room temperature and then with ethanol: benzene (2: l, v: v). Lignin is
heat-sensitive, so extractions should not be at the boiling point of the
solvents. For this reason also, the extracted wood should never be oven-
dried, but rather dried in a vacuum desiccator over phosphorus pentoxide
or other efficient desiccant.
The dry, extracted wood is then milled either in a vibratory ball mill 3 or
3 Siebtechnik vibrating ball mills may be obtained from Tema, Inc., 11584 Goldcoast Drive,
Cincinnati, Ohio 45249. We have successfully used mill type USM 12 to grind 200 g of
wood at one time. For smaller amounts of wood ( l - 1 0 g), we have used the smaller
[ 1] ISOLATION OF LIGNIN 7
in a conventional rotating jar ball mill. The time of milling can vary from
1 hr in a very efficient vibratory ball mill, such as the NBS-type mill first
used by BjOrkman, 4 to 12 hr in a large Siebteehnik vibratory mill, to 3
weeks using a rotating jar mill. Thus, milling time and the amount of
material milled must be optimized for each type of mill and grinding
medium. Because the temperature of the milling jar will increase, it is
desirable to cool the mill to avoid damaging the lignin. 3
Often the ball milling is done in a nonswelling solvent such as toluene.
The solvent excludes oxygen, and the milled wood may be recovered by
centrifugation. However, the wood may be milled dry, preferably under
carbon dioxide or nitrogen. In this case, the milled wood may conveniently
be removed from the balls after conditioning in a high humidity environ-
ment by shaking the balls on screens in a Ro-Tap sieve shaker. 5
The milled wood is dispersed in dioxane:water (96:4, v:v) and me-
chanically stirred; the ratio of wood to solvent is chosen to be convenient,
for example, 10 g of milled wood and 250 ml of dioxane:water. After 1
day, the suspension is centrifuged, and the residue is redispersed in fresh
dioxane:water and stirred for an additional day. Although lignin would
continue to be extracted for many subsequent extractions, the bulk of the
MWL is removed in the first two. The extracts are combined and then
freeze-dried (or simply dried in a rotary vacuum evaporator) to give a
crude MWL in about 20-30% yield; this lignin contains up to about 10%
residual carbohydrate. (The milled wood may also be extracted with 9:1
dioxane:water, giving a higher yield of MWL but with more carbohy-
drate.) As is, this crude MWL is useful for many experiments.
In most cases, it is desirable to purify the crude MWL. This is accom-
plished by dissolving the lignin (the dioxane: water is removed by vacuum
evaporation) in 90% acetic acid, using 20 ml of solvent for each gram of
lignin. The acetic acid solution is then added dropwise, with stirring, to
water (about 220 ml of water per gram oflignin). The precipitated lignin is
centrifuged and then freeze-dried or air-dried, followed by drying in a
vacuum oven. It is then dissolved with stirring in a mixture of 1,2-dichloro-
Siebtechnik mill or a custom-made mill patterned after the NBS mill. 4 The temperature
increase of the miU jars on the large mill is minimized by milling for no longer than 1 hr at
a time, followed by 1 hr of cooling. A large fan is used to cool the jars during the entire
process. The smaller mill may be placed in a cold room.
4 A. BjOrkman, Sven. Papperstidn. 59, 477 (1956).
5 For the preparation of large amounts of milled wood, we have found that mechanical
shaking on a sieve is a convenient way to remove the wood from the balls. We use a No. 7
(7-mesh) stainless-steel screen with a custom-made stainless inner collar to minimize ash
contamination. Sieves and the R o m p may be obtained from W. S. Tyler, Inc., Mentor,
Ohio.
8 LIGNIN [1]
ethane:ethanol (2: 1, v:v) and centrifuged to remove solids. The lignin
solution is added dropwise to anhydrous ethyl ether to precipitate the
lignin. About 20 ml of solvent and 230 ml of ether are used for 0.5- 1 g of
lignin. After centrifugation, the insoluble MWL is washed three times with
fresh ether. The yield of the purified MWL may be half that of the crude
preparation, but its residual carbohydrate content is about 4%.4 When
prepared from light-colored woods, MWL is cream colored.
There are several ways significantly to reduce the carbohydrate content
of MWL. However, yield losses may be substantial. Two such methods are
those of Freudenberg and Neish 6 and Lundquist and Simonson. 7
Milled Wood Enzyme Lignin
The wood is extracted and then ball milled as for milled wood lignin
(see above).
For digestion of the carbohydrate in 100 g of milled wood, 3 g of
Cellulysin (Calbioehem-Behring Corp., La Jolla, California 92307), which
is a mixture of polysaccharidase enzymes, or a comparable preparation, s is
dissolved in 40 ml of 0.5 M acetate buffer (pH 4.6) and 200 ml of distilled
water. The enzyme solution is centrifuged to remove undissolved mate-
rials. Water is added to the suspension of milled wood in the enzyme
solution to bring the total volume to about 1.5 liters. Several drops of
toluene are added as a preservative. The digestion is carried out with
stirring in a suitable glass container for a week to 10 days at 48 °. The
suspension is then centrifuged; the residue is washed with water and redi-
gested in the same way two more times. The final residue is thoroughly
washed and then freeze-dried. Yields based on the lignin in the wood are
6 K. Freudenberg and A. C. Neish, "Constitution and Biosynthesis of Lignin," p. 52.
Springer-Verlag Berlin and New York, 1968.
7 K. Lundquist and R. Simonson, Sven. Papperstidn. 78, 390 (1975).
s Solubilization of the polysaccharides in milled wood and other lignocellulosic materials
requires the concerted action of the ceUulase system (endo- and exo-l,4-glucanases) plus
hemicellulose-depolymerizing enzymes. The latter include enzymes that hydrolyze substi-
tuted 1,4-xylans and substituted glucomannans, also 1,4-1inked.Such mixtures of enzymes
are produced commercially with the fungus Trichoderma reesei (Trichoderma viride)
grown on delignified fignocellulosic substrates such as newsprint or on finely milled ligno-
cellulosics, which have the necessary constituents to induce the enzymes. By suitable
experimentation, the researcher should be able to produce the enzyme mixture without
undue difficulty. General references to the production of cellulases and hemicellulases by
T. reesei are as follows: K.-E. Eriksson and T. M. Wood, "Biosynthesis and Biodegradation
of Wood Components" (T. Higuchi, ed.), pp. 469-503. Academic Press, San Diego,
California, 1985; and R. F. H. Dekker, in "Biosynthesis and Biodegradation of Wood
Components" (T. Higuchi, ed.), pp. 505-533. Academic Press, San Diego, California,
1985.
[ 1] ISOLATION OF LIGNIN 9
over 95%. The MWEL will have a carbohydrate content of approximately
1 0 - 12%. 9
The MWEL may be fractionated to remove some of the residual carbo-
hydrate and to give a solvent-soluble lignin with lower molecular weight,
termed CEL. ~° To prepare CEL, MWEL is twice extracted with 96% (or
90%) dioxane. This extract may be purified in the same manner as milled
wood lignin as described above. The residual carbohydrate content of the
CEL-96 is about 4%, whereas that of the CEL-90 is higher. The extracted
residue may be further extracted with 50% dioxane. However, this extract
is not completely soluble in dichloroethane:ethanol and cannot be puri-
fied by the M W L procedure.~°
Brauns' Native Lignin (Brauns" Lignin, Native Lignin)
The wood is ground in a Wiley mill to pass a 100- to 150-mesh screen
and extracted first with cold water and then with ethyl ether for 48 hr to
remove extraneous components. The wood is then extracted by percola-
tion with 95% ethanol at room temperature for 8 - 1 0 days or until the
extract is colorless. A small amount of calcium carbonate is added to the
extract to neutralize wood acids, and the alcohol is removed under reduced
pressure. Water is added to the residue, and the evaporation continues to
remove traces of the alcohol. The lignin residue is triturated alternatively
with water and ether until it becomes solid. The solid is filtered and dried
over an efficient desiccant. The dry lignin is extracted with anhydrous
ether in a Soxhlet apparatus. The residue is dissolved in dioxane to give a
10% solution, and it is precipitated by dripping into stirred distilled water
(about 15 times the volume of the dioxane). If a colloidal solution forms
instead of a precipitate, a little sodium sulfate is added and the solution
vigorously stirred to coagulate the lignin. The precipitate is filtered, washed
with water, and dried in a desiccator. It is then dissolved in dioxane to give
a 10% solution, centrifuged, and filtered. The solution is slowly dripped,
with stirring, into anhydrous ethyl ether. The Brauns' native lignin sepa-
rates as a fine tan-colored powder. It is washed sequentially with ether,
high-boiling petroleum ether, and low-boiling petroleum ether and then
dried in a desiccator over sulfuric acid and paraffin shavings. Precipitation
into ether may be repeated until the methoxyl content of the Brauns'
native lignin is constant. The yield is about 8% based on the lignin in the
wood. ~i
Similar lignin preparations can be obtained from ground samples with
9 j. R. Obst, Tappi 65, 109 (1982).
1oH.-M. Chang, E. B. Cowling, and W. Brown, Holzforschung 29, 153 (1975).
u F. E. Brauns, "The Chemistry of Lignin," p. 51. Academic Press, New York, 1952.
10 LIGNIN [1]
other lignin solvents, including acetone:water, 9:1 (v:v), 12 and aqueous
dioxane.
Brown Rot Lignin [Enzymatically Liberated Lignin (ELL)]
The wood is decayed in soil block chambers (ASTM D 2017-81) by a
brown rot fungus, such as Gleophyllum trabeum, Lentinus lepideus, or
Poria vaillantiL to weight losses of about 60-70%. 13 The dried, decayed
wood is ground in a Wiley mill to pass a 60-mesh screen, Brown rot lignin
is then obtained, employing the purification methods used for milled wood
lignin or Brauns' native lignin. Alternatively, the Wiley-milled decayed
wood may be extracted with 50% aqueous p-dioxane and the lignin puri-
fied by gel permeation chromatography on Sephadex G-25) 3
Chemical Lignins (Kraft Lignin and Lignosulfonate)
Two types of commercially available lignin are kraft lignin and ligno-
sulfonate) 4 Although the lignins from commercial sources may be ade-
quate for many studies, it is recommended that this type of lignin be
isolated from laboratory pulping experiments whenever possible. In this
way, the entire history of the lignin is known and controlled.
Kraft Lignin (Thiolignin, Sulfate Lignin)
Kraft pulping is accomplished by degrading and dissolving the lignin in
hot alkaline sodium sulfide solution ("white liquor"). Kraft white liquor is
prepared by dissolving 16 g of sodium sulfide per liter of 1 N sodium
hydroxide. The extracted wood, either in chip form or Wiley-milled form,
plus white liquor at a 4:1 (w: w) liquor-to-wood ratio are sealed in a
stainless-steel bomb. Cooking temperature for most hardwoods (angio-
sperm woods) is about 155 °, whereas 170- 180" is required for softwoods
(gymnosperm woods). The bomb is usually heated in an oil bath and
rotated, end over end, to ensure mixing. If industrial conditions are to be
mimicked, the time to raise the bath from room temperature to the maxi-
m u m cooking temperature should be about 90 rain. Time at temperature
will depend on the pulp yield desired; 1 - 2 hr is typical.
t2 T. K. Kirk and H.-M. Chang, Holzforschung28, 217 (1974).
13T. K. Kirk, Holzforschung29, 99 (1975).
t4 Kraft lignin, and modified kraft lignins, may be obtained from Westvaco, Chemical Divi-
sion, Box 70848, Charleston Heights, South Carolina 29415. Lignin sulfonates may be
obtained from Reed Lignin, Inc., 100 Highway 51 South, Rothschild, Wisconsin 54474,
and from Crown Zellerbach Corp., P.O. Box 4266, Vancouver, Washington 98662.
[ 1] ISOLATION OF LIGNIN 11
The bomb is then cooled with water, and the "black liquor" containing
the lignin, some hemicellulose, carbohydrate degradation products, and
inorganic chemicals is filtered from the pulp. The lignin may be precipi-
tated by the addition of acid. Generally it is better to use acetic acid rather
than mineral acids. Traces of sulfuric acid or hydrochloric acid are hard to
remove, and they may cause the lignin to undergo condensation reactions
when it is dried. Carbon dioxide may also be used to precipitate the lignin,
but yields are usually lower than with acetic acid. The precipitated lignin
should be washed thoroughly with distilled water and freeze-dried.
The kraft lignin may be purified through solvent (pyridine:acetic
acid: water) fractionation. ~5 Alternatively, kraft lignin may be purified by
repetitive dissolution in 0.1 N sodium hydroxide and precipitated with
acetic acid. Finally, it is washed with distilled water and freeze-dried.
Lignosulfonate (Lignin Sulfonate, Sulfite Lignin)
The sulfite pulping of wood is accomplished by treating wood at high
temperatures with aqueous sodium sulfite. The cook may be acid, neutral,
or alkaline. An example of neutral sulfite cooking conditions is as follows.
The wood chips are heated from ambient temperature to 175 ° over 90 rain
in sulfite liquor at a 3 : 1 (w: w) liquor to wood ratio. The liquor contains
15% sodium sulfite and 1.5% sodium carbonate, based on the dry wood.
Time at temperature is 1 hr or more.
The sulfonated, water-soluble lignin (lignosulfonate) cannot be isolated
by precipitation from the spent liquor with acid as are kraft lignins. How-
ever, this lignin may be purified by complexing with amines. For example,
spent sulfite liquor, 408 ml containing 175 g of solids, is heated to 70-85 °
with mild stirring. N,N-Dimethylhexadecylamine (Armak, Chicago, Illi-
nois) is added, and then the solution is adjusted to pH 3.5 with 10 N
sulfuric acid. Four hundred grams of 1-octanol is added, and the solution is
stirred for 5 rain more, then left to stand. One hour is required for the
layers to separate. The bottom, aqueous acidic layer is discarded.
To the alcohol layer is added 122 g of water, with stirring, and 28 g of
50% sodium hydroxide (the solution should be about pH 9.5). The solution
is heated at 60-70 ° with stirring. The layers are allowed to separate over
30 min. The bottom, aqueous layer contains about 100 g of sodium lignin
sulfonate and a trace of octanol. The alcohol is removed by vacuum
evaporation. ~s
15K. Lundquist and T. K. Kirk, Tappi63, 80 (1980).
16S. Y. Lin (Reed Lignin, Inc., Rothschild, Wisconsin), personal communication.
12 LIGNIN [2]
Acknowledgment
The use of trade, firm, or corporation names in this publication is for the information
and convenience of the reader. Such use does not constitute an official endorsement or
approval by the U.S. Department of Agriculture of any product or service to the exclusion o f
others which may be suitable.
[2] L i g n i n - C a r b o h y d r a t e Complexes from
Various Sources
B y J U N - I C H I A Z U M A a n d KOSHIJIMA T E T S U O
Some polysaccharides in the cell walls of lignified plants are linked to
lignin to form lignin-carbohydrate complexes, t-4 Three types of evolu-
tionally different plants, softwoods (angiosperm), hardwoods (gymno-
sperm), and graminaceous plants (grass), contain structurally different
molecular species of hemicellulose and lignin. 5-s This implies the existence
of variations in the sugars linking to lignin in these different types of plants.
The major obstacle in the characterization of lignin-carbohydrate com-
plexes is the difficulty in isolating these complexes in a homogeneous state.
A simple procedure described below has been developed for the isolation
and fractionation of water-soluble lignin-carbohydrate complexes from
various types of lignified plants. 9- t3
t A. BjOrkman, Sven. Papperstidn. 60, 243 (1957).
20. P. Grushnikov and N. N. Shorygina, Russ. Chem. Rev. (Engl. Transl.) 39, 684 (1970).
3 y. Z. Lai and K. V. Sarkanen, in "Lignins" (K. V. Sarkanen and C. H. Ludwig, eds.), pp.
165- 240. Wiley (Interscience), New York, 1971.
4 E. Adler, WoodSci. Technol. 11, 169 (1977).
5 K. V. Sarkanen and H. L. Hergert, in "Lignins" (K. V. Sarkanen and C. H. Ludwig, eds.),
pp. 43-94. Wiley (Interscience), New York, 1971.
6 K. C. B. Wilkie, Adv. Carbohydr. Chem. 36, 215 (1979).
7 G. O. Aspinall, in "The Biochemistry of Plants" (J. Preiss, ed.), Vo|. 3, pp. 473-500.
Academic Press, New York, 1980.
s T. Higuchi, in "Plant Carbohydrates II" (W. Tanner and F. A. Loewus, eds.), pp. 194-224.
Spdnger-Vedag, Berlin and New York, 1981.
9 j. Azuma, N. Takahashi, and T. Koshijima, Carbohydr. Res. 93, 91 (1981).
to S. Mukoyoshi, J. Azuma, and T. Koshijima, Holzforschung 35, 233 (1981).
tt j. Azuma, N. Takahashi, and T. Koshijima, Mokuzai Gakkaishi 31, 587 (1985).
12 j. Azuma, T. Nomura, and T. Koshijima, Agric. Biol. Chem. 49, 2661 (1985).
13A. Kato, J. Azuma, and T. Koshijima, Holzforschung 38, 141 (1984).
Copyright© 1988by AcademicPress,Inc.
METHODS1N ENZYMOIXX~Y,VOL. 161 Allrightsof reproductionin any formreserved.
