Die Angewandte Makromolekulare Chemie 268 (1999) 59–68 (Nr.

4667) 59

Mild enzymatic isolation of mannan and glucan from yeast

Saccharomyces cerevisiae

Franziskus Kath, Werner-Michael Kulicke*

Institut für Technische und Makromolekulare Chemie, Universität Hamburg,
Bundesstrasse 45, D-20146 Hamburg, Germany
(Received 9 February 1999)

SUMMARY: An enzymatic procedure to isolate polysaccharides from the yeast saccharomyces cerevisiae
was developed which would both be environmentally friendly and preserve the native structure of the poly-
mers. Two proteases and three enzyme cocktails were employed.
To isolate the cell wall polysaccharides, the cell walls were first separated from the internal components by
mechanical cell disruption. The quality of cell disruption was investigated for four types of glass beads with
diameters from 0.25 – 1.5 mm and monitored via the inner cellular proteins released. With large glass beads
(Ø = 0.75 – 1.5 mm) both brewer’s and baker’s yeast cells exhibited more than 90% cell disruption after as
little as 12 min, whereas disruption was still incomplete with smaller beads. The isolated cell wall material
was purified on microporous membranes, freeze-dried and then incubated with enzymes. Proteases caused
protein lysis in the outer cell wall and released soluble mannan. The insoluble glucan was separated by cen-
trifugation and the mannan was purified by dialysis or chromatography.
In the digestion with proteases, complete conversion was achieved at concentrations higher than
240 mg g–1 of cell wall material within 26 h. Cell walls from baker’s yeast had to undergo a preliminary
extraction with petroleum ether. Complete conversion was only possible with one of the enzyme cocktails
investigated, and this was achieved after as little as 6 h for cell walls from both baker’s and brewer’s yeast.
The activity of the two other enzyme cocktails investigated was weaker, with brewer’s yeast cell walls gener-
ally reacting more readily than those of baker’s yeast.

ZUSAMMENFASSUNG: Im Hinblick auf die Entwicklung umweltfreundlicher Verfahren und den Schutz
der nativen Polymerstruktur wurde ein enzymatisches Verfahren zur Isolierung von Polysacchariden aus der
Hefe Saccharomyces cerevisiae entwickelt. Eingesetzt wurden zwei Proteasen sowie drei Enzymcocktails.
Zur enzymatischen Gewinnung der Zellwand-Polysaccharide wurden zunächst die Zellwände durch einen
mechanischen Zellaufschluß vom Zellinneren getrennt. Die Aufschlußgüte wurde für vier Glasperlensorten
mit Durchmessern von 0,25 – 1,5 mm untersucht und anhand der freigesetzten innerzellulären Proteine kon-
trolliert. Sowohl für Brauereihefezellen als auch Bäckerhefezellen zeigte sich mit großen Glasperlen
(Ø = 0,75 – 1,5 mm) bereits nach 12 min ein mehr als 90proz. Zellaufschluß, während bei kleineren Glasper-
len der Aufschluß noch unvollständig war. Das gewonnene Zellwandmaterial wurde an mikroporösen Mem-
branen gewaschen, gefriergetrocknet und anschließend mit Enzymen umgesetzt. Proteasen zerstörten in der
äußeren Zellwand die Proteinmatrix und setzten lösliches Mannan frei. Das unlösliche Glucan wurde durch
Zentrifugieren abgetrennt und das Mannan durch Dialyse oder Chromatographie gereinigt.
Bei Umsetzung mit Proteasen war vollständiger Umsatz ab einer Konzentration von 240 mg g–1 Zellwand-
material in 26 h möglich. Zellwände aus Bäckerhefe mußten zuvor mit Petrolether extrahiert werden. Von
den untersuchten Enzymcocktails war nur mit einem ein vollständiger Umsatz möglich, der aber sowohl für
Bäckerhefezellwände als auch für Brauereihefezellwände schon nach 6 h erreicht wurde. Die Aktivität der
beiden anderen untersuchten Enzymcocktails war schwächer, wobei sich Brauereihefezellwände generell bes-
ser umsetzen ließen als Bäckerhefezellwände.

Introduction mental influences or ageing, the immune system is wea-

The World Health Organisation (WHO) is expecting an
increase of epidemic proportions in cancer and other dis-
eases of civilization over the next two decades1). The
cause of this is the dramatic increase in life expectancy
and the continuous rise in environmental pollution. If a
host organism is exposed to stress, the strains of environ-
* Correspondence author.
kened2). This enables pathogens to reproduce and cause
damage. Active ingredients are thus being sought that are
able to modify the body’s own defence mechanism and
may be termed ‘biological response modifiers’ 3). These
include mannans and b-1,3-D-glucans, the action of which
has been the subject of intensive investigation in recent

Die Angewandte Makromolekulare Chemie 268 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 1999 0003-3146/99/0607–0059$17.50+.50/0
60
decades4). They occur in microorganisms as exopolysac-
charides or cell wall polysaccharides5). Today there is
already on the market a substantial range of immune-
stimulating fungi glucans. In the western world they have
hitherto only been employed for healing wounds and to
combat viral and bacterial infections6) as classical school
medicine is still preferred in these field. However, in
Japan these products are already being used as adjuvants
in cancer therapy7). Numerous in vitro tests on isolated
immune cells as well as in vivo tests on mice, rats and
higher mammals have in some cases demonstrated a
stronger stimulating effect of the glucan6, 8). b-1,3-D-glu-
can from the cell wall of baker’s yeast (Saccharomyces
cerevisiae), in particular, exhibits a strongly stimulating
effect9). This glucan is extracted chemically. However, as
it is insoluble in water, numerous procedures for manu-
facturing a water-soluble derivative have been devel-
oped10), which have enabled side-effects to be eliminated
and the activity to be increased9). One side-effect of the
process of glucan extraction is the release of mannan, for
which there is also documentary evidence of an immuno-
modulatory potential11–14).
By employing drastic reaction conditions in the chemical
extraction and in derivatization, the glucan is in some cases
severely degraded15, 16). However, as the molar mass has a
significant effect on the immunological activity8), this study
will attempt to reduce the drastic conditions in isolating the
polysaccharides. In addition, an examination will be under-
taken as to whether brewer’s yeast is suitable as a substrate
for extracting biologically active polysaccharides. Brew-
er’s yeast is also a species of Saccharomyces cerevisiae and
as a waste product of the brewery industry it is available as a
bulk raw material at very reasonable prices.
Structure of the yeast cell
The precise structure and composition of the yeast is
strongly dependent on the culture conditions17). A shortage
of amino acids, for instance, reduces the protein content in
the cell wall. Yeast is a unicellular organism with a rigid
cell wall made of polysaccharides. The cell is oval to
round and has a diameter of 5 – 13 lm. Scheme 1 shows a
picture of the yeast cell, as described in the literature.
The cell wall, which is ca. 70 nm thick, accounts for
approx. 20% of the cell’s weight18). A layered structure is
assumed on the basis of electron micrographs19). The
main components of the cell wall are mannan 31%, pro-
tein 13%, lipids 9%, glucan 29% and chitin 1 – 2%19, 20).
The chitin here is located entirely in the budding scar21).
The mannoprotein complex
By definition, mannan is a polymer that is exclusively com-
posed of mannose units. In yeast, mannan is associated with
protein in both the external yeast cell wall as a mucigenous
F. Kath, W.-M. Kulicke
Scheme 1: Structure of a yeast cell The cell wall consists of
two layers: on the outside there is a mannoprotein complex, on
the inside is the b-1,3-D-glucan as a structural polymer.
polysaccharide and in the inner cell membrane and
accounts for 20 – 50% of the mass22). First studies in 1937
were only able to detect the a-1,6 linkages of the main
chain23), whereas it later became possible by means of
methylation analysis to identify a-1,3 and a-1,2 linkages as
well23). The many individual aspects relating to the structure
of the mannoprotein complex have been compiled and pub-
lished in two review articles by Ballou et al.22, 24). Mannan is
linked to a peptide basic chain as an oligomer or polymer25).
The complex consists of 5% to 50% protein. Oligomeric
mannan is bonded directly to serine and threonine26),
whereas polymeric mannan is bonded to asparagine via N-
acetylglucosamine22).
In 1937 the molar mass of mannan was determined as
100 000 g mol–1 with osmometry23) and as 76 000 g mol–1
with ultracentrifugation25). After enzymatic digestion a
mannan with a molar mass of 180 000 g mol–1 was iso-
lated, but which had a protein content of 10%27). Phos-
phate groups were also found at the C6 position of the
mannose25), and they were found to occur on average at
every 15th mannose unit28). The picture of the inner struc-
ture of the mannan component is published in the two
review articles by Ballou et al.22, 24) and shown in
Scheme 2.
Overall, the mannoprotein can be divided into three
different regions: the peptide chain, the core region, and
the external chain in which the polymeric fraction is
located.
The glucan of the cell wall
Glucan accounts for 30 – 60% of the dry matter. The
majority had 1,3-glycosidic linkages and only 10 – 20%
1,6-glycosidic linkages29). 1,2 linkages could not be
Mild enzymatic isolation of mannan and glucan 61

Scheme 2: Sequence of the mannoprotein complex: the polymer mannan consists of

an a-1,6-glycosidic basic chain that carries a-1,2- and a-1,3-glycosidically linked oli-
gomannans as side groups. On the peptide chain oligomeric mannans are bonded
directly to the amino acids serine and threonine.

Scheme 3: The possible structure of the glucan; 1,3-glycosidic basic chains are
linked with one another via 1,6-glycosidic intermediate chains to form a three-dimen-
sional network.

found30). If glucan is treated with ca. 3% caustic soda at dic linkages34). Using electron micrographs it was possi-
75 8C, a maximum of one-third of the glucan goes into ble to demonstrate a fibrillar structure for the b-1,3 com-
solution31). The glucan can be subdivided into two com- ponent and an amorphous structure for the b-1,6 compo-
ponents: one that is insoluble in alkali (glucan A) and one nent35).
that is soluble (glucan B). Glucan A accounts for ca. 80 –
85% of the entire content and contains primarily 1,3-gly-
cosidic linkages as well as 3% 1,6-glycosidic linkages. Its Chitin and lipid in the cell wall
molar mass was determined as 240 000 g mol–1 32). At a
The chitin is located exclusively in the budding scar,
molar mass of 250 000 g mol–1, 15 – 20% of the entire glu-
where it forms a ring36). Tanner and Jung isolated the lipid
can is soluble in alkali (glucan B)33). 80 – 85% of the gly-
dolichol phosphate37), its yield is 3 mg kg–1 weight of wet
cosidic linkages are b-1,3, 8 – 12% are b-1,6-linked, and
yeast38). The lipid is linked to mannose via phosphate
3 – 4% of the glucose rings are branchings. The 1,6 lin-
groups.
kages are selectively hydrolysed by acetolysis. After this
the 1,3-glucan is soluble in DMSO. Consequently, the
1,3-glucan chains must be linked to one another via 1,6
intermediate chains31). Scheme 3 shows a proposed struc- Enzymes
ture for the glucan of the yeast cell wall. This study employed, on the one hand, specific enzymes
Enzymatic degradation of the 1,3-glycosidic linkages (proteases P1 from SIGMA and P2 from Merck) with pre-
yields a water-soluble b-1,6-glucan with a degree of poly- cisely defined lytic activity and, on the other hand, non-
merization, P, of 130 – 140 (molar mass L
21 000 – specific enzymes, so-called enzyme cocktails. Several
23 000 g mol–1) and a small proportion of b-1,3-glycosi- enzyme activities are required for complete hydrolysis of
62
Fig. 1. Monitoring the quality of the disruption broth by means of the intracellular
proteins liberated as a function of the time and the diameter of the glass beads.
a yeast cell: a protease and a b-1,3-glucanase are abso-
lutely necessary, mannanases and chitinases are also fre-
quently found39). Amongst the non-specific enzymes,
three enzyme cocktails were employed: EC1, an enzyme
cocktail from Helix pomatia (Merck), EC2 an enzyme
cocktail from Cytophaga sp. (Merck) and EC3, Lyticase
(SIGMA). These enzyme systems have been used to
extract mannan and glucose.
Results and discussion
Separation of the cell components
Diverse disruption methods were developed to extract the
yeast contents. One feature they all have in common is
separation of the two main components, glucan and man-
nan. In recent times the procedure has been specially
directed towards isolation of glucans. Due to its rheologi-
cal properties and its indigestibility in the human organ-
ism, the particulate b-1,3-glucan can be employed in diet-
ary foodstuffs as a thickener40) or fat substitute41). As
knowledge of the immune-stimulating properties of glu-
cans increases, work on synthesizing pharmaceutically
pure glucan from yeast is forging ahead, particularly in
the research group around Williams9, 16, 42, 43).
Mechanical yeast cell disruption
With the growing need for biotechnological products,
biological methods have been developed in addition to
the chemical and physical in order to obtain valuable pro-
ducts from microorganisms. Particular attention has been
focussed on isolating enzymes and proteins, e. g. from
yeast. In chemical disruption methods, proteins or
enzymes are often denatured. This is the reason why mild
procedures are required, such as the biological or
mechanical. The biological procedures include for
instance autolysis or digestion with enzyme systems that
break down yeast cells. The various disruption methods
F. Kath, W.-M. Kulicke
for obtaining proteins have been compiled in several
review articles44, 45).
Various homogenizers are generally employed in order
to extract large quantities of intracellular enzymes46–49);
Schütte et al., however, developed a method to disrupt
yeast cells on an analytical scale50, 51). This involves mix-
ing a suspension of yeast cells with glass beads and shak-
ing them at high frequency in a vibration mill. Very high
shear forces are generated between the glass beads which
result in the yeast cells being torn apart50). The cell walls
can be separated by centrifugation or filtration on micro-
porous membranes. Intracellular protein is found in the
supernatant and the cell walls are found in the sediment.
The proteins liberated from the contents of the cell can
be detected quantitatively by staining them with Coomas-
sie Blue (Bradford test52)). Fig. 1 shows the photometric
curves of the cell broths for brewer’s and baker’s yeast
cells, which were disrupted with glass beads of various
diameters. As the duration of disruption increases, the
concentration of proteins in the cell supernatant rises and
with it the depth of colour. A plateau value is established
after a certain time, which is designated as the maximum
disruption quality. In order to monitor the disruption qual-
ity, samples were taken out of each of the mill containers
at various times and the protein content was determined
in accordance with the Bradford test52).
The glass beads with the smallest diameter (0.25 –
0.5 mm) are not suitable for the disruption, as even after
60 min 90% of the maximum protein quantity still had
not been released. Almost identical curves are found for
the glass beads with diameters of 0.75 – 1.0 mm and 1.0 –
1.5 mm. After as little as 12 min a disruption quality of
more than 90% was achieved. Hence, isolation of the
yeast cell walls was carried out exclusively with glass
beads of at least 0.75 mm diameter and disruption was
continued for at least 15 min. As the autolytic activity of
yeast sets in at higher temperatures33), the cell suspension
and the mill beaker were cooled to 5 – 10 8C. After a dis-
ruption time of 60 min the temperature inside the mill
Mild enzymatic isolation of mannan and glucan 63

Fig. 2. Electron micrographs of yeast cells; 1. baker’s yeast, killed off with methanol; 2. brewer’s
yeast; 3. brewer’s yeast after mechanical cell disruption; 4. mechanically disrupted cells after
freeze-drying.

beaker was only 20 8C, so that there is no fear of damage
to the polysaccharide by autolysis or heat generation.
Electron micrographs were taken in order to determine
the effects of the mechanical disruption on the structure
of the yeast cellsa. Fig. 2 shows various photographs of
yeast cells.
The first picture shows baker’s yeast which was killed
off with methanol. The second shows cells from the
ready-to-pitch yeast. The baker’s yeast cells are firm and
round, whereas the brewer’s yeast clearly exhibits defor-
mations on the cells. This is presumably due to the brew-
ing process, in which the pH value falls during fermenta-
tion, which leads to a deterioration in the physiological
condition of the yeast. The third shows the sediment of a
mechanical cell disruption, in which there is no recogniz-
able cell structure whatsoever. However, freeze-drying
this material yields the fourth picture. The cells may
appear somewhat “shrivelled” because the water has been
drawn off, but they still have their full shape. Conse-
quently, the cells were only torn up but not broken down
into small fragments. Hence, in terms of the quality of the
polysaccharides that can be isolated from this procedure,
the deterioration in native structure is negligible.
Further processing of the cell walls in enzymatic diges-
tion was carried out exclusively with the freeze-dried cell
wall material.
Enzymatic digestion of yeast cell walls
The cell wall material obtained was reacted with different
enzymes to obtain the polysaccharides. Scheme 4 is a
schematic picture of the procedure employed. Protease is
employed to isolate the glucan and thus destroys the
matrix of the mannoprotein complex. This results in man-
nan detaching itself from the cell wall and going into
solution. Polymeric glucan, which is insoluble, is left
behind. It can be easily separated by centrifugation. In
addition to the enzyme, the supernatant also contains
small peptide residues as well as oligomeric and poly-
meric mannan. Dialysis or chromatography can be
employed to free the mannan or supernatant resulting

a
The yeast cell suspension is located to a large extent in the interstitial volume of the glass beads. This is why the sum of the
percentages is greater than 100%.
64
Scheme 4: Principle of enzymatic digestion; protease action
yields glucan and mannan, glucanase action yields the mannan
or mannoprotein.
from centrifugation from the oligomeric components and
salts.
If a glucanase is employed, it is able to hydrolyse the
glucan from the inner side of the cell wall. Provided the
glucanase is free of protease activity, oligomeric glucan
fragments and intact soluble mannoprotein are obtained.
Mannoprotein is in fact only insoluble when linked to the
glucan of the cell wall.
Separation of the cell wall components with specific
enzymes
Proteases and glucanases are enzymes with a specific
action. The action of proteases will be described first.
The two proteases P1 and P2 are used for this purpose.
The substrate is provided by the cell walls of brewer’s
and baker’s yeast as well as, in individual cases, the intact
living yeast cells. Progress of the reaction was monitored
photometrically by means of the reduction in optical den-
sity at a wavelength of 600 nm53, 54). As the conversion
increased, the extinction value decreased.
The first objective was to determine the optimal reac-
tion conditions. After specification of the pH value and
the temperature, the most important variables are the time
and enzyme quantity. The influence of these two param-
eters on the reaction rate is shown in Fig. 3, where the
reduction in extinction as a function of time was moni-
tored for various enzyme quantities.
In order to be able to compare the different reactions
with one another, the curves were normalized. This
involved dividing each extinction value by the value at
the start of the reaction. Hence all the curves begin at an
extinction of 1.0. As the enzyme quantity increases, the
curve is found to drop more rapidly. However, beyond an
enzyme quantity of 240 mg/g of cell walls no further
increase can be achieved, even when 480 mg of P2 is
F. Kath, W.-M. Kulicke
Fig. 3. Influence of the enzyme quantity in mg on the reduc-
tion in optical density during the reaction of 1 g of brewer’s
yeast cell walls with enzyme cocktail P2.
Fig. 4. Decrease in the optical density of the reaction mixture
during reactions of baker’s yeast cell walls (bacw) with enzyme
cocktail P1.
employed, the pattern of the curve is identical. After ca.
24 h a plateau value is more or less attained. With sub-
stantial quantities of enzymes the reaction should have
reached completion whereas complete conversion will
not yet have been achieved with smaller quantities of
enzymes. Therefore, in order to ensure complete reaction,
240 mg/g of cell walls was employed.
If only mannans are to be extracted, which are in fact
located in the external cell wall, it is also conceivable to
react proteases with the intact yeast cells. In the case of
baker’s yeast absolutely no reduction in the extinction
was observed. For this reason baker’s yeast was investi-
gated in more detail and only the cell walls extracted
mechanically were reacted with P1. Fig. 4 shows the
result of the reaction. Even if the cell walls of baker’s
yeast are reacted with P1, no reduction in the extinction
is found. Apparently, interference with the attack of the
enzyme P1 has occurred. Lipids37) are located in the cell
wall that disrupt the enzyme attack55). Hence reaction
should be possible after extraction of the lipids.
Mild enzymatic isolation of mannan and glucan 65

In order to confirm this, the cell walls of the baker’s

yeast were stirred at room temperature in petroleum ether
and then reacted with the P1 again. But here too no
decrease in extinction over time was observed. Not until
the cell walls have been extracted for 20 h with petroleum
ether at 70 8C is any clear reduction found in the extinc-
tion during the enzyme action. The lipid layer located on
the baker’s yeast would therefore appear to be responsible
for the resistance to P1. However, if the lipids are
removed by extraction, reaction is then possible in an
analogous manner to that of the brewer’s yeast cell walls.
Presumably this problem does not occur in the case of
Fig. 5. Reaction of brewer’s (brcw) and baker’s (bacw) yeast
brewer’s yeast because alcohol is formed during the fer- cell walls as well as reactivated (reac.) baker’s and brewer’s
mentation process, which results in the cell wall lipids yeast cells with different batches of the EC2 enzyme system.
being transferred to the beer.

Separation of the cell wall components with enzyme

cocktails
Apart from the specific enzymes, enzyme cocktails can
also be employed. They contain mixtures of various pro-
teases and glucanases and are thus able to completely
hydrolyse the yeast cells. When enzyme cocktail EC1
was reacted with baker’s yeast cell walls, only a slight
reduction in the optical density was observed, and this
could not even be accelerated by degreasing the cell walls
(not shown in a figure here). The best conversion rates
were achieved with cell walls from brewer’s yeast as a
substrate. However, even after 27 h no plateau value had
been attained, which is why two further enzyme cocktails
were used.
EC2 is another enzyme cocktail. The decrease in opti-
cal density during the reaction of yeast cells and cell
walls with this enzyme system is shown in Fig. 5.
First, baker’s yeast cells were reacted with EC2, with
no decrease whatsoever being observed in the optical
density of the suspension (Batch 1). To examine whether
the enzyme is active at all or whether the enzymatic
activity has possibly been destroyed by incorrect storage,
for example, a second reaction was carried out with the
enzyme from a different batch (Batch 2). No decrease in
optical density was observed here, either.
As aged yeast cells are known to form a thicker and
more resistant cell wall structure, attempts were made to
improve the physiological condition of the baker’s yeast.
Compressed baker’s yeast is in a physiologically poor
condition with a very low metabolic output56). For this
reason a sample of baker’s yeast was reactivated (aerobi-
cally) for 24 h in a nutrient solution to which oxygen was
added. The improvement in physiological condition was
shown by the metabolic output. The ability to liberate car-
bon dioxide was distinctly higher after 24 h, as demon-
strated by the greater degree of bubble formation and
frothing of the suspension on stirring.
Fig. 6. Decrease in the optical density during the reaction of
brewer’s and baker’s yeast cell walls with EC3.
The reactivated yeast cells were centrifuged and reacted
immediately with the enzyme system. If the resistance of
the cells was attributable to the poor physiological state of
the yeast, then the yeast cells would have to successfully
react with the enzyme. But here too no decrease in the opti-
cal density was seen over a period of 30 h.
When only the isolated cell walls were reacted with
EC2, a weak decrease in the optical density was found,
which could not even be improved by prior degreasing.
Therefore the reaction was presumably attributable to the
glucanase activity because in the case of the cell walls, in
contrast to the intact cells, an attack on the glucan can
take place from the inner side of the cell wall.
Only in the case of brewer’s yeast cells did the reaction
proceed without any problems, even though the level of
the plateau value is relatively high. The decrease in opti-
cal density was only 30%, which is less than in protease
reactions, where the decrease was 75%.
EC3 is another enzyme cocktail. The decrease in optical
density during the reaction of cell walls from brewer’s and
baker’s yeasts with this enzyme system is shown in Fig. 6.
An identical curve pattern is found for the cell walls
from both yeasts. The plateau value is attained in as little
66
Fig. 7. Decrease in optical density during reactions of baker’s
yeast or 1 g each of cell walls from brewer’s (brcw) and baker’s
(bacw) yeast with different quantities of cellulase in mg.
as 5 h. In this enzyme system there are either lipases or
the protease activity is not disrupted by adhering lipids on
the baker’s yeast cell walls, as was found for reactions
between baker’s yeast cell walls and P1 (cf. Fig. 6).
The final example of an enzyme cocktail investigated
was industrial cellulase. Reaction of yeast cells with cel-
lulase is conceivable because industrial enzymes fre-
quently do not have a particularly high degree of purity.
Consequently, there are always various secondary activ-
ities that might be exploited for reactions. The advantage
of the enzyme is its low price. The reaction of baker’s
yeast and yeast cell walls from brewer’s and baker’s yeast
with cellulase is shown in Fig. 7.
The curves for both intact cells and cell walls from
baker’s yeast display identical patterns. Consequently,
protease activity must also be present in addition to the
known b-1,3-glucanase activity, otherwise the intact cells
would not be hydrolysed. Overall the decrease in optical
density was only weak even over a period of more than
60 h, and in addition no plateau value is attained.
The influence of the enzyme quantity is also illustrated
by reactions with brewer’s yeast cell walls in Fig. 7. If
the quantity of enzyme is increased, then no rise in the
reaction rate is observed above 200 mg/g of cell walls.
However, a plateau value is not attained until after
approx. 50 – 60 h. Yet the reactions with cell walls from
brewer’s yeast proceeds much better than with those from
baker’s yeast. Consequently, reaction with industrial cel-
lulase is in fact possible but time-consuming.
Summary of the enzyme activities
The reactions of yeast cells and cell walls with the
enzymes and enzyme cocktails investigated may be sum-
marized as follows:
F. Kath, W.-M. Kulicke
1. In the case of the specific enzymes, complete reac-
tion of cell walls from brewer’s yeast with P1 and P2 is
possible within 24 h. Baker’s yeast cell walls can only be
reacted after prior degreasing.
2. The EC1 enzyme system displays very little lytic
activity. The EC2 enzyme cocktail displays a similarly
low lytic activity with brewer’s yeast cell walls, which
comes to a standstill in reaction with baker’s yeast and
baker’s yeast cell walls. Even degreasing the baker’s
yeast cell walls does not produce any success in this case.
3. High lytic activity is only found for the EC3 enzyme
cocktail, which enables almost complete lysis of brewer’s
and baker’s yeast cell walls after only a few h, even with-
out degreasing.
4. If a long reaction period is allowed, it is possible to
achieve lysis of cell walls with certain industrial cellu-
lases, which apparently have b-1,3-glucanase and pro-
tease secondary activities.
5. In general the cell walls from brewer’s yeast can be
digested more easily than those from baker’s yeast, which
first have to be degreased. In terms of completeness of
reaction within an acceptable time, reactions with P1 and
EC3 are preferable. With the other enzyme systems the
reaction within a period of 24 h are too low to practicably
achieve disintegration.
However, photometric monitoring of the reaction alone
is not sufficient to assess the quality of enzymatic conver-
sion, as, for instance, it is not possible to assess the level
of purity and the molar masses with which the polysac-
charides can be isolated. Therefore, the isolated fractions
were characterized by methods of polymer analysis, the
report of which will also be published shortly.
Experimental
Preliminary purification of the brewer’s yeast
In order to obtain the polysaccharides, spent brewer’s yeast
were primarily employed in addition to baker’s yeast. This
comes from the brewing cycle as ready-to-pitch yeast or
compressed surplus yeast and therefore first had to be freed
of beer and hop residues. For this purpose the yeast was first
sieved (mesh width 125 lm, Retsch analytical sieve). The
sieved yeast was centrifuged, the supernatant discarded and
the sediment resuspended in water. The procedure was
repeated three times. The content of dry matter of the reacti-
vated yeast was 12 – 15%, of which ca. 1 – 2% are accounted
for by hop residues. Baker’s and compressed yeast had a dry
matter content of ca. 27 – 30%, the centrifuge sediment of ca.
20%.
Mechanical cell disruption
The mechanical cell disruption is based on an analytical
method by Schütte et al.50). For this purpose a 30% cell sus-
pension was prepared. With reference to the centrifuge sedi-
Mild enzymatic isolation of mannan and glucan
ment this corresponds to an initial weight of 1 g sediment in
2.5 mL suspension. For the baker’s yeast 1 g was weighed
into 2.5 mL, which corresponds to a 40% suspension. A
phosphate buffer was used in order to keep the pH value neu-
tral (13.7 g KH2PO4 , adjusted to pH = 7.5 with NaOH). The
total volume of the milling container was 86 mL.
3
73 mL 112.8 g glass beads (85% of the volume) were
mixed with 51 mL yeast suspension (60% of the volume)a.
The suspension was placed into the mill beaker through a
screw opening with a funnel, the beakers were suspended in
the vibration mill (Type MM 2000, Retsch) and agitated at
the highest power setting for 15 min. Afterwards the contain-
ers were opened and the glass beads were separated via a
sieve, they were then rinsed with water and the filtrate was
centrifuged. The solid was resuspended and centrifuged sev-
eral times and filtered on microporous membranes (pore size
0.65 lm; Minitan-S system, Millipore). The cell walls were
washed with deionized water until the conductivity was less
than 20 lS mL–l. Approximately three times 500 mL were
needed for this. After freeze-drying, 7.4 g of cell walls were
obtained by means of washing and centrifugation and 5.6 g
by separation on microporous membranes.
Detection of proteins
The progress of mechanical cell disruption can be monitored
by quantitative detection of the proteins liberated from the
cell interiors, which were stained with Coomassie Blue. The
depth of colour increases as the concentration of the proteins
rises. In order to monitor the quality of disruption, at various
intervals 100 lL samples were taken from the mill container,
diluted with 1 800 lL buffer, centrifuged for 5 min at
10 000 rpm and mixed with 2 700 mL of Coomassie blue
solution in accordance with the Bradford test for determining
protein content in the supernatant. After a waiting period of
at least 3 min the extinction was measured at a wavelength
of 595 nm52).
Enzymatic digestion
5.0 g yeast cell walls from mechanical disruption were sus-
pended in 115 mL tris buffer pH = 7.5, 10.6 mg Protease (S-
treptomyces griseus, SIGMA (P1), Merck (P2)) were added
and the suspension was stirred at 37 8C for 6 h. The sediment
was centrifuged and the supernatant was dialysed against
water. Freeze drying of the solution yielded mannan, while
2.9 g glucan were found in the sediment. Digestion runs of
EC1 (enzyme cocktail from Helix pomatia, Merck) were car-
ried out at pH = 5.0, 50 – 100 mg enzyme were used to digest
400 mg cell walls. EC2 (from Cytophaga sp., Merck) was
carried out at pH = 7.5, 200 to 400 mg enzyme were used to
digest 1 000 mg cell walls. Digestion with lyticase (EC3,
SIGMA) was carried out at varying pH. Tris buffer was used
for the alkaline range and citrate buffer for the acidic range.
10 000 to 50 000 u were used for 1 000 mg cell walls.
a
All the electron micrographs were taken in Dr. Grunwald’s research group, Institut für Physikalische Chemie, University of Ham-
burg and kindly placed at our disposal.
67
Enzymatic digestion with cellulase
250 mg of cell walls from brewer’s or baker’s yeast were
suspended in 10 mL citrate buffer, pH = 4.9 and conditioned
to 37 8C. Up to 1 g/g of enzyme was added. Using chromato-
graphic separation it was possible to isolate 61 mg of a high-
molar-mass mannan.
Monitoring the enzyme reactions
The progress of enzyme reactions was monitored by obser-
ving the reduction in optical density of the reaction mixture.
100 lL of the reaction suspension was removed, placed in a
cuvette and diluted with 2 mL of water. The extinction was
then determined immediately afterwards at 600 nm.
This report was based on a project funded by the Federal Ger-
man Ministry of Education, Science, Research and Technology
under project code 0311141. Responsibility for the content of
this publication rests with the author.
1) World Health Organization, Fifty facts from the World Health
Report 1997, WHO (Ed.), Internet (WWW) 1997
2) Sir G. J. V. Nossal, Spektrum der Wissenschaft Spezial: Das
Immunsystem 1993(2), 8
3) G. H. Werner, P. Jollès, Eur. J. Biochem. 242 (1996) 1
4) J. A. Bohn, J. N. BeMiller, Carbohydr. Polym. 28 (1995) 3
5) G. Franz, “Struktur und biologische Funktionen von Polysac-
chariden”, in: Polysaccharide, W. Burchard (Ed.), Springer-
Verlag, Berlin, Heidelberg, New York, Tokyo (1985), p. 1
6) H. Eggensperger, M. Wilker, Seifen, Oele, Fette, Wachse 123
(1997) 542
7) H. Furue, Med. Actual. (Drugs Today) 23 (1987) 335
8) W.-M. Kulicke, A. I. Lettau, H. Thielking, Carbohydr. Res.
297 (1997) 135
9) D. L. Williams, I. W. Browder, Polym. Adv. Technol. 5
(1994) 529
10) D. L. Williams, R. B. McNamee, E. L. Jones, H. A. Pretus,
H. E. Ensley, I. W. Browder, N. R. Di Luzio, Carbohydr. Res.
219 (1991) 203
11) T. Mikami, T. Nagase, T. Matsumoto, M. Suzuki, S. Suzuki,
N. Kumano, Microbiol. Immunol. 26 (1982) 913
12) T. Matsumoto, M. Takanohashi, Y. Okubo, M. Suzuki, S.
Suzuki, Carbohydr. Res. 83 (1980) 363
13) S. Suzuki, H. Hatsukaiwa, H. Sunayama, M. Uchiyama, F.
Fukuoka, M. Nakanishi, S. Akiya, Gann 60 (1969) 65
14) S. Suzuki, M. Suzuki, T. Matsumoto, Y. Okawa, Gann 62
(1971) 343
15) A. Müller, “Experimentelle Studien zur Struktur und zu aus-
gewählten Eigenschaften von Polysaccharidfraktionen aus
Hefezellwänden”, Dissertation, Humboldt-Universität, Berlin
(1993)
16) A. Müller, H. Ensley, H. Pretus, R. McNamee, E. Jones, E.
McLaughlin, W. Chandley, W. Browder, D. Lowman, D. Wil-
liams, Carbohydr. Res. 299 (1997) 203
17) R. Bonaly, H. Moulki, A. Touimi Benjellouen, M. Pierrefitte,
Biochim. Biophys. Acta 244 (1971) 484
18) J. S. D. Bacon, V. C. Farmer, D. Jones, I. F. Taylor, Biochem.
J. 114 (1969) 557
68 F. Kath, W.-M. Kulicke

19) D. H. Northcote, R. W. Horne, Biochem. J. 51 (1952) 232 41) U.S. Pat. 4122196 (1978), Anheuser-Busch, Inc., Invs.: E. A.
20) E. D. Korn, Northcote D. H, Biochem. J. 75 (1960) 12 Robbins, R. D. Seeley, Chem. Abstr. 90 (1979) 53150p
21) E. Cabib, R. Roberts, B. Bowers, Ann. Rev. Biochem. 51 42) D. L. Williams, H. A. Pretus, R. B. McNamee, E. L. Jones,
(1982) 763 H. E. Ensley, I. W. Browder, Carbohydr. Res. 235 (1992) 247
22) C. E. Ballou, Adv. Microbiol. Physiol. 14 (1976) 93 43) US Pat. 4739046 (1988), Di Luzio, Invs.: D. L. Williams, I.
23) W. N. Haworth, E. L. Hirst, F. A. Isherwood, J. Chem. Soc. W. Browder, Chem. Abstr. 111 (1989) 5945g
139 (1937) 784 44) H. Schütte, M.-R. Kula, Biotechnol. Appl. Biochem. 12
24) C. E. Ballou, Adv. Enzymol. 40 (1974) 239 (1990) 599
25) R. Sentandreu, D. H. Northcote, Biochem. J. 109 (1968) 419 45) H. Hustedt, K. H. Kroner, H. Schütte, U. Menge, N. Papami-
26) R. Sentandreu, D. H. Northcote, Carbohydr. Res. 10 (1969) chael, BioEngineering 1986(1), 12
584 46) T. N. Cawley, Harrington M. G., R. Letters, Biochem. J. 129
27) K. Kitamura, Agric. Biol. Chem. 46 (1982) 2093 (1972) 711
28) T. S. Stewart, E. C. Ballou, Biochemistry 7 (1968) 1855 47) D. Brady, A. D. Stoll, L. Starke, J. R. Duncan, Biotechnol.
29) S. Peat, J. R. Turvey, J. M. Evans, J. Chem. Soc. 1958, 3868 Bioeng. 44 (1994) 297
30) D. J. Manners, J. C. Patterson, Biochem. J. 98 (1966) 19c– 48) U.S. Pat. 3867554 (1975), Anheuser-Busch, Inc., Invs.: R. W.
20c Sucher, E. A. Robbins, D. R. Sideti, E. H. Schuldt, R. D. See-
31) J. S. D. Bacon, V. C. Farmer, D. Jones, Biochem. J. 114 ley, Chem. Abstr. 81 (1974) 103261n
(1969) 557 49) P. J. Hetherington, M. Follws, P. Dunnill, M. D. Lilly, Trans.
32) D. J. Manners, A. J. Masson, J. C. Patterson, Biochem. J. 135 Inst. Chem. Eng. 49 (1971) 142
(1973) 19 50) H. Schütte, M.-R. Kula, Enzyme Microb. Technol. 10 (1988)
33) G. H. Fleet, D. J. Manners, J. Gen. Microbiol. 94 (1976) 180 552
34) D. J. Manners, A. J. Masson, J. C. Patterson, Biochem. J. 135 51) W. Hummel, M.-R. Kula, J. Microbiol. Methods 9 (1989)
(1973) 31 201
35) M. Kopecká, J. Basic Microbiol. 25 (1985) 161 52) M. M. Bradford, Anal. Biochem. 72 (1976) 248
36) E. Cabib, B. Bowers, J. Biol. Chem. 246 (1971) 152 53) T. Obata, K. Fujioka, S. Hara, Y. Namba, Agric. Biol. Chem.
37) P. Jung, W. Tanner, Eur. J. Biochem. 37 (1973) 1 41 (1977) 671
38) J. Burgos, F. W. Hemming, J. F. Pennock, R. A. Morton, Bio- 54) K. Kitamura, T. Kaneko, Y. Yamamoto, J. Gen. Appl. Micro-
chem. J. 88 (1903) 470 biol. 20 (1974) 323
39) B. A. Andrews, J. A. Asenjo, Trends Biotechnol. 5 (10) 55) K. Kitamura, Y. Yamamoto, Agric. Biol. Chem. 45 (1981)
(1987) 273 1761
40) U.S. Pat. 4810646 (1989), Massachusetts Institute of Tech- 56) L. Narziß, “Die Technologie der Gärung”, in: Abriß der Bier-
nology, Invs.: S. Jamas, C. K. Rha, A. J. Sinskey, Chem. brauerei, 5. Aufl., Enke Verlag, Stuttgart (1986), Ch. 3,
Abstr. 110 (1989) 191290s S. 199