Note: Descriptions are shown in the official language in which they were submitted.
~. 1 1 336~
54-344.501
Process for extracting and purifvinq
epidermin
The present invention relates to a process for
obtaining the antibiotic polypeptide epidermin.
Epidermin, which is disclosed in EP-A-181578, acts
as an antibiotic against skin infections, such as
eczema, impetigo, cellulitis and acne.
EP-A-181578 describes a process for obtaining,
isolating and purifying epidermin starting from a
culture broth of a resistant strain of Staphylococcus
epidermidis. Such a strain was deposited on 26th
October 1984 at the "Deutsche Sammlung von
Mikroorganismen" under the Number DSM 3095.
To isolate epidermin, the active component is
concentrated by extracting the culture filtrate, freed
from cells and lime, with n-butanol, followed by
evaporating the butanol extract, dissolving the residue
in methanol and stirring it into an excessive quantity
of cold diethyl ether in order to separate off the
lipidic contaminants, the activity remaining in the
precipitate; alternatively in order to concentrate
epidermin, the centrifuged culture filtrate is adsorbed
on the polymer Amberlite XAD-8 (available from Serva) or
on related types of acrylic ester based polymer,
adsorbed epidermin is eluted from the resin with
methanol/conc. hydrochloric acid (99:1) and isolated by
evaporation from the hydrochloric acid methanol
solution, after neutralisation with ammonia. Subsequent
chromatography of the Amberlite XAD eluate or of the
butanol extract freed from lipids, using Sephadex LH-20
with methanol/acetic acid (95:5), separates a large
number of small peptides, amino acids and salts from
epidermin in the medium. In subsequent multiplicative
counter-current distribution using the Craig method,
epidermin is left behind at the start in a first liquid-
f c~Je ma~k ~,
I 336896
liquid distribution using the system n-butanol/ethyl
acetate/0.1 N acetic acid (3:1:3). In a second Craig
distribution with the neutral system 2-butanol/0.05 N
ammonium acetate (1:1) epidermin is in the centre of the
apparatus. Ammonium acetate is removed by
lyophilisation under high vacuum, and, after freeze-
drying, epidermin is obtained as a white powder which is
uniform in all the thin layer systems used. In this
way, 2.6 g of lyophilised epidermin can be obtained from
80 litres of culture filtrate (with adsorption on
Amberlite XAD-8, gel chromatography on Sephadex LH-20
and multiplicative distribution according to Craig).
As described in EP-A-181578, the producing organism
StaphYlococcus epidermidis DSM 3095 is grown aerobically
at 37C in a complex medium made up of 2 to 4% meat
extract, 1 to 3% malt extract and 0.25 to 1% CaCO3 or
0.25 to 0.5% Ca(OH) 2 (the percentages being by weight).
um antibiotic activity is reached after 18 to 23
hours.
There is accordingly a need for a simpler and more
efficient method of extracting and purifying epidermin
and it has now surprisingly been found that this can be
achieved by a procedure involving adsorbing epidermin
from a culture of Staphylococcus epidermidis onto a
styrene divinyl copolymer resin, eluting it onto a
cation exchanger, eluting the epidermin from the cation
exchanger and adsorbing it onto a further styrene
divinyl copQlyme~ resin and then releasing the purified
epidermin thcrcfor~ and, if desired, further purifying
it by preparative high performance liquid chromatography
(HPLC).
According to one aspect, the invention thus
provides a process for extracting and purifying
epidermin from a culture of an epidermin - producing
strain of Staphylococcus epidermidis, said process
comprising:
a) adsorbing epidermin from said culture onto a first
- 3 ~ 9 ~
styrene-divinyl copolymer resin;
b) eluting epidermin from said resin and applying the
eluate to a cation exchanger;
c) eluting epidermin from said exchanger and adsorbing
epidermin from the resultant eluate onto a second
styrene-divinyl copolymer resin; and
d) eluting epidermin from said second resin.
Epidermin obtained using the process of the
invention may conveniently be further purified by HPLC
and obtained as a solid by evaporation or freeze-drying.
Using the process of the invention, the antibiotic
epidermin can be obtained in substantially higher yields
than before and by a simpler method.
Thus in one particularly preferred embodiment of
the process of the invention the antibiotic in the
culture filtrate or in the culture broth, still
containing the microorganism, is adsorbed on Amberlite
XAD-1180 or Amberlite XAD-16 or a related type of
styrene-divinyl copolymer based resin either by passing
the epidermin containing culture through a column
containing the resin or by adding the resin in small
amounts to the epidermin containing culture, e.g. to the
culture broth.
In a particularly preferred embodiment, the process
of the invention comprises the following steps:
i) contacting a culture filtrate or broth of an
epidermin producing strain of StraphYlococcus
epidermidis with a styrene-divinyl copolymer resin;
ii) releasing the active component from the resin by
elution with methanol and diluted hydrochloric
acid;
iii) adjusting the pH of the eluate to between 5.3 and
5.8;
iv) contacting the eluate with a cation exchanger;
v) washing out non-bound substances with a buffer
solution at pH 7;
vi) eluting the active component from the cation
- 1 ~3689~
-
exchanger with a solution comprising buffer
substance, sodium chloride and methanol at pH 6.0
to 8.0;
vii) adsorbing epidermin from the resulting eluate onto
a second styrene-divinyl copolymer resin;
viii)eluting epidermin from said second resin with
methanol and acetic acid; and
ix) evaporating or freeze drying the resulting eluate
and if desired subjecting the epidermin thus
obtained to further purification by HPLC.
In the process of the invention the active
component is conveniently released from the first resin
by elution with methanol diluted especially with 0.01 N
hydrochloric acid (9:1,v:v), the pH of the eluate is
adjusted to 5.3 to 5.8 and the eluate is conveniently
placed on a weak cation exchanger such as Amberlite
IRC-50 or a related resin such as Amberlite IRC-84, for
example. The ion exchange may be effected by passage of
the eluate through a column containing the ion exchange
resin or by addition of ion exchange resin in small
amounts to the eluate.
Where ion exchange resin is added to the eluate,
this is preferably effected by two additions of a
quantity of 3% by volume of the ion exchange resin
within one hour. After adsorption the resin is then
preferably introduced into a column, and non-bound
substances may then be washed out with a 0.05 N sodium
phosphate buffer solution in water at pH 7.0, the
epidermin is then eluted with a solution of 80% of the
above-mentioned sodium phosphate buffer, which is 1.5 N
with regard to common salt and contains 20% by volume of
methanol, at a pH between 6.0 and B.0, preferably at pH
7Ø
To remove the salt from the epidermin containing
eluate from the ion exchanger, the eluate is preferably
adjusted to a pH of 6.0 and then added in small amounts
for adsorption to a second styrene-divinyl copolymer
3 3 ~
~ 5
resin, e.g. Amberlite XAD-1180. The salts may then be
washed out with water and the bound epidermin may then
be eluted with methanol/50% acetic acid (9:1, v:v).
After removal of the solvent and freeze drying, a
lyophilised crude epidermin is obtained. From this,
pure epidermin can be isolated by preparative HPLC for
example using Nucleosil~100 C-18 (10 ~m) or Lichro-Sorb
RP Select B. For this purpose the epidermin is
preferably eluted in stepwise fashion, first with a
solvent A consisting of water containing 1~ by weight of
formic acid, then with a solvent B, consisting of
methanol/water (80:20, v:v) with 1~ by weight of formic
acid.
The process of the invention will now be further
described with reference to the accompanying drawings,
in which:
Figure 1 is a graph showing the effect of 3% sodium
chloride and varying concentrations of ferric chloride
in the fermentation medium on epidermin production,
Figure 2 is a graph showing the variation with time
in the course of batch fermentation in a 20 litre
fermentor of
the number of living cells (colony forming units)
( ~);
the epidermin concentration ( ~ ),
the sugar concentration ( 0 ),
the nitrogen concentration ( ~ ),
the phosphate concentration ( ~ ), and
the pH ( ~ ),
Figure 3 is a graph showing the variation with time
in the course of feeding fermentation in a 20 litre
fermentor of
the number of living cells (colony forming units)
( ~ ),
the epidermin concentration ( ),
the sugar consumption ( ~ ),
the nitrogen consumption ( ~ ), and
~r6lde- ~n q~k
- 1 336896
. -6
the phosphate consumption ( ), and showing, with
an arrow, the start of nitrogen and phosphate feeding,
Figure 4 is a graph showing the variation with time
in the course of feeding fermentation with discontinuous
adsorption of
the number of living cells (colony forming units)
the concentration of epidermin in the culture broth
( O ), and
the concentration of resin-bound epidermin ( ~ ).
Figure S is a graph showing the influence of
ventilation and stirring rates on epidermin production
in a 20 litre bioreactor,
Figure 6 is a graph showing the influence of
glucose concentration on epidermin production,
Figure 7 is a graph showing the influence of
initial phosphate concentration on epidermin production,
Figure 8 is a graph showing the influence of
initial concentration of ammonium ions (as ammonium
chloride) on epidermin production,
Figure 9 is a flow diagram of a discontinuous
adsorption process for epidermin extraction, and
Figure 10 is a flow diagram for the extraction of
epidermin by continuous on-line adsorption batch and
column processes.
According to the invention epidermin may
conveniently be produced by batch fermentation, or a
related feeding fermentation in which individual
substances are added during fermentation, and extracted
by continuous or discontinuous on-line adsorption.
In batch fermentation it has also been found that
the epidermin yield can be improved by modifying the
chemical composition of the culture medium, and the best
results were achieved with a nutrient solution having
the following composition: 3.3% of meat extract, 3% of
malt extract, 0.37% of calcium hydroxide (% by weight).
The yield is advantageously affected by the addition of
~ 7 1 336896
1 to 6% by weight of alkali metal chlorides (such as
sodium chloride and potassium chloride), 0.001 to 0.002%
by weight of iron ions (e.g. in the form of FeCl3 and/or
FeS04), and also ammonium chloride or sulphate (between
57 and 200 mM); the preferred concentrations being 3% by
weight of sodium chloride and 0.00125% by weight of
ferric chloride.
Thus in another aspect the invention also provides
a method of producing epidermin comprising culturing an
epidermin-producing strain of StaPhylococcus epidermidis
in a medium comprising 2 to 4% by weight of meat
extract, about 3% by weight of malt extract or maltose,
0.25~ to 1% by weight of calcium hydroxide and/or
calcium carbonate e.g. about 0.37% of calcium hydroxide,
1 to 6% by weight of sodium chloride and/or potassium
chloride, 0.001 to 0.002% of iron ions, and 57 to 200 mM
ammonium ions, and, optionally, potassium or sodium
dihydrogen phosphate at a concentration of up to 10 mM,
at a pH of 6.0 to 7Ø
In the epidermin producing method of the invention,
which is preferably performed in conjunction with the
epidermin extracting and purifying process of the
invention, iron ions are preferably provided by ferric
chloride and/or ferrous sulphate and ammonium ions by
ammonium chloride and/or sulphate.
The influence of initial concentrations of 3%
common salt and different concentrations of ferric
chloride on epidermin production is illustrated in
Fig. 1. Of all the C-sources for the culture medium,
maltose gave the best results after malt extract. It is
advisable to use glucose only in conjunction with other
C-sources. A combination of lactose with maltose or
galactose with maltose gave equally good results. A
combination of glucose with maltose or lactose or
galactose gave the same yield as the malt extract. All
the other conventional C-sources resulted in either
minimal or no epidermin production.
1 336896
Fermentation is conveniently carried out with good
ventilation at temperatures of between 34 and 37C,
preferably 36C. The best pattern of production is
obtained if the pH value is 6.0 to 7.0 before
fermentation. In the absence of carbonates or
hydroxides of divalent cations, such as calcium
carbonate or calcium hydroxide, only a little production
occurs. After the addition of calcium carbonate, for
example, the pH value shows a characteristic pattern,
falling into the acid range, in which case only slight
production occurs. When the pH value subsequently
climbed into the alkaline range production initiated.
Instead of calcium carbonate it is also possible to use
magnesium carbonate whilst calcium hydroxide yielded
better results than calcium carbonate. The production
was increased somewhat by using 50 mM calcium hydroxide
rather than 25 mM calcium carbonate. When the C-sources
(sugar) are utilised by the strain, organic acids such
as acetic acid are formed, which are complexed by
divalent cations whilst at the same time the medium is
buffered.
The increase in epidermin production using
procedures according to the invention may be illustrated
by the activity at the time of maximum production by the
strain DSM 3095, determined by the plate diffusion test
(determining the diameter of the inhibitory area in mm
against Micrococcus luteus ATCC 9341), using a
calibrated line, with the activity in a brain-heart
infusion nutrient solution according to EP-A-27 710
being taken as 100%. For the following culture media,
using the prior art processes or the process according
to the invention, the activities determined were as
follows:-
by the processes according to the prior art:
g t 336896 -`
Run Medium ActivitY
A brain-heart-infusion-agar
(according to EP-A-27 710) 100%
B 3% meat extract, 2% malt extract,
25 mM calcium carbonate
(according to EP-A-181 578) 200%
C 3% meat extract, 2% malt extract,
50 mM calcium hydroxide
(according to EP-A-181 578) 320%
by the processes according to the invention:
D 3.3% meat extract, 3% malt extract,
50 mM calcium hydroxide 440%
E 3.3% meat extract, 3% malt extract,
50 mM calcium hydroxide, 3% sodium chloride,
75 ~M iron(III)chloride 1,720%
F 3.3% meat extract, 3% malt extract,
50 mM calcium hydroxide, with the
further addition of glucose, KH2 P4
and ammonium chloride 1,950%
G 3.3% meat extract, 3% malt extract,
50 mM calcium hydroxide, with
additional on-line adsorption of
the epidermin produced after the
first isolation step, and for
other additives see Run F2,780%.
Fig. 2 shows the course of fermentation for Run D
above; Fig. 1 shows the dependency of epidermin
production on the addition of 3~ sodium chloride and of
- 1 336896
--
various quantities of iron(III)chloride as specified for
Run E. All the percentages given above are percent by
weight. Fig. 3 shows the course of fermentation in Run
F, Fig. 4 shows the same course when on-line adsorption
is included, i.e. in Run G.
A comparison of the activity values achieved by the
plate diffusion test or HPLC shows that the process
according to the invention as compared with the
processes known per se enables a significant increase in
the epidermin yields, namely from 320% up to 2,780%, to
be achieved.
The individual steps and preferred conditions for
carrying out the process according to the invention are
described more fully hereinafter.
The epidermin producing strain is best stored by
deep-freezing (-18C) in a medium contAin;ng 3.3% by
weight of meat extract, 3% by weight of malt extract,
0.37% by weight of calcium hydroxide and 40% by weight
of glycerol (the balance being water). For each
fermentation, a pre-culture is grown for 18 hours at
36 C on an agar medium (pH 7.2 to 7.4) which contains
per litre 8 g of Lab Lemco powder (available from
Oxoid), 10 g of peptone, 3 g of common salt, 2 g of
Na2HPO4, 15 g of agar and 10 g of sterilised glucose.
Fermentation can conveniently be carried out in
suitable shaking flasks and, in order to produce larger
quantities of the substance, fermenters with capacities
of 200 litres or more may be used.
For flask tests, 500 ml Erlenmeyer flasks with a
lateral opening may be used. The flasks are
conveniently filled with 100 ml of nutrient solution and
autoclaved for 20 minutes at 121C. The inoculant used
is conveniently 1~ of an 8 hour old preculture.
Incubation is conveniently at 36C on a shaking machine
rotating at 160 revolutions per minute (rpm).
For fermentation on the 15 litre scale, a 20 litre
fermentor (type b 20 Braun/Melsungen or Giovanola
~-~
~ 11 1 336896
Freres, Monthey, Switzerland; with a recirculating
system) was charged with 15 litres of nutrient solution
with the addition of 0.5 ml of a polyol
(polypropyleneglycol) and sterilisation was carried out
in situ at 121CC for 30 minutes. The inoculant used was
150 ml of an 8 hour old preculture. Fermentation was
carried out at between 35 to 37C, 0.2 to 0.6 vvm and 700
to 1000 rpm, especially at 36C, 0.4 vvm and 900 rpm.
Production of epidermin by batch fermentation:
In the bioreactors used, the best growth and yield
of epidermin were obtained at 36C, a ventilation rate
of 0.4 vvm and stirring at 900 rpm. Reduced ventilation
and slower stirring reduced the yield of epidermin
(as shown in Fig. 5). More powerful ventilation and
stirring resulted in a slight improvement in the yield
but excessive foaming also occurred. This is because of
the powerful surface activity of the antibiotic; the
foam could not be suppressed mechanically or chemically
without some loss of activity.
Fig. 2 shows the course of a batch fermentation on
the 20 litre scale with the vigorously epidermin
producing strain DSM 3095 in a nutrient solution
containing 33 g of meat extract, 30 g of malt extract,
3.8 g of calcium hydroxide in 1 litre. This strain
produces epidermin in a quantity up to 80 mg/l.
Intensive growth combined with the utilisation of the C-
sources provided, with the formation of acetate, can be
recognised by the drop in the pH value. A maximum
number of cells is achieved after 30 hours. After this
time, the chief fermentable C-sources such as glucose
and maltose are exhausted. The phosphate added is used
up after 8 hours. The maximum concentration of the
antibiotic is achieved after 48 hours and amounts to
80 mg/l.
Both the addition of chlorides such as sodium or
1 336896
- 12
potassium chloride in a quantity up to 70 g per litre
and the addition of FeCl3 or FeSO4 up to 150 ~M increased
the yield of epidermin without lengthening the
fermentation process (see Fig. 1). The maximum yield of
epidermin of 310 mg/l was obtained by simultaneously
adding 3% sodium chloride and 75 ~M iron(III)chloride to
the production medium.
This rapid epidermin production constitutes a good
method of developing a continuous fermentation process
with high throughputs of the medium used.
Epidermin production bY feeding fermentation:
In order to be able to increase the yield of
epidermin by extending the growth phase and achieving a
higher cell density, higher concentrations both of the
C-sources and also of the added phosphate were
necessary. Epidermin production is subject to strong C-
catabolic repression (see Fig. 6) and is also regulated
by the phosphate content of the medium (see Fig. 7).
Throughout the entire fermentation process the
nitrogen concentration is above 150 mM and the majority
of this content cannot be used by microorganisms. In
flask cultures, the addition of ammonium salts up to 150
mM, indeed up to 190 mM, and preferably between 75 and
175 mM, more preferably between 100 and 150 mM and
especially preferably between 125 and 150 mM resulted,
as shown in Fig. 8 (for ammonium chloride), in a
significant stimulation of epidermin production.
Glucose is primarily metabolised to form acetate,
as shown by the acidification of the medium. If there
is a shortage of sugars, organic acids are used as
additional C-sources, whilst the pH of the medium
changes to the alkaline range. Glucose may therefore be
added in accordance with the pH during the course of
fermentation, such that the pH is maintained at about
6.0 and acidification is not suppressed. Glucose may
13 1 3 3 6 8 9 6 -
conveniently be added at a concentration of 20 mM.
Phosphates may be added continuously, e.g. to a
concentration of up to 11 mM, preferably 5 to 10 mM.
The rate of addition was calculated by means of the rate
of consumption of the phosphates in batch fermentations
in which 10 mM of phosphate were added.
Neither the addition of glucose or phosphate alone
nor together resulted in a significantly higher yield of
epidermin during fermentation. The higher content of
epidermin which was found with the pH-regulated addition
of glucose during fermentation, does not depend on
additional C-sources but is caused by the slower
decomposition of the antibiotic in the acidic medium.
This can be demonstrated by fermentations in which the
pH value is kept constant by the addition of sulphuric
acid. Only the development of combined feeding
fermentation with pH-dependent addition of glucose and
continuous addition of phosphates and ammonium nitrogen
resulted in both a significant increase in the biomass
and also an increase in the yield of antibiotic. Fig. 3
shows the course of a combined feeding fermentation.
The cell mass grows by three times the amount to
2xl01l cells per ml; the maximum epidermin yield is
350 mg/l. Although the maximum cell concentration is
reached after 24 hours, the maximum epidermin yield is
only achieved during the stationary phase after 72
hours; in the course of feeding fermentation the
identity of the growth and production phase which is
characteristic of batch fermentation disappears.
Nevertheless, epidermin production remains closely
linked with the growth of the organism. Firstly, 80% of
the total yield are produced during the log phase and
secondly the biomass is produced by the number of living
cells, which means that the stationary phase can be
regarded as a balanced state produced by the growing and
lysing cells.
The addition of sodium chloride during feeding
- 1 336896
14
fermentation does not produce the same stimulating
effect as occurs in batch fermentation. During the
first 24 hours the production rate was 40~ higher and
the growth rate was 70~ higher than in the case of
feeding fermentation. The specific productivity of the
microorganism is therefore 20% lower; after 24 hours the
epidermin production ceases, which can be put down
either to limitation of nutrients by a hitherto unknown
factor or inherent poisoning of the organism by
metabolic by-products.
The combined feeding fermentation was also
transferred to the 200 litre scale of a pilot plant,
using the same ventilation rate r stirrer speed and the
same conditions of addition. Without any additional
optimisation, epidermin yields of the order of 80 to 90%
of those achieved on the 20 litre scale were obtained.
Obtaining ePidermin with on-line adsorption of the
antibiotic produced:
Discontinuous adsorption:
In order to prevent feedback inhibition and other
possible influences on the producing organisms and in
order to protect the epidermin formed from the
decomposing influence of proteases and heat, the
epidermin was discontinuously removed from the
fermentation liquor during fermentation.
In order to do this, the entire fermentation liquor
including the producing organisms were sprayed under
pressure into a spherical adsorption chamber filled with
Amberlite XAD-1180. This effectively made the resin
highly turbulent and resulted in rapid adsorption of the
epidermin. The free-flowing resin pellets were filtered
off with a screen (diameter 0.25 mm), whilst the
fermentation liquor with the biomass was recycled into
the reactor (see Fig. 9). The course of feeding
1 33689~
fermentation with discontinuous adsorption of the
epidermin is shown in Fig. 4. The fermentation
parameters and conditions of addition were identical to
those described above. The maximum cell mass was
achieved after 46 hours, at 4 x 101 cells and the
maximum epidermin yield after elution from the resin was
500 mg/l. The total fermentation time was the same as
in the feeding fermentation.
The first purification or crude purification is
simplified by including the first isolation step in the
fermentation process; the epidermin yield after this
first purification step is 50% higher compared with the
yield in feeding fermentation before purification.
Continuous adsorption:
The continuous on-line adsorption of the epidermin
during the fermentation process is carried out using a
cross-flow filtration apparatus. The material retained
is recycled into the reactor whilst the filtrate is
adsorbed on an Amberlite XAD-1180 column. The eluate is
also recycled into the reactor (see Fig.10). Adsorption
is set in operation after 12 to 15 hours' fermentation.
Maximum epidermin yields after the antibiotic has been
released from the adsorber resin are achieved with
quantities of up to 500 mg/l after 80 to 90 hours.
The optimised isolation plan, as shown in Fig. 10,
constitutes a major achievement in the attempt to
increase the production of epidermin. Finally, a
product with a purity of 80~ is obtained from the
combination of adsorption according to the invention
with ion exchange chromatography. The use both of batch
and on-line adsorption processes results in a rapid
method of purifying epidermin.
In order to monitor the course of fermentation,
samples are taken under sterile conditions at various
times during fermentation. The samples were evaluated
.. 16 1 336896 --
as follows:
a) pH values: ~
Measurement with a laboratory pH-meter (Knick pH-mV
meter).
.
b) Growth pattern:
Growth was monitored by means of the increase in
the number of living bacteria. 0.5 ml of culture
taken under sterile conditions were diluted in
saline and 0.1 ml of this was plated out onto
plates (medium: peptone 10 g, meat extract 8 g,
common salt 3 g, disodium hydrogen phosphate 2 g,
glucose 10 g to 1 litre). After 18 hours'
incubation at 37C the individual colonies could be
counted.
c) Concentration of antibiotic:
The samples were centrifuged in an Eppendorf
centrifuge 3200 for 2 minutes and 10 ~1 of the
supernatant were tested either in the plate
diffusion test or by HPLC as described below. In
parallel, a calibrating curve was plotted using
known concentrations.
_ ~ 17 1 336896
HPLC system for measuring epidermin concentration:
Spray volume: 10 ~1
Eluent: A : water cont~in;ng 0.05% 70% perchloric
acid
B : acetonitrile
Gradient: Minutes A B
0 77.5 22.5
8 63.0 37.0
8.5 0 100
9.5 o 100
77.5 22.5
14 77.5 22.5
Flow rate: 2 ml/min.
Detection: 210 nm ~
Column: Nucleosil 7 C-18 with associated
C preliminary column.
d) Phosphate measurement:
The phosphate content in the culture filtrate was
measured using the method of Itaya and Ui, Clin.
Chim. Acta 14: 361-366 (1966).
e) Measurement of nitrogen:
The nitrogen concentration was measured by the
Micro-Kjeldahl method using an automatic
distillation apparatus (of the Kjeldahl system II
type made by Tecator~. -
f) Measurement of glucose and maltose:
The content of glucose and maltose in the medium
was determined enzymatically using a Test Kit made
by Boehringer Mannheim.
g) Acetate measurement:
This was carried out by gas chromatography
18 1 33689~
following the instructions of Platen and Schink
Arch. Microbiol. 149: 136-141 (1987).
h) Epidermin measurement:
The epidermin concentration in the culture broth
was measured once by bioassay and another time by
HPLC according to Fiedler et al., Chromatographia
24: 433-438 (1987).
After the production peak had been reached the
culture liquid was centrifuged off by continuous
centrifugation (centrifuge type LA 71b-4, Loher & Sohne,
Ruhstorf/Rott) at 1380 rpm. In order to achieve optimal
separation of the cells the flow rate had to be kept
very slow. A first concentration of the active
components was achieved by adsorption on styrene-divinyl
copolymers as described hereinbefore.
Other methods of adsorption of epidermin, for
example batch adsorption, discontinuous and continuous
adsorption processes with and without separation of the
cell mass, have already been described hereinbefore.
The following non-limiting Examples are intended to
illustrate the invention:
Example 1
Batch fermentation with a strain of Staphylococcus
epidermidis DSM 3095
he culture medium contained:
3.3% by weight Lab Lemco powder
3.0% by weight malt extract
0.38% by weight calcium hydroxide
pH adjusted to 6.5 with 3 N H2SO4.
The reactor used was:
Type b 20 (Giovanola) with 15 1 of medium
Ventilation: 0.4 vvm
Stirring rate: 900 rpm
- 1 3 3 6 8 96
. 19
Temperature: 36C.
It was found that the maximum epidermin yield, 80 mg/l,
occurred after 48 hours (see Fig. 2).
Example 2
Feeding fermentation with a strain of
StaPhylococcus ePidermidis DSM 3095
The culture medium contained:
3.3% by weight Lab Lemco powder
3.0% by weight malt extract
0.38~ by weight calcium hydroxide
pH adjusted to 6.5 with 3 N H2S04.
The bioreactor was
Type b 20 (Giovanola) with 15 1 of medium
Ventilation: 0.4 vvm
Stirring rate: 900 rpm
Temperature: 36C.
The conditions of addition of nutrients were:
Solution 1:
Glucose 1 kg, water 1 1, pH 6Ø This solution was
added in accordance with the pH and the pH of the
fermentation solution was adjusted to 6Ø
Solution 2:
NH4Cl 500 g, KH2PO4 120 g, water 1.5 1, pH 6.0 (adjusted
with anhydrous NaOH). The solution was fed in
constantly at a flow rate of 1 ml per litre per hour.
The addition began after 4 hours.
It was found that the maximum epidermin yield, 350 mg/l,
occurred after 72 hours (see Fig. 3).
~ 20 1 336896
ExamPle 3
Feeding fermentation with on-line adsorption of the
antibiotic
The culture medium and bioreactor were as in Example 2.
Epidermin was adsorbed onto Amberlite XAD-1180, 150 g
dry weight.
The maximum epidermin yield, 500 mg/l, occurred after 72
hours. Epidermin yield was measured after the first
isolation step (see also Example 4). For results see
Fig. 4.
Example 4
Isolation and purification of the epidermin from a
15 1 culture broth after using on-line adsorption
After on-line adsorption of epidermin during the
fermentation process, the resin (see Example 3) was
washed with 150 1 of water and, if an adsorption chamber
was used, transferred into a column for further washing
and elution.
The resin was washed with 5 1 methanol/H20 1:1 (v:v)
and eluted with 5 1 methanol/0.01 N hydrochloric acid
9:1 (v:v).
The pH of the eluate was adjusted to 5.5;
epidermin was adsorbed onto the ion exchange resin by
the addition of two batches of 45 g of Amberlite IRC-50
over a period of 1 hour to the eluate. The ion exchange
resin was transferred into a column for washing with 2 1
0.05 N Na phosphate buffer pH 7.0 and subsequent elution
with 10 1 0.05 N Na phosphate, 1.5 N common salt in
water/methanol 8:2 (v:v), pH 7Ø
In order to remove the salt the pH of the eluate
was adjusted to 6.0 and epidermin was adsorbed in
batches on Amberlite XAD-1180, in 2 batches each
consisting of 75 g of dry weight within 1 hour. The
.
21 1 3 3 6 8 9 6
resin was packed into a column for washing with 20 1 of
water, and subsequent elution with 2.5 1 of
methanol/0.01 N hydrochloric acid 9:1 (v:v).
After evaporation of the solvent and freeze-drying
of the eluate, 6500 mg of a substance were obtained
containing 5200 mg of epidermin (80% purity). Final
purification was carried out by preparative HPLC with
gradient elution.
The column used was Nucleosil 100 C-18 (10 ~)
The solvents were A: water containing 1% by weight
formic acid
B: methanol/water 8:2 containing 1%
formic acid.
After preparative HPLC and freeze-drying, 4.94 g of
pure epidermin were obtained, which were uniform in all
the tests carried out.
The flow diagram for the procedure of Examples 3
and 4 is shown in Fig. 10.
During the preparation and isolation of epidermin,
the plate diffusion test was used for monitoring and
biological characterisation. Further information on
this subject can be found in EP-A-181578, which
describes the strain StaPhylococcus epidermidis DSM-3095
in more detail.
The culture broth produced by the strain DSM-3095
can also be exchanged for a culture broth produced by
the strain NCIB 11536 (deposited at the National
Collection of Industrial Bacteria in Aberdeen), in order
to produce epidermin therein, for subsequent isolation
and purification. However, in terms of epidermin
production, the strain NCIB 11536 is inferior to the
strain DSM 3095.
