Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
"IMPROVED PROCESS FOR
CLAVULANIC ACID PRODUCTION"
Scope of the Invention
This invention is related to important improvements in the
clavulanic acid fermentation using strains of Streptomyces clavuligerus, low
cost
complex media and strategies of easy industrial implementation. Clavulanic
acid
is used in medicine in association with antibiotics that are inactivated by (3-
lactamases.
Background to the Invention
Clavulanic acid (3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabi-
cyclo[3.2.0]heptane-2-carboxylic acid) is a molecule that has the structural
formula
CHzC~H
H I
H~
'C-C~O\C%C~
H'~ I I I H
,C-N C.
CC!OH
H
This acid has weak antibacterial activity. However, it is a potent
inhibitor of ~3-lactamase enzymes produced by many strains of Staphylococcus
aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and
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Haemophilus influenzae. ~3-Lactamases, through the hydrolysis of the (3-lactam
ring, inactivate several antibiotics, and make the microorganisms, which
produce
them, resistant to those antibiotics.
As potent inhibitor of ~i-lactamases, clavulanic acid is able to avoid
this mechanism of resistance, widening the antibacterial activity spectrum of
several antibiotics. Clavulanic acid presents good synergetic activity when
associated with antibiotics such as amoxycillin, ampicillin, carbenicillin,
ticarcillin, benzylpenicillin or cephaloridine, against (3-lactamase-producing
organisms.
There are several microorganisms which produce clavulanic acid,
namely Streptomyces clavuligerus, Streptomyces jumonjinensis (ES Patent
543 854) and Streptomyces katsurahamanus (JP Patent 53-104796, Takeda
Chemical Industries, Ltd.). There are various processes described for
clavulanic
acid production by Streptomyces clavuligerus, for example {a) discontinuous
fermentation using complex or chemically defined media (BR Patent 1 508 977,
Beecham Group Ltd.); (b) fermentation with automatic control of pH between 6.3
and 6.7 (BR Patent 1 571 888, Glaxo Laboratories Ltd.); (c) fermentation with
continuous or semicontinuous feeding of a carbon source (for example maltose
or
glycerol) (ES Patent 537 157, Antibioticos, S.A.); and {d) fermentation with
control of soluble phosphate in the medium, at the beginning and throughout
the
fermentation (EP Patent 0 811 689, Antibioticos, S.A.).
In discontinuous fermentations of Streptomyces clavuligerus using
soluble media, it is generally observed that the time course of clavulanic
acid titre
is closely related to the dry weight concentration until a maximum value is
reached, followed by a decay of both. Decay of dry weight concentration is
ascribed to sporulation and/or mycelium lysis. Decay of clavulanic acid titre
can
be due to its degradation rate being higher than its production rate.
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Description of the Invention
The present invention concerns a process for clavulanic acid
production by a technique of fermentation which includes the aerobic submerged
culture using selected and/or culture collection strains of Streptomyces
clavuligerus, or mutants thereof. Accordingly, the culture is carried out with
continuous or semicontinuous feeding of one or more organic nitrogen complex
sources, preferably soybean meal, so as to control the protein concentration
in the
filtered broth within certain limits during the time course of the
fermentation.
The conditions described in this invention successfully led to the
following important improvements in clavulanic acid fermentation: (a) a
significant increase in the clavulanic acid production; (b) the prevention of
mycelium lysis throughout the fermentation; (c) a continuous increase in the
clavulanic acid titre and in the dry weight concentration throughout the
fermentation (without partial discharges of fermentation broth); and/or (d) a
continuous increase in the clavulanic acid titre, maintaining the dry weight
concentration approximately constant from a certain point in time (with
partial
discharges of fermentation broth).
Therefore, this invention describes new process strategies for
clavulanic acid production by cultivating the producing microorganism, as for
example Streptomyces clavuligerus ATCC 27064, or mutants thereof, in aerobic
submerged culture using low cost complex media, in distinct conditions from
those patented or reported to date, leading to important improvements in
clavulanic acid fermentation. These conditions consist in the continuous or
staggered feeding of one or more organic nitrogen complex sources, in order to
control the protein concentration in the filtered broth within certain limits
during
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the fermentation, since very high values may inhibit/repress the biosynthesis
of
the antibiotic, and very low values may be limiting for the desired
biosynthesis.
The organic nitrogen complex sources can be seed protein such as soybean meal,
peanut meal, cottonseed meal and linseed meal, fish meal, hydrolysates and
filtrates of such proteins, meat extracts and hydrolysates such as peptones,
being,
preferably, soybean meal. The amount of organic nitrogen complex source to be
fed in a continuous or semicontinuous mode can be in the daily concentration
of
0.1-1.5%, preferably between 0.18 and 1.0%, and/or may be such that the
protein
concentration in the filtered broth is between 200 and 3500 mg/L, preferably
400-
1500 mg/L throughout the fermentation.
According to this process, the initial culture medium can be
composed by one or more organic nitrogen complex sources and, additionally,
one or more carbon sources. The concentration of the organic nitrogen complex
source may fall preferably between 1.3 and 1.8%. The carbon sources may be
glycerol and/or carbohydrates like starch, starch hydrolysates, dextrins and
maltose. It was observed that the simultaneous use of glycerol and dextrin in
the
initial culture medium, preferably in the concentration ranges of,
respectively,
0.9-1.3% and 1.8-2.2%, improve the production of clavulanic acid.
Additionally, one or more carbon sources may be fed continuously
or semicontinuously in daily concentrations between 0.18% and 1 %, and/or so
that the glycerol concentration in the filtered broth is between 0.2 and 12
g/L,
and/or the dextrin or maltose concentrations in the filtered broth are in the
range
4-22 g/L or 2-12 g/L, respectively, during the time course of the
fermentation. It
was observed that the simultaneous feeding of glycerol and dextrin or maltose
improve the production of clavulanic acid. .
The fermentation can be performed with continuous or
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semicontinuous partial discharges of fermentation broth on such way as to
maintain the volume of fermentation broth between 3$ and 6$% of the total
fermenter capacity. The agitator speed can be progressively increased,
according
to the increase in volume, broth viscosity and dry weight concentration during
the
fermentation, so as to improve the mixing and the dissolved oxygen levels in
the
culture.
The culture can be carried out at a temperature between 26 and
29°C and the pH can be controlled between 6.5 and 6.8 by automatic
addition of
acid and base, such as a hydrochloric acid solution and a sodium hydroxide
solution. Foam can be controlled by addition of antifoam, as for example, a
silicone suspension.
The fermentation vessel should be a typical tank for aerobic
fermentation with agitation and aeration devices. The aeration volumetric flow
rate per unit of broth volume may be 0.6 to 1.3 vvm. These tanks should be
supplied with aseptic systems for continuous or semicontinuous feeding of
several nutrients in the form of solution and/or suspension, with aseptic
systems
for continuous or semicontinuous partial discharges of fermentation broth, and
possibly with variable agitator speed.
To carry out the analysis, the samples of culture broth may be
vacuum filtered, preferably through filter paper previously dried in an oven
at
86°C for 12 h. After the filtered broth has been collected, the filter
paper with
the mycelium is washed with distilled water and subsequently dried in an oven
at
86°C for 24 h. The dry weight concentration of the sample is thus
obtained.
The clavulanic acid, protein, glycerol, dextrin or maltose
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concentrations can be measured in the filtered broth by spectrophotometric
methods or, preferably, by high pressure liquid chromatographic methods, for
example using the methods described in (Bird, A.E. et al. 1982, Analyst 107:
1241-1245; Foulstone, M. and Reading, C. 1982, Antimicrob. Agents Chemother.
22: 753-762), (Bradford, M.M. 1976, Anal. Biochem. 72: 248-254), (Bok, S.H.
and Demain, A.L. 1977, Anal. Biochem. 81: 18-20), (Nelson, N. 1944, J. Biol.
Chem. 153: 375-380; Somogyi, M. 1952, J. Biol. Chem. 195: 19-23) respectively.
A summary of the results obtained is presented through the various
examples that follow.
EXAMPLE I
A spore suspension of Streptomyces clavuligerus ATCC 27064 was
prepared from agar slants containing hydrated dextrin 10 g, yeast extract 1 g,
meat extract 1 g, bacteriological peptone 2 g, CaC03 2 g, agar 20 g, per litre
of
distilled water. The pH was corrected to 7.1 with 1 M NaOH and 1 M HCI.
This spore suspension was used to inoculate various 500 mL
conical flasks containing 50 mL of culture medium A (soybean meal 15 g, (87%)
glycerol 10 g, hydrated dextrin 10 g, KH2P04 1 g, per litre of distilled
water). The
pH was corrected to 7.2 with 1 M NaOH and 1 M HCI. The flasks were sterilized
at 121°C for 15 min and incubated at 30°C and 110-140 rpm during
2 days. The
content of 5 flasks was then mixed, giving the vegetative inoculum.
Separately, 3.1 L of a culture medium B composed by soybean meal
15 g, (87%) glycerol 13 g, hydrated dextrin 20 g, {50%) silicone suspension 1
g,
per litre of tap water, was introduced into a 8 litre STR fermenter and
sterilized
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for 20 min at about 120°C. The fermenter was inoculated with the
vegetative
inoculum prepared previously.
The fermentation was carried out with automatic control of pH at
6.60,05 by addition of a 5% (v/v) HCl solution and 1M NaOH solution. Foam
was controlled by addition of a SO% silicone suspension. The temperature was
kept between 26 and 29°C, and the aeration between 0.7 and 1.2 vvm.
At 24 h the staggered feeding of a culture medium C composed by
soybean meal 65 g and (87%) glycerol 100 g, per litre of tap water, was
started,
using a peristaltic pump of variable rate. The volumetric flow rate was
controlled
manually according to the result of the daily analysis of protein and glycerol
concentrations in the filtered culture broth. Thus, the volumetric flow rate
varied
throughout the time course of the fermentation as follows:
TABLEI
Time Flow
h mL/h
24-79 5
79-101 8
101-159 18
159-166 0
Since the volume, broth viscosity and dry weight concentration
increased throughout the course of the fermentation, the agitator speed was
manually increased, on such way as to improve the mixing and the dissolved
oxygen levels in the culture. Thus, the agitator speed and the dissolved
oxygen
concentration varied in the following way during the fermentation:
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TABLE II
Time Agitator Speed Dissolved Oxygen
(h m
0-24 500 100.0-75.6
24-56 600 89.6-52.5
56-72 700 56.8-48.5
72-79 800 51.3-49.9
79-96 1000 48.5-29.7
96-1 O 1100 41.7-34.3
1
1 O 1-1171200 36.9-39.2
117-166 1300 45.9-21.6
The following values of volume of fermentation broth, dry weight
concentration, clavulanic acid titre and protein, glycerol and dextrin
concentrations in the filtered broth, throughout the time course of the
fermentation, were obtained:
TABLE III
Time Volume ClavulanicDry WeightProteinGlycerolDextrin
(h) (L) Acid (g/L) (mg/L) (g/L) (g/L)
/mL
0 3.34 0 5.3 3064 10.9 21.9
24 3.34 90 7.0 860 9.0 19.3
48 3.50 441 9.6 511 8.6 13.4
72 3.67 698 11.2 494 7.0 8.4
96 3.83 857 12.2 674 3.9 7.7
120 4.20 1021 13.8 854 4.4 7.4
144 4.65 1096 14.7 911 4.5 5.9
166 4.92 1224 15.6 1154 2.8 7.2
Sporulation was observed under the microscope throughout the
fermentation, but mycelium lysis was not detected.
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At 166 h the culture broth attained a volume of 4.92 L, a clavulanic
acid titre of 1224 ~g/mL and a dry weight concentration of 15.6 g/L. In other
words, 1943 ~,g of clavulanic acid and 24.8 mg of dry weight per mL of culture
medium B were obtained.
EXAMPLE 2
The fermentation was carried out as described in example 1, except
for the changes described next.
The staggered feeding of a culture medium D composed by
soybean meal 65 g, (87%) glycerol 56.3 g, hydrated dextrin 86.6 g and (50%)
silicone suspension 2 g, per litre of tap water, was carried out from 24 h
fermentation time until the end of the fermentation. The volumetric flow rate
was
manually controlled according to the result of the daily analysis of protein,
glycerol, and dextrin concentrations in the filtered broth. Thus, the
volumetric
flow rate varied in the following way throughout the time course of the
fermentation
TABLE IV
Time Flow
h mL/h
24-107 11
107-131 19
131-143 11
The agitator speed and the dissolved oxygen concentration varied
throughout the fermentation time as follows:
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TABLE V
Time Agitator Speed Dissolved Oxygen
h m
0-24 S00 99.4-71.9
24-5 S 600 7 8.9-54.0
55-75 700 66.6-53.2
75-107 800 59.0-44.4
107-131 900 51.4-21.0
131-143 1000 29.2-3 5.6
Partial discharges of fermentation broth were carned out (8.3% v/v)
at 100 and 123 h, using a peristaltic pump with manual control, according to
the
increase in volume of culture broth in the fermenter.
The following values of volume of fermentation broth, volume of
partial discharges of fermentation broth, dry weight concentration, clavulanic
acid titre and protein, glycerol and dextrin concentrations in the filtered
broth
along the fermentation process were obtained:
TABLE VI
Time Volume Partial ClavulanicDry WeightProteinGlycerolDextrin
(h) (L) DischargeAcid (g/L) (mg/L) (g/L) (g/L)
Volume (~,g/mL)
L
0 3.24 - 0 5.5 1673 11.4 20.8
24 3.26 - 91 6.8 718 9.4 18.3
48 3.56 - 426 10.6 634 8.2 19.0
75 3.88 - 801 14.2 318 7.8 16.1
100 4.20 0.35 1035 16.2 464 4.2 17.5
123 4.21 0.35 1264 17.3 674 2.2 24.6
143 4.15 - 1374 18.7 647 0.6 22.8
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Sporulation throughout the fermentation, and the beginning of
mycelium lysis at the end of the fermentation (at 143 h) were observed under
the
microscope.
At 143 h the culture broth attained a volume of 4.15 L, a clavulanic
acid titre of 1374 ~,g/mL and a dry weight concentration of 18.7 g/L. In other
words, 1839 ~,g of elavulanic acid and 25.0 mg of dry weight per mL of culture
medium B were obtained. Considering the partial discharges, a total of 2099
~,g
of clavulanic acid and 28.8 mg of dry weight per mL of culture medium B were
obtained.
EXAMPLE 3
The fermentation was carried out according to example 1, except
for the changes described next.
Culture medium B was replaced by culture medium E composed by
soybean meal 1 S g and (50%) silicone suspension 2 g, per litre of tap water.
The staggered feeding of a culture medium F composed by soybean
meal 65 g, (87%) glycerol 70 g and monohydrated maltose 65 g, per litre of tap
water, took place from 0 h of fermentation time. The volumetric flow rate was
manually controlled according to the result of the daily analysis of the
protein,
glycerol and maltose concentrations in the filtered broth. Thus, the
volumetric
flow rate varied throughout the fermentation time as follows:
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TABLE VII
Time Flow
h mL/h
0-24 S
24-129 11
129-153 22
153-216 11
The agitator speed and the dissolved oxygen concentration varied during
the fermentation as follows:
TABLE VIII
Time Agitator Speed Dissolved Oxygen
h m
0-24 500 98.6-68.9
24-48 600 74.4-79.9
48-72 700 82.0-73.1
72-146 800 80.6-51.7
146-216 900 54.4-37.9
Partial discharges of fermentation broth were performed through a
peristaltic pump of manual control, according to the increase in volume of
culture
broth in the fermenter. Thus, partial discharges of fermentation broth of
8.3%,
7.2%, 10.3%. 10.5% and 7.3% (v/v) at 96, 120, 144, 168 and 192 h,
respectively,
were carried out.
The following values of volume of fermentation broth, volume of
partial discharges of fermentation broth, dry weight concentration, clavulanic
acid titre and protein, glycerol and maltose concentrations in the filtered
broth
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throughout the time course of the fermentation were obtained:
TABLE IX
Time Volume Partial ClavulanicDry WeightProteinGlycerolMaltose
(h) (L) DischargeAcid (g/L) (mg/L) (g/L) (g/L)
Volume (~tg/mL)
L
0 3.14 - 0 5.4 3137 0.3 2.5
24 3.32 - 123 7.0 962 0.7 2.7
48 3.62 - 496 10.2 742 2.0 3.8
72 3.93 - 759 11.9 625 2.3 4.6
96 4.20 0.3 5 904 13 .2 770 1.9 5.3
120 4.16 0.30 1074 13.9 862 1.2 4.7
144 4.35 0.45 1164 15.7 850 3.6 7.8
168 4.28 0.45 1295 16.1 853 5.1 8.2
192 4.10 0.30 1475 16.3 810 6.0 7.9
216 4.02 - 1607 16.6 936 6.4 8.2
Sporulation was observed under the microscope throughout the
fermentation, but no mycelium lysis was detected.
At 216 h the culture broth attained a volume of 4.02 L, a clavulanic
acid titre of 1607 ~g/mL and a dry weight concentration of 16.6 g/L. In other
words, 2084 ~,g of clavulanic acid and 21.5 mg of dry weight per mL of culture
medium E were obtained. Taking into consideration the partial discharges, a
total
of 2790 ~g of clavulanic acid and 30.6 mg of dry weight per mL of culture
medium E were attained.
