U.S. patent application number 10/994268 was filed with the patent office on 2005-04-28 for fermentation process for lipstatin and method of extracting lipstatin from a fermentation broth.
This patent application is currently assigned to Teva Pharmaceuticals USA, Inc.. Invention is credited to Balogh, Gabor, Csorvasi, Andrea, Erdei, Janos, Gulyas, Eva, Keri, Vilmos, Toth, Laszlo.
Application Number | 20050089978 10/994268 |
Document ID | / |
Family ID | 26990598 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089978 |
Kind Code |
A1 |
Erdei, Janos ; et
al. |
April 28, 2005 |
Fermentation process for lipstatin and method of extracting
lipstatin from a fermentation broth
Abstract
The present invention provides a fermentation process for
producing lipstatin comprising the steps of: a) preparing a
fermentation medium containing a lipstatin-producing microorganism
comprising an oil and an assimilable carbon source, wherein the
wt/wt ratio of oil and assimilable carbon source is regulated to
achieve an optimal lipstatin biosynthesis by the microorganism; and
b) feeding the fermentation medium with an emulsifier, wherein the
emulsifier provides an optimal viscosity for the fermentation
medium and optimal pH during the fermentation to permit
fermentation for lipstatin production. The disclosed process also
provides a process for extracting a lipstatin from a fermentation
broth.
Inventors: |
Erdei, Janos; (Debrecen,
HU) ; Gulyas, Eva; (Debrecen, HU) ; Balogh,
Gabor; (Debrecen, HU) ; Toth, Laszlo;
(Balmazujvaros, HU) ; Keri, Vilmos; (Debrecen,
HU) ; Csorvasi, Andrea; (Debrecen, HU) |
Correspondence
Address: |
KENYON & KENYON
ONE BROADWAY
NEW YORK
NY
10004
US
|
Assignee: |
Teva Pharmaceuticals USA,
Inc.
|
Family ID: |
26990598 |
Appl. No.: |
10/994268 |
Filed: |
November 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10994268 |
Nov 23, 2004 |
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10313603 |
Dec 4, 2002 |
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6844174 |
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60337218 |
Dec 4, 2001 |
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60394566 |
Jul 9, 2002 |
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Current U.S.
Class: |
435/134 ;
435/125 |
Current CPC
Class: |
C07D 305/12 20130101;
C12P 17/02 20130101 |
Class at
Publication: |
435/134 ;
435/125 |
International
Class: |
C12P 007/64; C12P
017/06 |
Claims
1-25. (canceled)
26. A process for separating lipstatin from a fermentation broth,
comprising the steps of: (a) extracting the lipstatin from a
fermentation broth with a first extraction solvent; (b)
concentrating the first extraction solvent; (c) extracting the
lipstatin from the first concentrated extraction solvent with a
second extraction solvent; (d) concentrating the second extraction
solvent; (e) adding a third extraction solvent; (f) washing the
lipstatin in the third extraction solvent with a fourth extraction
solvent; and (g) separating the washed lipstatin from the third
extraction solvent.
27. The process for extracting lipstatin from a fermentation broth
as in claim 26, wherein the separating step (g) comprises: (h)
concentrating the third extraction solvent; (i) adding a fifth
extraction solvent; (j) extracting the lipstatin from the fifth
extraction with a fourth extraction solvent; (k) concentrating the
fourth extraction solvent; (l) diluting the extraction solvent with
a lower alkyl alcohol; and (m) applying the diluted lower alkyl
alcohol solvent to an anion-exchanger to obtain lipstatin.
28. The process of extracting the lipstatin from a fermentation
broth as in claim 26, after the step (f) and before step (g),
further comprising the steps of: (n) extracting lipstatin from the
fourth extraction solvent into a third extraction solvent; (o)
combining the washed third extraction solvent of step (f) with
third extraction solvent of step (n); and (p) concentrating the
combined third extraction solvents of step (O).
29. The process of extracting the lipstatin from a fermentation
broth as in claim 27, after the step (k) and before step (l),
further comprising the steps of: (q) extracting lipstatin into the
fifth extraction solvent; and (r) concentrating the extracted fifth
extraction solvent.
30. The process of claim 26, wherein the first extraction solvent
is a water immiscible solvent.
31. The process of claim 26, wherein the first extraction solvent
is selected from the group consisting of ethyl acetate, i-butyl
acetate, butyl acetate and methyl ethyl ketone.
32. The process of claim 26, wherein the first extraction solvent
is i-butyl acetate.
33. The process of claim 26, wherein the first extraction is done
at a pH about 2 to about 10.5.
34. The process of claim 26, wherein the first extraction is done
at a pH about 5 to about 8.
35. The process of claim 26, wherein the first extraction is done
at a pH about 6 to about 7.
36. The process of claim 26, wherein the concentrating step of the
first extraction solvent is carried out under reduced pressure.
37. The process of claim 26, wherein the concentrating step of the
first extraction solvent is carried out at a maximum temperature of
about 80.degree. C.
38. The process of claim 26, wherein the second extraction solvent
is a lower alkyl alcohol.
39. The process of claim 26, wherein the second extraction solvent
is selected from the group consisting of methanol, ethanol,
1-propanol, 2-propanol, 1-butanol, i-butanol, tert-butanol,
acetonitrile, and acetic acid.
40. The process of claim 26, wherein the second extraction solvent
is methanol.
41. The process of claim 26, wherein the concentrating step of the
second extraction solvent is carried out under reduced
pressure.
42. The process of claim 26, wherein the concentrating step of the
second extraction solvent is carried out at a maximum temperature
of about 80.degree. C.
43. The process of one of claims 26 and 27, wherein the third
extraction solvent is acetonitrile.
44. The process of one of claims 26 and 27, wherein the fourth
extraction solvent serves to wash lipstatin present in the third
extraction solvent.
45. The process of one of claims 26 and 27, wherein the fourth
extraction solvent is a hydrocarbon solvent.
46. The process of one of claims 26 and 27, wherein the fourth
extraction solvent is selected from the group consisting of
pentane, hexane, cyclohexane and heptane.
47. The process of one of claims 26 and 27, wherein the fourth
extraction solvent is hexane.
48. The process of one of claims 26 and 27, wherein the fourth
extraction solvent is heptane.
49. The process of one of claims 26 and 27, wherein the fourth
extraction solvent serves to extract lipstatin from the fifth
extraction solvent.
50. The process of one of claims 27, 28 and 29, wherein the fifth
extraction solvent is a lower alkyl alcohol.
51. The process of one of claims 27, 28 and 29, wherein the fifth
extraction solvent is a mixture of lower alkyl alcohol and
water.
52. The process of claim 51, wherein the lower alkyl alcohol is
methanol.
53. The process of claim 52, wherein the volume to volume ratio of
methanol to water is about 70:15.
54. The process of claim 27, wherein the lower alkyl alcohol is
methanol.
55. The process of one of claims 27, 28 and 29, wherein the
extracting step is carried out under reduced pressure.
56. The process of one of claims 27, 28 and 29, wherein the
extracting step is carried out at a temperature of less than about
80.degree. C.
57. The process of claim 27, wherein the anion-exchanger is an
anion-exchanger resin.
58. The process of claim 56, wherein the anion-exchanger resin is
Amberlite.TM. IRA.
59-63. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
1.119(e) of Provisional Applications Ser. No. 60/337,218 filed Dec.
4, 2001 and No. 60/394,566 filed Jul. 9, 2002, the disclosure of
which is incorporated by reference in its entirety herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a fermentation process for
producing lipstatin and a method of extracting lipstatin from a
fermentation broth.
BACKGROUND OF THE INVENTION
[0003] Obesity and hyperlipidemia are often associated with insulin
resistance, diabetes mellitus, hypertension, and cardiovascular
diseases, resulting in a high morbidity rate and early mortality.
Little is known about the etiology of obesity and hyperlipidemia.
The current strategy involves weight control. Several alternate new
therapeutic approaches include the use of .beta..sub.3-adrenoceptor
agonists, CCK-A/cholecystokinin-A-receptor agonists, neuropeptide
Y-receptor antagonists and pancreatic lipase inhibitors. The
pancreatic lipase inhibitors (orlistat/lipstatin) are among the
best candidates.
[0004] Lipstatin is a precursor for Orlistat. Upon hydrogenation,
lipstatin is converted to form Orlistat. Orlistat has the chemical
name (2S, 3S,
5S)-5-[(S)-2-formamido-4-methylvaleryloxy]-2-hexy-3-hydroxyhexad-
ecanoic acid lactone [a/k/a "N-formyl-L-leucine ester with (3S,
4S)-3-hexyl-4-[(2S)-2-hydroxytridecyl]-2-oxetanone",
(-)-tetrahydrolipstatin, tetrahydrolipstatin, and orlipastat].
Orlistat has the following chemical formula: 1
Orlistat
[0005] U.S. Pat. No. 4,598,089 is directed to orlistat and its use
in treating obesity. Orlistat is currently available for the
treatment of obesity. It inhibits lipase enzymes that are
responsible for breaking down ingested fat [Borgstrom, B. (1988).
Biochem. Biophy. Acta. 962 (3): 308-316], resulting in lipids being
digested in the feces rather than being absorbed. Orlistat's
inhibition has valuable therapeutic uses, such as control or
prevention of hyperlipaemia, atherosclerosis and
arteriosclerosis.
[0006] Although lipstatin can be synthesized chemically,
fermentation to produce lipstatin is more economical. Weibel et al.
disclose generally the fermentation process for lipstatin (Journal
of Antibiotics Vol XL, No.8 pp 1086-1091) and E. Hochuli, et al.
disclose the structural chemistry for lipstatin (Journal of
Antibiotics Vol XL, No.8 pp 1081-1085).
[0007] U.S. Pat. No. 4,598,089 and Eur. Pat. Appl. 129,748 disclose
the cultivation and fermentation of Streptomyces toxytricini. The
'089 patent discloses the use of a specific Stretomycetes strain
(i.e., Streptomycin toxytricini [NRRL 15443]) in producing
lipstatin.
[0008] According to the examples of the U.S. Pat. No. 4,598,089,
the vegetative mycelium of Streptomyces toxytricini was used for
seeding of the inoculum fermentation. After three days incubation,
the inoculum fermentation is seeded into a pre-culture medium, and
after three additional days incubation this culture serves for
seeding the production medium. In the production medium the main
carbon sources were potato starch, glucose, ribose and glycerine,
and the main nitrogen sources were peptone, soya bean meal, and
ammonium-sulphate. During incubation the temperature was maintained
at 28.degree. C., aeration was 1 vvm and the mixing speed was 150
rpm. In some examples, lard oil was used in a small quantity (0.1%)
as the lipase inductor and anti-foaming agent.
[0009] EP 0 803 567 A2 discloses an improved process wherein
selected lipstatin precursors were included in the fermentation.
These ingredients were linoleic acid, caprylic acid and
N-formyl-L-leucine or preferably L-leucine. The yield of the
fermentation was low because of the toxicity of the two fatty acids
and the amounts of feed solutions were very small. This
fermentation process uses a medium that is substantially free of
fats and oils because they result in uncontrolled fatty acid
liberation during the fermentation and high oil residue at the end
of fermentation.
[0010] An additional problem with using fats and oils is the
question of the emulsion formation. Emulsion formation is important
for the microbial consumption of fats but there is no emulsion
formation without emulsifier. Eisenreich discloses the use of
lecithin as an emulsifier (Journal of Biological Chemistry, Vol
272. No.2, pp. 867-874, 1997). Lecithin may produce a strong
emulsion and result in undesirably high viscosity of the
fermentation broth which may hinder mixing and aeration during the
fermentation. The use of lecithin in lipstatin fermentation may
therefore be disadvantageous. There is a continuing need for a
desirable emulsifier for use in fermentation processes for
producing lipstatin.
[0011] U.S. Pat. No. 4,598,089 and Eur. Pat. Appl. 129,748 also
disclose the preparation of lipstatin from a fermentation broth of
Streptomyces toxytricini in the preparation of orlistat. The
purification of lipstatin involves multiple chromatographies. The
repeated purification processes are costly and impractical for
large scale manufacturing. There is a continuing need to improve
the isolation and purification of lipstatin from a fermentation
broth.
OBJECT AND SUMMARY OF THE INVENTION
[0012] The present invention provides a fermentation process for
producing lipstatin, comprising the steps of:
[0013] a) preparing a fermentation medium containing a
lipstatin-producing microorganism and further comprising an oil and
an assimilable carbon source, wherein the wt/wt ratio of oil and
assimilable carbon source is adjusted to regulate lipstatin
biosynthesis by the microorganism; and
[0014] b) introducing an emulsifier to the fermentation broth to
regulate the fermentation broth viscosity to regulate the
production of lipstatin production.
[0015] Preferably, the wt/wt ratio of oil and assimilable carbon
source is at least 2:1. More preferably, the wt/wt ratio of oil and
assimilable carbon source is at least 3:1 Most preferably, the
wt/wt ratio of oil and assimilable carbon source is at least
5:1.
[0016] Preferably, the quantity of oil fed into the fermentation
broth is not less than 5% (wt/wt) and not more than 15%
(wt/wt).
[0017] Preferably, the oil is selected from the group consisting of
a natural oil, a synthetic oil and a mixture thereof. The natural
oil is selected from the group consisting of Soya been oil, palm
oil, sunflower oil, flax oil, rape seed oil, and corn germ oil.
Preferably, the synthetic oil is a synthetic fatty acid glyceride.
Preferably, the synthetic oil is selected from the group consisting
of AGRIMUL GTO 39 G/glycerol trioleate and AGRIMUL FAC 18
SB/unsaturated C-18 vegetable fatty acid.
[0018] Preferably, the emulsifier is a natural emulsifier. More
preferably, the natural emulsifier is lecithin. Preferably, the
emulsifier is a synthetic, non-consumable emulsifier. More
preferably, the synthetic, non-consumable emulsifier is selected
from the group consisting of Triton-X-100, Triton-X-45, Brij.35,
Igepal/octoxynol as the Triton-X-100 and a mixture thereof.
[0019] Preferably, the amount of synthetic emulsifier fed into the
fermentation broth is 0.01% (vol/vol) to 0.6% (vol/vol) of the
fermentation broth. More preferably, the amount of synthetic and
natural emulsifier of emulsifier is equal to or less than 1.3%
(vol/vol).
[0020] Preferably, the assimilable carbon source is selected from
the group consisting of glucose, fructose, saccharose, maltose, and
glycerol.
[0021] Preferably, the viscosity of the fermentation broth is
controlled at least below 1,000 mPascal secundum. More preferably,
the viscosity of the fermentation broth is controlled at 300-400
mPascal secundum. Preferably, the feeding of emulsifier is
performed at the beginning of the fermentation process.
[0022] Preferably, the present invention provides a fermentation
process of lipstatin, comprising the step of regulating pH of the
fermentation broth. More preferably, the pH of the fermentation
broth is regulated between about 6.0 to about 7.5. Most preferably,
the pH of the fermentation broth is regulated between about 6.5 to
about 7.5.
[0023] Preferably, the pH is regulated by feeding the fermentation
broth with at least one of an acid, a base, or an assimilable
carbon source. Preferably, the lipstatin-producing microorganism is
Streptomyces toxytricini.
[0024] The present invention provides a process for separating a
lipstatin from a fermentation broth, comprising the steps of:
[0025] (a) extracting the lipstatin from a fermentation broth with
a first extraction solvent;
[0026] (b) concentrating the first extraction solvent;
[0027] (c) extracting the lipstatin from the first concentrated
extraction solvent with a second extraction solvent;
[0028] (d) concentrating the second extraction solvent;
[0029] (e) adding a third extraction solvent;
[0030] (f) washing the lipstatin in the third extraction solvent
with a fourth extraction solvent; and
[0031] (g) separating the washed lipstatin from the third
extraction solvent.
[0032] Preferably, after the step (f) and before step (g), the
present invention further comprises the steps of:
[0033] (n) extracting lipstatin from the fourth extraction solvent
into a third extraction solvent;
[0034] (o) combining the washed third extraction solvent of step
(f) with third extraction solvent of step (n).
[0035] The present invention further provides extracting the
lipstatin from a fermentation broth wherein the step (g) comprises
the additional steps of:
[0036] (h) concentrating the third extraction solvent;
[0037] (i) adding a fifth extraction solvent;
[0038] (j) extracting the lipstatin from the fifth extraction with
a fourth extraction solvent;
[0039] (k) concentrating the fourth extraction solvent;
[0040] (l) diluting the extraction solvent with a lower alkyl
alcohol; and
[0041] (m) applying the diluted lower alkyl alcohol solvent to an
anion-exchanger to obtain lipstatin.
[0042] Preferably, after the step (k) and before step (l) of the
additional steps, the present invention further comprises the steps
of:
[0043] (q) extracting lipstatin into the fifth extraction solvent;
and
[0044] (r) concentrating the extracted fifth extraction
solvent.
[0045] Preferably, the first extraction solvent is a water
immiscible solvent. Preferably, the first extraction solvent is
selected from the group consisting of ethyl acetate, i-butyl
acetate, butyl acetate and methyl ethyl ketone. Most preferably,
the first extraction solvent is i-butyl acetate.
[0046] Preferably, the first extraction is done at a pH about 2 to
about 10.5. More preferably, pH is about 5 to about 8. Most
preferably, the pH is about 6 to about 7. Preferably, the
concentrating step of the first extraction solvent is carried out
under reduced pressure and at a temperature less than about
80.degree. C.
[0047] Preferably, the second extraction solvent is a lower alkyl
alcohol. Preferably, the second extraction solvent is selected from
the group consisting of methanol, ethanol, 1-propanol, 2-propanol,
1-butanol, i-butanol, tert-butanol, acetonitrile, and acetic acid.
More preferably, the second extraction solvent is methanol.
Preferably, the concentrating step of the second extraction solvent
is carried out under reduced pressure and at a temperature less
than about 80.degree. C.
[0048] Preferably, the third extraction solvent is
acetonitrile.
[0049] Preferably, the fourth extraction solvent serves to wash
lipstatin present in the third extraction solvent. Preferably, the
fourth extraction solvent is a hydrocarbon solvent. More
preferably, the fourth extraction solvent is selected from the
group consisting of pentane, hexane, cyclohexane and heptane. Most
preferably, the fourth extraction solvent is hexane or heptane.
[0050] Preferably, the fourth extraction solvent serves to extract
lipstatin from the fifth extraction solvent.
[0051] Preferably, the fifth extraction solvent is a mixture of
lower alkyl alcohol and water. More preferably, the fifth
extraction solvent is a mixture of lower alkyl alcohol. More
preferably, the fifth extraction solvent is a mixture of methanol
and water. More preferably, the volume to volume ratio of methanol
to water is about 70:15.
[0052] Preferably, the steps (l) and (m) are performed using a
lower alkyl alcohol. More preferably, the lower alkyl alcohol is
methanol. Preferably, the extracting step is carried out under
reduced pressure and at a temperature of less than about 80.degree.
C.
[0053] Preferably, the anion-exchanger is an anion-exchanger resin.
More preferably, the anion-exchanger resin is Amberlite.TM. RA.
DETAILED DESCRIPTION OF THE INVENTION
[0054] Definitions:
[0055] As used herein, the terms: "vvm" refers to
volume/volume/min; "rpm" refers to revolution per min; "ppm" refers
to part per million; and "mPa.s" refers to millipascal
secundum.
[0056] As used herein, the term "fermentation broth" refers to the
fermentation culture medium containing a lipstatin producing
microorganism. Preferably, the microorganism is a bacterium.
Preferably, the lipstatin producing bacteria include Streptomyces
(a genus of gram-positive spore forming bacteria that grow slowly
in soil or water as a branching filamentous mycelium similar to
that of fungi. A preferred lipstatin producing Streptomyces is
Streptomyces toxytricini, which also include many different
Streptomyces toxytricini strains (e.g., Streptomycin toxytricini
NRRL 15443).
[0057] As used herein, the term "lipstatin" refers to a precursor
of orlistat. Orlistat, also known as tetrahydrolipstatin, has four
more hydrogens than lipstatin Thus, the conversion of lipstatin to
orlistat requires hydrogenation.
[0058] As used herein, the term "oil" is used interchangeable with
"fat". Oil is referred to as a carbon source or a precursor of
carbon source.
[0059] As used herein, the term "emulsifier" refers to a substance
which can be used to produce an emulsion from two liquids that
normally cannot be mixed together (such as oil and water). An
emulsion refers to a preparation of one liquid distributed in small
globules, i.e., a discontinuous phase throughout the body of a
second liquid, i.e., a continuous phase.
[0060] As used herein, the term "Triton X-100" refers to
octylphenol ethylene oxide condensate which is a nonionic
detergent. It was a registered trademark formerly owned by Rohm and
Haas Co., but now owned by Union Carbide. The "X" series of Triton
detergents are produced from octylphenol polymerized with ethylene
oxide. The number ("-100") relates only indirectly to the number of
ethylene oxide units in the structure. X-100 has an "average of
9.5" ethylene oxide units per molecule. A nonionic detergent refers
to a detergent in which the hydrophilic head group is
uncharged.
[0061] As used herein, the term "extraction solvent" may refer to a
solvent used to extract lipstatin from the fermentation broth. Of
course, all extraction solvent function as solvent and may also be
used as washing solvent, e.g., fourth extraction solvent.
[0062] Extraction solvent is a hydrophobic solvents may be a
hydrophobic or hydrophilic solvent. Exemplary hydrophobic solvents
include i-butyl acetate and ethyl acetate and exemplary hydrophilic
solvents include alcohols.
[0063] As used herein, the term "alcohol" refers to alkyloxides.
Examples for alcohol include methanol, ethanol, propanol or
butanol.
[0064] As used herein, the term "under reduced pressure" refers to
less than atmospheric pressure (760 mmHg).
[0065] As used herein, the term "ion-exchanger" preferably refers
to an anion-exchanger.
[0066] As used herein, the term "extraction yield %" refers to the
% of the ratio of the amount of lipstatin present in the
fermentation broth before and after the extraction step.
[0067] As used herein, the term "overall yield" refers to the
average yield.
[0068] As used herein, the term "linoleic acid" refers to an
impurity or precursor that is present in the fermentation broth and
after the addition of second extraction solution is an
impurity.
[0069] As used herein, the term "impurity" refers to compounds that
are present in the fermentation broth other than lipstatin before,
during or after the purification steps. An example of impurity
includes linoleic acid.
[0070] Supplying the fermentation broth with precursors (e.g.,
linoleic acid) is beneficial to the lipstatin biosynthesis. Because
of the toxicity of fatty acids it has to be fed very carefully.
Many natural oils contain linoleic acid in its glycerol ester form.
Streptomyces toxytricini also produces lipase, and depending on the
pH of broth the lipase can liberate linoleic acid in different
quantities.
[0071] Preferably, the fermentation broth is fed with fat in
sufficiently high amount to permit good fermentation and lipstatin
production. Typically, the amount of fat is in excess than that of
that amount of assimilable carbon. Preferably, the wt/wt ratio of
oil and assimilable carbon source in the fermentation broth is at
least 2:1, more preferably, the ratio is 3:1 and most preferably,
the ratio is 5:1 or higher.
[0072] Preferably, the fat includes natural fat, synthetic fat or a
mixture thereof. The natural fat may include, but not limited to,
soya bean oil, palm oil, sunflower oil, flax oil, rape seed oil,
and corn germ oil. Synthetic oil includes, but are not limited to,
AGRIMUL GTO 39G/glycerol trioleate and AGRIMUL FAC 18 SB/C-18
unsaturated vegetable fatty acids and mixtures thereof. Preferably,
the amount of fat is not less than 5% (wt/wt) and not more than 15%
(wt/wt) of the fermentation broth.
[0073] The relationship between the pH of the fermentation broth
and the actual concentration of linoleic acid which is liberated
from the oil content of the fermentation broth is illustrated in
Table 1, below.
1 TABLE 1 pH Concentration of Linoleic Acid 6.0 200 .+-. 50 ppm 6.5
600 .+-. 100 ppm 7.0 1,000 .+-. 200 ppm 7.5 1,700 .+-. 300 ppm 8.0
3,500 .+-. 500 ppm
[0074] With Streptomyces toxytricini, the toxic level of linoleic
acid in liquid culture is about 2,300-3,000 ppm. We found that if
the pH of lipstatin fermentation broth was not higher than about
7.0 to about 7.5, the linoleic acid liberation and linoleic acid
consumption were balanced, and the actual concentration of linoleic
acid was not more than about 0.15 to about 0.2%, consequently not
reaching the toxic level. For this reason, the pH of lipstatin
fermentation was controlled so that the linoleic acid being
liberated from fats and oils was enough for the lipstatin
biosynthesis but not raised to a toxic level.
[0075] Preferably, the pH of the fermentation broth is regulated at
about 6.0 to about 7.0. Most preferably, the pH of the fermentation
broth is regulated at about 6.5 to about 7.0.
[0076] Preferably, the pH of the fermentation broth is regulated by
feeding the fermentation broth with an acid, a base or an
assimilable carbon source. Preferred assimilable carbon sources
include, but are not limited to, glucose, fructose, saccharose,
maltose, and glycerol.
[0077] Preferably, oils or fats are used as an assimilable carbon
source. It is beneficial to use an emulsifier for enhancing the oil
consumption. In the presence of an emulsifier, the oil forms a
macroscopically homogenous emulsion with the aqueous phase. In
addition, the emulsifier increases the bioavailability of oil as a
lipstatin precursor for the microorganisms. The efficiency of the
emulsification could be monitored by the lipstatin intermediates
(e.g., linoleic acid) and lipstatin formation.
[0078] We found that a properly selected emulsifier was an
important factor in regulating the viscosity of the fermentation
broth. It is known that the soya lecithin (see in Journal of
Biological Chemistry, Vol. 272. No. 2. Pp. 867-874, 1997) results
in a highly viscous emulsion. We found that if a synthetic
emulsifier was used instead of lecithin, a lesser amount was enough
for the emulsification and regulating the viscosity of the broth,
e.g., about five times less than the lecithin needed. When lecithin
emulsifier was used it produced extremely high viscosity and the
whole aerobic process was stopped by the third day of fermentation
and lipstatin biosynthesis stopped as well. Also the dissolved
oxygen concentration went to zero, oxygen consumption stopped, and
the broth became like a solid foam. As a result, the lipstatin
yield was only the half of the expected value.
[0079] Among the many main parameters that can influence the
emulsification, mixing rate for the fermentation broth is a key
factor. Preferably, the mixing rate is increased during the
fermentation process. The exact mixing rate may be adjusted
according to the dissolved oxygen level present in the fermentation
broth. The dissolved oxygen level was monitored with an
amperometric oxygen electrode (Mettler Toledo GmbH, Urdorf,
Switzerland). The oxygen electrode was calibrated to the air
saturated medium (set to be 80%) and to a zero point which was set
electronically. The dissolved oxygen level during the fermentation
is expected to decrease relative to the saturated oxygen level due
to the metabolic oxygen consumption and uptake by the
microorganisms. Hence, the mixing rate is preferably increased
during the fermentation process to compensate the decrease in
dissolved oxygen level and to maintain the dissolved oxygen level
at an optimal level.
[0080] A preferred viscosity for the fermentation broth is less
than 1,000 mPascal secundum. A more preferred viscosity for the
fermentation broth is less than 400-500 mPascal secundum. The most
preferred viscosity for the fermentation broth is less than 300-400
mPascal secundum. The viscosity of the fermentation broth was
measured with a RHEOLAB MC 1 (Paar Physica, Stuttgart, Germany)
rotational, shear stress and creep rheometer equipped with a Z3 DIN
standard measuring system.
[0081] We tested different conventional synthetic emulsifiers
including Triton-X-100, Triton-X-45, Brij-35, and Igepal. We found
that they were all suitable for the emulsion formation. Optimal
concentrations of these synthetic emulsifiers were determined for
achieving a condition whereby the maximum viscosity was low enough
but the dissolved oxygen level and aerobic metabolic activity were
maintained. Ideally, emulsifier is used at a concentration to
provide sufficient emulsification such that the particle size of
emulsion is small and a homogeneous emulsion is formed. Preferably,
agitation is adjusted by changing the mixing rate. Agitation is
used to enhance mixing of fermentation broth and allow emulsifier
to achieve maximum emulsification effect. Agitation therefore
allows adequate oxygen level while maintaining a homogeneous
emulsion.
[0082] Preferably, the amount of synthetic emulsifier fed into the
fermentation broth is 0.01% (vol/vol) to 0.6% (vol/vol) of the
fermentation broth. Preferably, the amount of natural and synthetic
emulsifier fed into the fermentation broth is equal to or less than
1.3% (vol/vol) of the fermentation broth. The introduction of the
emulsifier can occur at the beginning of the fermentation process
or during the course of the fermentation process. Preferably, the
feeding of emulsifier occurs at the beginning of the fermentation
process.
[0083] The following alternative embodiments are based on the
extraction processes of lipstatin as set forth in the claims.
[0084] According to one alternative embodiment of the present
invention, lipstatin present in the fermentation broth is extracted
using the first extraction solvent. Preferably, the first
extraction solvent is a hydrophobic solvent selected from the group
consisting of ethyl acetate, i-butyl acetate, butyl acetate and
methyl ethyl ketone. More preferably, the first extraction solvent
is i-butyl acetate.
[0085] Preferably, the first extraction is carried out at a pH of
about 2 to about 11. More preferably, the first extraction is
carried out at a pH of about 5 to about 8. Most preferably, the
first extraction is carried out at a pH of about 6 to about 7.
[0086] According to another alterative embodiment, the present
invention provides a process for extracting lipstatin from a
fermentation broth wherein the concentrated fermentation broth is
diluted with the second extraction solvent. Preferably, the second
extraction solvent is selected from the group consisting of
methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, i-butanol,
tert-butanol, acetonitrile and acetic acid. More preferably, the
second extraction solvent is methanol.
[0087] Preferably, the step of concentrating the second extraction
solvent is carried out under reduced pressure and preferably at a
temperature of less than about 80.degree. C.
[0088] According to another alternative embodiment, the present
invention provides a process for extracting lipstatin from a
fermentation broth wherein the concentrated fermentation broth is
diluted with the third extraction solvent. Preferably, the third
extraction solvent is acetonitrile.
[0089] According to another alternative embodiment, the present
invention provides a process for extracting lipstatin from a
fermentation broth wherein the concentrated lipstatin fermentation
broth is washed with the fourth extraction solvent. Preferably, the
fourth extraction solvent is a hydrocarbon solvent. More
preferably, the fourth extraction solvent is selected from the
group consisting of pentane, hexane, cyclohexane and heptane.
[0090] According to another alternative embodiment, the present
invention provides the fourth extraction solvent can function both
as a washing solvent and an extraction solvent. While the fourth
extraction solvent serves to wash the lipstatin present in the
third extraction solvent, it may also serve to re-extract lipstatin
that is present in the fifth extraction solvent.
[0091] According to the present invention, the extracted lipstatin
at this stage attains a high degree of purity. In an alterative
alternative embodiment of the present invention, the extraction
process of lipstatin can be optionally repeated to improve the
purity.
[0092] According to yet another alternative embodiment, the present
invention provides a process for extracting lipstatin from a
fermentation broth wherein the lipstatin present in the third
extraction solvent is diluted with fifth extraction solvent.
Preferably, the fifth extraction solvent is a mixture of lower
alkyl alcohol and water. The fifth extraction solvent can be used
without water. Preferably, the fifth extraction solvent is a
mixture of methanol and water. More preferably, the volume to
volume ratio (v/v) of methanol to water is 70:15.
[0093] According to another alterative embodiment, the present
invention provides a process for extracting lipstatin from a
fermentation broth wherein the extracted lipstatin from the fifth
extraction solvent is further extracted using the fourth extraction
solvent.
[0094] According to another alternative embodiment, the present
invention optionally provides a process for extracting lipstatin
from a fermentation broth wherein the extracted lipstatin from the
fourth extraction solvent is further extracted in the fifth
extraction solvent.
[0095] According to another alterative embodiment, the present
invention optionally provides a process for extracting lipstatin
from a fermentation broth wherein the extracted lipstatin from the
fifth extraction solvent is concentrated.
[0096] According to another alternative embodiment, the present
invention provides a process for extracting lipstatin from a
fermentation broth wherein the concentrated lipstatin in the fifth
extraction solvent is diluted with methanol and the lipstatin in
the diluted methanol solvent extracted by subject the methanol
solvent to chromatography. Preferably, the chromatographic column
used is an anion-exchanger. More preferably, the anion-exchanger is
an anion-exchanger resin. Exemplary resin includes Amberlite.TM.
IRA. It is useful to remove the impurities (e.g., lineolic acid)
that are present in the fermentation broth.
[0097] Preferably, the dilution steps [i.e., additional steps of
(l) and (m) is carried out using a lower alkyl alcohol. More
preferably, the lower alkyl alcohol is methanol.
[0098] According to another alternative embodiment, the extraction
processes involving multiple extraction solvents as disclosed
herein can be generally used in other fermentation processes, in
additional to the lipstatin fermentation process as disclosed
herein. One of ordinary skill in the art would appreciate that some
conditions for extraction can be modified without affecting the
lipstatin obtained.
[0099] The present invention is described in further detail with
reference to the following examples. However, the scope of the
present invention is by no means restricted by these specific
examples.
[0100] Fermentation Process for Lipstatin
EXAMPLE 1
[0101] a) Preparation of a Seed Culture
[0102] A seed medium was prepared containing Soya bean flour 10.0
grams, glycerol 5.0 grams, Soya peptone 5.0 grams, Soya oil 10 mL,
Triton X-100 0.5 grams in water (1 litre). The pH of the seed
medium was adjusted to 6.5.+-.0.1 with a NaOH solution. An inoculum
medium (50 mL) was filled into a 500 mL Erlenmeyer flask and closed
with a cotton plug and sterilized. Sterilization was performed at
121.+-.2.degree. C., 100.+-.10 kPa for 25 minutes. The sterilized
inoculum medium was inoculated with a spore suspension of
Streptomyces toxytricini and incubated at 28.+-.2.degree. C. for
20-40 hours under aerobic conditions.
[0103] b) Main Fermentation Process
[0104] About 2-5 vol % of the above seed culture was used for the
inoculation of a 500 ml Erlenmeyer flask, which contained 50.+-.5
ml fermentation medium (F-1). The fermentation medium contained
Soya bean flour 30 grams, glycerol 7.0 grams, cotton seed meal 1.0
gram, polypropylene glycol 0.20 gram, Soya oil 80 ml, Triton X-100
1.0 gram as an emulsifier in 1 litre tap water. The pH of the
fermentation medium was adjusted before sterilization to 7.0.+-.0.1
with NaOH. Sterilization was at 121.+-.2.degree. C., 100.+-.10 kPa
for 25 minutes. Fermentation was carried out at 28.+-.2.degree. C.
for 6-7 days under aerobic conditions.
[0105] After fermentation for 144-168 hours, the concentration of
lipstatin was 1,700-1,800 mg/l.
[0106] Isolation of Lipstatin
[0107] The lipstatin (active ingredient) was isolated from the
fermentation broth using a standard purification method. 50 ml
fermentation broth was extracted with the organic solvents (150 ml
acetone and 100 ml hexane). After the separation of the organic
layer, the aqueous layer was extracted three times with 100 ml of a
1:1 mixture of acetone and hexane The combined organic extracts
were dried with sodium sulfate and concentrated to oil form. The
oil was dissolved in hexane (35 ml) and chromatographed using
silica gel column. The column was developed with hexane 40 ml and
300 ml hexane ethyl acetate mixture (20:1), 200 ml hex ane ethyl
acetate mixture (10:1) and finally 200 ml hex ane ethyl acetate
mixture (5:1). After evaporation of solvent, the yield was 95 mg
crude material containing 60 mg lipstatin (63% purity).
[0108] The viscosity of the fermentation broth was measured with a
RHEOLAB MC 1 (Paar Physica, Stuttgart, Germany) rotational, shear
stress and creep rheometer equipped with a Z3 DIN standard
measuring system. The thermostated (25.degree. C.) measuring cup
was filled with 10 ml of fermentation broth and the rheological
curve (by gradually increasing shear rate) and viscosity (at D=10
s-1 speed) were determined. The viscosity of the fermentation broth
was obtained in mPascal secundum and was determined to be 300-400
mPascal secundum.
EXAMPLE 2
[0109] The seed culture (80 ml) of Example 1 was used to inoculate
on a laboratory scale. The stirred fermentor with a vessel size of
7 litres contained 4.0 litres of the main fermentation medium
(F-2). Composition of the main fermentation medium included Soya
bean flour 120.0 grams, glycerol 28.0 grams, cotton seed meal 4.0
grams, polypropylene glycol 1.0 gram, Soya oil 320 ml, Triton X-100
6.0 grams as an emulsifier in 4 litres. The pH of the fermentation
medium was adjusted to 7.0.+-.0.1 with NaOH before sterilization.
Incubation was carried out at 28.+-.2.degree. C. for 6-7 days under
aerobic condition (1,500 rpm, 1 vvm).
[0110] The amount of lipstatin was 1,500-1,600 mg/l after an
incubation period of 144-168 hours. The viscosity of the
fermentation broth was 400-500 mPascal secundum as determined with
the instrument: Anton Paar Rheolab MCI MS Z3 DIN D=10 s-1.
EXAMPLE 3
[0111] About 2-5 vol. % of the seed culture of Example 1 was used
to inoculate a 500 ml Erlenmeyer flask which contained 50.+-.5 ml
of fermentation medium (F-2). The fermentation medium (F-2)
contained the following ingredients: Soya bean flour 30 grams,
glycerol 7.0 grams, cotton seed meal 1.0 gram, polypropylene glycol
0.20 gram, Soya oil 80 ml, Brij 35 S 3.0 grams as an emulsifier in
1 litre tap water. The pH of the fermentation medium was adjusted
to 7.0.+-.0.1 with NaOH. Sterilization was done at 121.+-.2.degree.
C., 100.+-.110 kPa, for 25 minutes. Fermentation was carried out at
28.+-.2.degree. C. for 6-7 days under aerobic conditions.
[0112] The amount of lipstatin was 1,300-1,400 mg/l after an
incubation period of 144-168 hours. The viscosity of the
fermentation broth was 600-700 mPascal secundum as determined with
the instrument: Anton Paar Rheolab MCI MS Z3 DIN D=10 s-1.
EXAMPLE 4
[0113] About 2-5 vol % of the seed culture of Example 1 was used to
inoculate a 500 ml Erlenmeyer flask, which contained 50.+-.5 ml of
fermentation medium (F-3). The fermentation medium (F-3) contained
the following ingredients: Soya bean flour 30 grams, glycerol 7.0
grams, cotton seed meal 1.0 gram, polypropylene glycol 0.20 gram,
Soya oil 80 ml, Soya lecithin 12 gram as an emulsifier in 1 litre
tap water. The pH of the fermentation medium was adjusted to
7.0.+-.0.1 with NaOH. Sterilization was done at 121.+-.2.degree.
C., 100.+-.10 kPa for 25 minutes. Fermentation was carried out at
28.+-.2.degree. C. for 6-7 days under aerobic conditions.
[0114] The amount of lipstatin was 1,200-1,300 mg/l after an
incubation period of 144-168 hours. The viscosity of the
fermentation broth was 1,600-1,700 mPascal secundum as determined
with the instrument: Anton Paar Rheolab MCI MS Z3 DIN D=10 s-1.
EXAMPLE 5
[0115] 80 ml of the seed culture of Example 1 was used to inoculate
a lab scale fermentor with a vessel size of 7 litres that contained
4.0 litres of fermentation medium F3. The fermentation medium
contained Soya bean flour 120.0 grams, glycerol 28.0 grams, cotton
seed meal 4.0 grams, polypropylene glycol 1.0 gram, Soya oil 320
ml, Soya lecithin 48 grams as emulsifier in 4 litres. The pH of the
fermentation medium was adjusted to 7.0.+-.0.1 with NaOH before
sterilization. Incubation was carried out under aerobic conditions
(1,000 rpm, 1 vvm) at 28.+-.2.degree. C. for 6-7 days.
[0116] The litre of lipstatin was 600-700 mg/l after an incubation
period of 144-168 hours. The viscosity of the fermentation broth
was 2,200-2,500 mPascal secundum as determined with the instrument:
Anton Paar Rheolab MCl MS Z3 DIN D=10 s-1.
[0117] The analysis of the samples was performed by HPLC, using RP
C 18 type, 5 micron column. The mobile phase was a mixture of
acetonitrile and 0.1% phosphoric acid in a v/v ratio of 8 to 2.
Detection was done by UV detector at 205 nm wavelength.
[0118] Extraction of Lipstatin from a Fermentation Broth
EXAMPLE 6
[0119] A fermentation broth (3.4 kg) containing lipstatin was
extracted with i-butyl acetate (step a) at a pH of about 2.0 to
about 10.5. The extraction achieved a yield of 94%.
[0120] The i-butyl acetate phase was further washed with water at a
pH of about 3.0 to about 8.0.
[0121] The washed i-butyl acetate phase was concentrated (step b)
in a vacuum at a maximum temperature of about 80.degree. C. The
concentrated i-butyl acetate phase (311.9 grams) contained the
lipstatin (1.305 gram) and was then extracted (step c) three times
with methanol. The methanol phases were combined. The combined
methanol phases contained 1,100 gram of lipstatin. The overall
yield of the steps was about 79%.
EXAMPLE 7
[0122] A methanol phase was produced according to Example 6.
[0123] The methanol phase (936 ml) was concentrated (step d) under
reduced pressure to an oily residue (20.39 grams). The oily residue
was diluted (step e) with acetonitrile (125 ml). The diluted
solution contained the lipstatin (1,100 gram).
[0124] The acetonitrile solution was washed (step f) three times
with hexane. The hexane phases were combined (step n) (300 ml) and
washed three times with acetonitrile (10 ml). The acetonitrile
phases were combined (step o). The combined acetonitrile phases
contained the lipstatin (952 mg) product to be separated (step g).
The combined solution was concentrated in vacuum at a maximum
temperature of about 60.degree. C. The mass of concentrate was 4.95
grams. The yield of this step was about 87%.
EXAMPLE 8
[0125] An acetonitrile concentrate was produced according to
Example 7.
[0126] The concentrate (4.95 grams) was diluted (step i) with a
methanol:water (70:15) mixture (85 ml) resulting in a diluted
solution containing lipstatin. The diluted solution containing
lipstatin (952 mg) was extracted (step j) five times with hexane
(85 ml). The hexane phases were combined and concentrated (step k)
in vacuum at a maximum temperature of about 60.degree. C. to a
volume of 85 ml.
[0127] The concentrated hexane solution was extracted (step q) five
times with methanol:water (70:15) mixture (85 ml). The combined
methanol:water phase was concentrated (step r) in vacuum at a
maximum temperature of about 80.degree. C. to an oily residue
(1.267 gram). The oily residue contained lipstatin (748 mg). The
yield of the step was about 86%.
EXAMPLE 9
[0128] An oily residue was produced according to Example 8.
[0129] The oily residue (603 mg) was diluted (step l) with methanol
(10 ml). The diluted solution contained lipstatin (356 mg) and was
passed through an anion-exchanger (step m) [15 ml]. The type of
anion-exchanger used was Amberlite.TM. IRA 67 resin in hydroxide
form. The resin was washed with methanol. Fractions containing
lipstatin were collected. Volume of the combined fractions was 20
ml. The combined fractions contained about 302 mg lipstatin and
about 142 mg other substances.
EXAMPLE 10
[0130] A lipstatin containing 302 mg active substance was produced
according to Example 9.
[0131] The lipstatin was hydrogenated in the presence of a catalyst
in a methanol solution. The hydrogenation of about 302 mg lipstatin
resulted in about 270 mg orlistat (tetrahydrolipstatin).
EXAMPLE 11
[0132] Lipstatin was extracted according to Example 6, but instead
of methanol, one of ethanol, 1-propanol:water mixture,
2-propanol:water mixture, 1-butanol:water mixture, i-butanol:water
mixture, tert-butanol:water mixture, acetonitrile, acetic acid was
used as the extraction solvent.
[0133] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the claims. Various
publications are cited herein, the disclosure of which are
incorporated by reference in their entireties.
* * * * *