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3,070,514
United States Patent 0 "ice
Patented Dec. 25, 1962
2
1
growth characteristics of strain C1. 2007, which is a wild
type, and C1. 7190, which is a mutant producing 6-de
3,070,514 ,
PROCESS FOR THE MANUFACTURE OF
I
methyitetracycline, on ‘various media incubated at
6-DEMETHYLTETRACYCLINE
27-293. C.
Abramo Virgilio and Carlo Hengeller, Naples, Italy, as
signors to Lepetit S.p.A., Milan, Italy
No Drawing. Filed May 4, 1961, Ser. No. 107,666
Claims priority, application Great Britain May 4, 1960
'
I
.
For the determination of colors the Os'twald tables were
used (Die kleine Farbmesstafel nach Ostwald, Ausgabe A, '
Wissenschattlicher Veriag, Gattingen, Frankfurt, Berlin).
8 Claims; (Cl. 195-80)
( l ) STARCH-AGAR“
This invention relates to a new fermentation process. 10
More particularly, the invention is concerned with a
Streptamyces prammoticua
process of preparations of the antibiotic 6-demethyltetra
cycline by fermentation.
It is known that while antibiotics in general have con
tinuously grown in importance in the ?eld of human 15
therapy in the last years, the class of tetracycline anti
biotics has got a preeminent position in the treatment of
infective disease, due to the very broad antibacterial spec
trum and to the comparatively low toxicity of its mem
20
bers.
6-demethy1tetracycline represents one of the most recent
steps in the studies concerning the antibiotics of the tetra
cycline class. It differs from tetracycline in lacking a
methyl group at position 6 of the tetracycline molecule.
R
OH
N(CH:)I
OH“
Strain C1. 2007
Growth _____________ _-
Poor ................ __
Aerial mycellurn ____ .. Very poor, white
becoming light
olive bu? (ie2)
Sporulation _______
-_
Very poor.
Very poor, white
becoming light
olive bull (ie2).
Slight _______________ -.
Diiiusible pigment _. Poor, lieht yellow
(ga2) to light olive
green (gel).
Poor.
Poor, light brown
(ge3).,
Reverse _____________ ._ Light olive buff (ie1).. Brown (gei).
Diastasic activity___..
Positive .............. _. Positive.
'
I Prepared in accordance with the directions given in “StreptomyceS
Conference, Stockholm, 1958."
25
(2) CZAPEK AGAR SOLUTIONb
OH
CONH:
Strain Cl. 7190
Streptomucea psammoticua
Strain C1. 2007
30
Strain C1. 7190
I
OH O
OH
Tetracycline: R=CHa
6 demethyltetracycline: R=H
Growth ............. _.
Very poor ........... .-
Aerial mycelium ____ .. Abs
t
Very poor.
Absent.
Sp‘rulati-m ......... ..
D0.
Di?usible pigment__..
Two ways have already been described for the produc 35
tion of 6-demethyltetracycline. The ?rst process consists
in fermenting certain mutant strains of Streptomyces
aureofaciens.
The species S. aureofaciens is known to produce, under
conventional fermentation conditions, the antibiotic chlor 40
tetracycline, while some carefully controlled modi?cations
-
Reverse ............. -.
D0.
Colorless.
*1 Prepared according to 'I‘. G. Pridham et al., “A Selection of Media
for Maintenance and Taxonomic Study of Streptomyeetes," Anti
biotics Annual, 1956-57, pp.‘ 947-953.
(3) YEAST EXTRACT, MALT EXTRACT, AGARh
Sireptomucea p-xa'm'moticua
of the culture media lead to the simultaneous production
of chlortetracycline and tetracycline.
A strict parallelism exists between the behavior of S.
aureojaciens and the above mentioned mutant strains.
These latter, when grown in a conventional culture medi
um, tend to produce the antibiotic substance o-demethyl
chlortetracycline, while under controlled conditions 6-de
Strain C1. 2007
Growth _____________ ..
Sporulntion _________ ..
methyltetracycline is simultaneously formed; in both cases,
Slight ............... __
Ditiusible pigmeut-_.- Poor. yellow (ga2) to
brown (ne3).
Brown (ie3).
.
‘I Prepared according to '1‘. G. Pridham et al., "A Selection of Media (or
Maintenance and Taxmo'nic Study oi Streptomycetcs," Antibiotics
Annual, 1956-57, DD. 947-953.
_
55
(4) OAT FLAKES AGAR ACCORDING
TO CARVAJALb
Streptomucea paammotlcua
cline, which must be highly puri?ed in order to isolate it
from the related antibiotics formed in the course of the 60
fermentation and then subjected to the hydrogenation.
The primary subject of this invention is the sole produc
tion of G-demethyltetracycline by fermentation in conven
(eel) to light olive
bu? (ie2)
Very poor.
Reserve ............. -. Dark brown (pg4)___._ Maroon brown (let).
and yield cutting procedure of separation from the related
Another known method for preparing 6-demethy1tetra
cycline consists in catalytically hydrogenating 6-demethyi
chlortetracycline, thus involving a preliminary fermenta
tion step for the production of 6-demethylchiortetracy
Good.
White to water green
water nreen (eel) to
light olive bu?‘ (ie2).
minor amounts of the antibiotics tetracycline and chlor
tetracycline are also formed. The sole production of
6-demethyltetracycline therefore implies a cumbersome
antibiotic substances.
Good_____________.--.
Aerial mycelium ____ .- Moderate. white to
Strain C1. 7190
Strain C1. 2007
Strain C1. 7190
Growth ............. ..
Abundant ........... ..
Abundant.
Aerial myceliuzn .... ..
White to water green
White to water green
tional nutrient media of some induced mutants of Strep
(eci) to green olive
(eel) to green olive
buil (ie2).
.
bu? (ie2).
tomyces psammoticus such as, for instance, a strain which 05 sp'trulfttion ......... -. Abundant
........... .. Abundant.
is indicated in Lepetit’s collection of culture as Cl. 7190.
Diii‘usible pigment__-_ Abundant, yellow
Brown (ng3).
(2:12) to mar-om
. .
The strain Cl. 7190 has been deposited with the American
.
green-yellow (ie2).
Type Culture Collection under the number 14125.
Reverse ............. __ Maroon green-yellow
Dark maroon (p14).
Although the mutant C1. 7190 in many characteristics
resembles the parent strain, however, a diiierentiation ap 70
b Prepared according to '1‘, G, Pridham et al., “A Selection 01’ Media tor
pears evident when they are parallelly grown on several
Maintenance and Taxonomic Study of Streptomyeetes," Antibiotics
media. We give in the following a comparison of the
Annual, 1956-57, pp. 947-953.
3,070,514
3
(s) OAT FLAKES TOMATO PASTE AGAR”
Streptomycn peammoticul
Streptomycu peammoticu:
Strain C1. 2007
Growth ............. ._
Moderate ........... _.
Aerial myceiium .... _. Poor. white to water
green (eel) to light
olive buff green (ie2)
Sporulatlon ......... .-
Poor ................ ..
Ditluslble pigment.... Moderate. yellow to
brown (203).
Strain Ci.
Strain C1.
2007
7100
Strain C1. 7190
Growth ................................. .- Very poor... Very poor.
Moderate.
Aerial myceiium ............... .._ ....... ._
Poor, white to water
Sporulation-_
Diilusible pigment
green (ecl) to light
olive bu? (lc2).
Poor.
Moderate, brown
10
Reverse _________________________________ ..
Calcium malate digestion ............... .-
Absent .... _.
Absent.
Colorless.-Negative..-_
Colorless.
Negative.
..__-do
-__-.do
Do.
Do.
‘
(ec3 .
Reverse ............. .. Brown (le5) ......... -- Light brown (ie'3) to
(10) GELATIN
dark brown (ie5).
Gelatin, Difco ________ ..__ _________ ..__--..-g..-
120
b Prepared according to T, G. Prldharn et al., "A Selection of Media for
3
Maintenance and Taxonomic Study 01 Streptomycetes," Antibiotics In-l 5 Meat, extract, Difco ____________________ .._g....
Peptone, Difco
8
5
Annual, 1956-57, pp. 947-953.
Dist. water, q.s. to _______________ -_....___ml_.. 1,000
Post sterilization, pH 6.7 to 6.8.
(6) GLYCEROL ASPARAGINE AGAR ‘
20
Strepiomycer pra'mmoticua
Strain C1. 2007
Poor growth. Vegetative my
Strain Cl. 2007
‘ Strain C1. 7190
Growth
Poor.
Poor.
Very poor, white ____ -. Absent.
Sporulatlon.
Absent
Do.
Di?'usibie pigment_--. Very poor, light
Very poor, pinkish
Aerial mycellum .... ..
yellow (la2
Reverse ............. .. Brown (103) ......... -.
brown (gc .
Light yellow (e03)
zviltlg
darker spots
p 4 .
- Prepared in accordance with the directions given in "Streptomyces
Conference, Stockholm, 1958."
25
strains was carried out on cultures in starch agar plates
35 after incubation of 20 days at 28° C. The morphological
characteristics were found to be identical and may be
summarized as follows:
Strain C1. 7100
40
Growth ............. .. Moderate to good _..-.
Good.
Aerial mycelium ____ -- Poor, white to water
Poor, white with
Sporulation ......... --
Very poor ........... ..
Poor.
Moderate brown (pill).
Reverse .............. _. Dark maroon ....... -_
Deep maroon (p15).
- Prepared in accordance with the directions given in "Streptomyoes
Conference, Stockholm, 1958."
(8) GLUCOSE ASPARAGINE MEAT
EXTRACT AGARh
Streptomycee peammolicua
Growth ............. .-
Moderate ........... ..
Aerial myeellum .... -- Poor, white with
Strain C1. 7190
Moderate.
Poor, white.
water green (ecl)
spots.
Sporuiation .... ..
Very poor ........... ..
Di?usible pigmen
R
Practically absent..__. Light brown (gel).
Brown (ge5).
Absent.
Sporophores: straight or slightly waved (Sectio: Rcctus
Flexibilis according to Pridham, Hesseltine ct al., Ap
plied Microbiology 6, 52-79 (1958)), sometimes with
open irregular bendings.
Spores: smooth, generally cylindrical (1.5-6 1: 1.0-1.3,u).
Vegetative mycelium: the breath of hyphae is about 0.51;.
spots from water
green (eel) to light
Diiluslbie pigment_-_. Poor (ue3) ____ ..
Strain 01.2007
pale brown. Aerial mycelium ab~
sent. Very poor ii ueiaciiomlimlted
to the zone imme iateiy below the
culture. Very poor light brown
soluble pigment.
The test for the production of melanoid pigment, car
ricd out according to Ettlinger et al., Archiv. fiir Mikro
biologic 31, 326 ( 1958), and the test for the production
30 of hydrogen sul?de, carried out according to Tresncr and
Danga, I. Bact. 76, 239 (1958), gave negative results for
both strains.
Strepio'mz/ces psammotz'cus
green (eel) to light
olive bu? (ie2).
Poor growth. Vegetative myoeiium
A microscopic morphological observation of the two
‘(7) GLYCEROL GLYCINE AGAR“
Strain Cl. 2007
ceiium hyaline. Aerial my
celium absent. Very poor
liquefaction, limited to the
zone immediately below the
culture.
Strain C1. 7190
45
One of the simplest procedure to carry out the process
of the invention is described in the following, although
any other conventional procedure may give comparable
results. The microorganism Sir. psammoticus Cl. 7190 is
grown in submerged culture under conditions practically
similar to ‘those which permit the production of tetracy
cline using Str. psammoticus, Cl. 2007. and which we de
scribed in “II Farmaco,” Sci. Ed. XV (3), 168, 1960.
The examples I to V are illustrative of the fermentation
process. At the end of the fermentation the broth is acidi
65 ?ed to a pH of about 1.5 to solubilize the antibiotic and
?ltered to free the clear solution from the mycelium. The
?ltrate is then made alkaline to pH about 8.5-9.0 and
extracted with a water immiscible lower aliphatic alcohol,
such as butanol. The organic extract is extracted in turn
60 with a dilute mineral acid, such as hydrochloric or sulfuric
acid, and the water extract is evaporated in vacuo at a
temperature not exceeding 25° C. to a small volume and
adjusted to pH about 5.0.
The yellowish precipitate which forms is collected,
bPrePared ncco'ding to T. G. Prldham at 111., "A Selection
of Med a tor Maintenance and Taxonomic Study of Strepto 65
washed and dried in vacuo. This product consists of
mycetes,” Antibiotics Annual, 1956-57, pp. 947-953.
(9) CALCIUM MALATE AGAR
Calcium malate
NH4C1
KQHPO‘
Agar
g
10.0
g
g-..
g__
0.5 70 by the descending technique using strips of paper What
man No. 1 and water saturated butanol as the solvent.
0-5
The development is carried out by allowing the strips to
18.0
Dist. water, q.s. to ___________ .._..-___--__ml_.. 1,000
Post sterilization, pH 6.4 to 6.6.
almost pure ?-demethyltetracycline.
For analytical purposes a sample of 1-2 drops may be
taken off from the ?ltered broth at the end of the fer
meniation and subjected to a chromatographic analysis
stand at 20° C. for 18-20 hours. After evaporation of
the solvent the strips are placed on plates containing nu
75 trient agar seeded with spores of B. cereus var. mycoides
3,070,514
5
6
ATCC 9634. The plates are incubated at 32° C. for
12-14 hours. A single zone of inhibition is always ap
ing G-demethyltetracycline, a 10 litres fermentor contain
ing 4 litres of the'following culture medium was inocu
parent with the Rf value 0.30, identical with the known
Rf value of G-demethyltetracycline. Under the described
conditions tetracycline hydrochloride has Rf 0.37, 6-de—
lated:
methylchlortetracycline hydrochloride has Rf 0.47 and
chlortetracycline hydrochloride has Rf 0.59.
g-..
g ‘_
MgSO4.7H2O
__________ __" _____________ __g__.
2804
KH2PO4
It will be appreciated that no provision is given in
Examples I to III for lowering the common chloro ion
concentration of the selected culture media.
-
Peanut meal
Cerelose
K2CO3
‘
9.0
0.1
g.__
0.8
racycline was always the sole antibiotic substance pro
duced.
Example I
9.0
Tap water, q.s. to _______________ ...; ____ __ml_.. 1,000
also been carried out in the‘presence of substantial
amounts of intentionally added chloro or brorno ions.
The result was in any case the same, and 6-demethyltet
1.0
g
g
However, 10 CaCOa _______________________________ --g.~...
as indicated in Examples IV and V, fermentations have
30
66
Post sterilization, pH 6.9 (sterilization 30 min
utes at 120° C.).
The
fermentation was carried out at 28° C. with stir
15
ring at 750 r.p.m. and an aeration of 0.75 v./v./min. The
duration of fermentation was 80-85 hours.
The antibiotic activity as determined on the harvest
A 500 ml. ?ask containing 100 ml. of the following
and expressed in 6-demethyltetracycline was 1080-y/ml.
culture medium:
The paper chromatography con?rmed that 6-demethyl
20
tetracycline was the only antibiotic present in the fermen
Peptone ______________________________ __g__ 10.0
tation broth.
Constantino meat extract ________________ __g__.
1.0
Example III
Dextrose
g__ 20.0
With 200 ml. of a subculture prepared as described in
K2HPO4
g
1.0
MgSO4.7H2O _______________ __'_ _______ __g__
0.2 25 Example I a 10 litres fermentor containing 4 litres of
Tap water, q.s. to _____________________ __ml_‘_ 1,000
the following culture medium was inoculated:
Before sterilization, pH 7.1 (sterilization 20 min
(NH4)2SO4
g-10
utes at 120° C.).
Cerelose
___g_..
55
was inoculated with a spore suspension of S. psammoticus
CaCO3
g__
8
C1. 7190 from a slant culture on Carvajal’s oak ?akes 30 KI-l2PO4
g-.. 0.15
agar. The culture was incubated for 36 hours at 28° C.
MgSO4.7H2O
on a reciprocating shaker operated at 100 cycles per
minute.
ZnSO4.7H2O
The whole content of the ?ask was used to inoculate a
10 litres glass prefermentor containing 4 litres of the fol
lowing culture medium:
Soybean meal
g
5.0
Corn steep liquor _____________________ __g__
15.0
Cerelose _____________________________ __g__
(NH4)aso4
, g“
35.0
6-0
Kl-I2PO4
MgSO4.7H2O
__
g__
_________________________ __g__.
0.2
0.2
__g__.
6.0
CaCO3 _
__
g..-
mg
FeSO4.7H2O _________________________ -_mg__
0.25
75
40
m<r
50
35 MnSO4.4H2O
Tap water, q.s. to _____________________ __ml__ 1,000
Post sterilization, pH 6.9 (sterilization 20 min
utes at 120° C.).
The fermentation was carried out at 28° C. with stir
ring at 750 r.p.m. and aeration of 0.75 v./v./min. The
duration of fermentation was 8085 hours. The anti
biotic activity in 6-demethyltetracycline was 260'y/ml.
The paper chromatography con?rmed that G-demethyl
tetracycline was the only antibiotic present in the fer
Tap water, q.s. to _____________________ __ml__ 1,000 45 mentation broth.
Before sterilization, pH 7.0 (sterilization 20 min
Example IV
utes at 120° C.).
To prove that the production of demethyltetracycline
This culture was inoculated at 28° C. with stirring at
from S. psammoticus, C1. 7190, is independent of the
750 rpm. and introducing sterile air at a rate of 0.75
concentration in chloro ion of the fermentation medium,
v./v./min. After 18-20 hours this subculture was ready 50 the following experiment was carried out.
for transfer. The mycelium showed short hyphae,
Under the same conditions as described in Example
slightly branched. Two hundred milliliters were used to
II, but with the addition to the fermentation medium of
inoculate a 10 litres fermentor containing 4 litres of fer
NaCl in a proportion of two grams/liter a produuction
mentation medium having the following composition:
of 950'y/ml. of 6-demethyltetracycline was obtained.
55
The chromatographic analysis on the harvest con
g.
?rmed that 6-demethyltetracycline was the only antibiotic
Corn steep liquor
..
30.0
(NH4)2SO4
Cerelose
9.0
____
MgSO4.7H2O
K2CO3
_
CaCO;;
Post sterilization, pH 6.8-6.9 (sterilization 20 min
utes at 120° C.).
present in the broth.
.
Example V
1.0 60
To
prove
that
the
production
of demethyltetracycline
0.8
from
St.
psammoticus,
C1.
7190,
is independent of the
9
__ 66.0
'
Y
concentration in. bromo ion of the medium the fol
lowing experiment was carried out: under the same con
ditions as described in Example II but with the addition
The fermentation was carried out at 28° C. with stirring 65 to the fermentation medium of NaBr in a proportion of
at 750 rpm. and aeration of 0.75 v./v./min.
The dura- 1
tion of fermentation was 80—85 hours.
The antibiotic activity as determined on the harvest and
2 g./litre a production of 3507/m1. of G-demethyltetra
cycline was obtained.
The chromatographic analysis on the harvest con?rmed
expressed in G-demethyltetracycline was 760'y/ml.
that 6-demethyltetracycline was the only antibiotic pres
6-demethyltetracycline was the only antibiotic present 70 ent in the broth.
in the fermentation broth.
Example VI
Example II
Extraction of 6-de-methyltetracycline from the fermen
tation broth. Ten litres of harvest broth containing
With 200 ml. of a subculture prepared in Example I
and using a strain of Str. psammoticus capable of produc 75 8l0-y/ml. of 6-demethyltetracycline were acidi?ed at pH
3,070,514
8
1.5 with concentrated HCl and ?ltered from the my
celium.
The mycelium was suspended twice in a water volume
corresponding to one-?fth of the harvest volume and
then ?ltered.
The washings were added to the ?ltered broth and the
myces psammoticus ATCC 14125, and its mutants and
variants in an aqueous medium having a pH between 4
and 8, containing a soluble carbon source, a source of
assimilable nitrogen and essential mineral salt at a tem
perature between 25° and 37° for a period of 36 to 120
hours and recovering G-demethyltetracycline from the
mixture (12.75 litres) was alkalized to pH 8.8 with 10%
medium.
‘
NaOH and then extracted with 2.5 litres of butanol.
4. A process for producing G-demethyltetracycline
1.5 litres of butanol having an activity of 30907/mi.
which comprises growing under aerobic submerged con
in 6-demethyltctraeycline were obtained.
10 ditions a culture of Streptomyces psammoticus, ATCC
The rich butanol was extracted four times at pH 1.5
14125, in an aqueous medium having a pH between 4
with 300 ml. portions of dilute sulfuric acid.
and 8 and containing a soluble carbon source, a source of
The aqueous extracts (1200 ml.) having a content of
assimilable nitrogen and essential mineral salts at a tem
31007/ml. of 6-demethyltetracycline were concentrated
perature of about 28° for a period of 70—90 hours and
in vacuo at 25° C. to a volume of 400 ml. and then 15 recovering 6~demethyltetracycline from the medium.
adjusted to pH 5 with 10% NaOH.
A yellow product precipitated and was ?ltered, washed,
5. A process for recovering é-demethyltetracycline
which comprises acidifying the fermentation medium of
and dried in vacuo at 50° C. A product (3.2 g.) was
a microorganism of the class consisting of Streptomyces
obtained assaying 9767/mg. as o-dernethyltetracyclinc
psammoticus, ATCC 14125, and its mutants and variants,
sulfate.
20 ?ltering to clear the solution from mycelium, adjusting
We claim:
the ?ltrate to alkaline pH, extracting with a water im
1. A process for producing 6-demethyltetracycline,
miscible organic solvent, extracting the organic phase
which comprises cultivating a microorganism of the class
With a dilute mineral acid, evaporating the water extract
consisting of Streptomyces psammoticus, ATCC 14125,
in vacuo at a temperature not exceeding 25° to a small
and its mutants and variants in an aqueous nutrient medi 25 volume, adjusting to pH about 5.0 and collecting the pre
um under aerobic submerged conditions until substantial
antibiotic activity is imparted to said mediumand re
cipitate so obtained.
6. A process as in claim 5 in which the organic extrac
covering the antibiotic activity from the medium.
tion solvent is butanol.
2. A process for producing é-demethyltetracycline, sub
7. A process as in claim 5 in which the fermentation
stantially free from 6-demethylchlorotetracycline, which 30 broth,
?ltered from the mycelium, is adjusted to pH
comprises cultivating a microorganism of the class consist
8.5-9.0.
ing of Streptomyces psammoticus, ATCC 14125, and its
8. A process as in claim 5, in which the mineral acid
mutants and variants in an aqueous nutrient medium
used to extract the organic phase is sulfuric acid.
under aerobic submerged conditions until substantial anti
biotic activity is imparted to said medium and recovering 35
References Cited in the ?le of this patent
the antibiotic activity from the medum.
3. A process for producing o-demethyltetracycline,
UNITED STATES PATENTS
which comprises growing under aerobic conditions a cul
2,878,289
McCormick et a1 _______ .... Mar. 17, 1959
ture of a microorganism of the class consisting of Strepto
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