Note: Descriptions are shown in the official language in which they were submitted.
~o50460
BACKGROUND TO THE INVENTION `
British Patent No. 1,363,075 disclosed that a useful
B- lactamase inhibitor could be obtained by the fermentation
of certain strains of Streptomyces olivaceus. Until our
present invention, it was believed that the material
disclosed in British Patent No. 1,363,075 was substantially
, pure. However, we have disclosed that this is not so and
- that a minor component of that material can be isolated and
' has potent antibacterial activity. This new material is
. ~ .,. . - .
designated MM 13902 and it is now believed to be responsible
for a part of any antibacterial activity present in the
material of British Patent No. 1,363,075 although it i~
responsible for only a very minor part of B-lactamase
inhibitory activity exhibited by that material. Naturally,
~ nothing in this specification should be construed as claiming
c~ mat~rial as dsiclosed in British Patent No. 1,363,075. Other
~-lactamase inhib;tors are known to be produced by strains of
Streptomyces, for example those disclosed in German Published
. ,. ~ ~ . .
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~ 50460
Patent Application No. 23400Q5, but such known materials
have not been demonstrated as having the potent antibacterial
activity of the kind possessed by MM 13902.
DESCRIPTION OF THE INVENTION
The present invention provides the antibacterial agent
designated herein MM 13902 and its salts.
The structural formula for MM 13902 is believed to be as
follows: CH3
HO350 ~ ; - NH - CO - CH3
O2H H
~,
MM 13902 is a solid carboxylic acid which in the form of a
substantially pure sodium salt has the following characteris-
tics:
i. It is highly soluble in wat:er, soluble in
methanol and substantially insoluble in chloro-
` form, diethylether and hydrocarbons.
.,
`j ~i. In a~ueous solution, it has a characteristic
ultraviolet spectrum with absorption maxima one
of which is at about 305 nm.
iii. When present at 0.4% w/w in a freshly prepared
KBr disc, it has a characteristic infra-red
spectrum which has absorption maxima at inter
'~ 3
,~ .
,
1~50460
alia about 3450, 2950, 1750, 1620~ 1510,
1400 and 1260 cm 1,
iv. It has a characteristic n.m.r. spectrum when
taken in D20 ~7hich spectrum possesses inter
alia (a) a pair
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~OS04~0
of low field doublets centred at approximately
2.85Z and 4.00Z with coupling constants of
approximately 14 Hz; (b) a doublet centred at
approximately 8.55 Zand (c) a sharp singlet at
approximately 8.00~.
v. It possesses antibacterial activity against various
species including inter alia, strains of StaPh
lococcus aureus, Bacillus subtilis, Escherichia
coli, Klebsiella aerogenes, Proteus mirabilis,
Salmonella typhi and Pseudomonas aeruginosa.
vi. When mixed with ampicillin it synergyses its
activity agains organisms including strains of
Escherichia coli, Klebsiella aerogenes,
Proteus mirabilis, Proteus morganii and
Staphy~ococcu~ aureus Russell.
The pure di-sodium salt of MM 13902 is a4 -lactamase
inhibitor and has an I50 (as hereinafter described) of
between 0.001 ~g/ml and 0.0001 ~g~ml against the ~-lactamase
of Escherichia coli B 11.
When run on cellulose in a thin layer chromatography
,` . system the pure di-sodium salt of MM 13902 has the
following approximate Rf values:-
ta~ n-butanol/isopropanol/water - 7:7:6 v/v;
Rf - 0.72
! (b) isopropanol/water - 7:3 v/v; Rf = 0.85
tc) n-butanol/ethanol/water - 7:7:6; Rf = 0.81
td) n-propanol/water - 4:1; Rf _ 0.75
I From another viewpoint, MM 13902 may be characterised
:.1 as a sulphur containing antibacterial agent, salts of which are
!
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1050460
produced during the cultivation of Streptomyces olivaceus
ATCC 31126 and in the form of an aqueous solution of
its di-sodium salt has UV absorption maxima at about 305 nm
and at about 225 nm substantially as shown in Fig. 1 herein. ;~
.
Alternatively, MM 13902 may be characterised as an anti-
bacterial agent which in the form of its substantially pure
di-sodium salt has an infra-red absorption spectrum sub-
stantially as shown in Fig. 2 when taken in a freshly
prepared 0.4% W/W KBr disc and which in the form of its
substantially pure di-sodium salt has n.m.r. spectrum
substantially as shown in Fig. 3 when taken in a freshly
prepared solutîon in D20.
. J~ .
The material MM 13902 tends to be unstable when in the
unsalted acid form. Aec~rdingly a preferred aspect of this
invention provides salts of MM 13902. Suitably the salts
of MM 13902 are di-basic salts. Most suitably these salts
are those formed with pharmace~ically acceptable ions such
as 8~dium, potassium, calcium, magnesium, aluminium,
conventional substituted ammonium ions and the like.
Particularly suitable salts are alkali metal salts such as
The sodium or potassium salts, for example 9 the di-sodium
or di-potassium salts.
Suitably the MM 13902 and its salts as provided by this
invention are at least 50~ pure, more suitably at least 70
pure and preferably 80% pure, for example 90-100~ pure.
. ~ .
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In a further aspect this provides a pharmaceutical composition
which comprises MM 13902 as hereinbefore described or a salt -~
thereof together with a pharmaceutically acceptable carrier.
Most suitably the compositions of this invention contain a
sodium or potassium salt of MM 13902, for example, the
di-sodium or di-potassium salt of MM 13902.
Such compositions may be in a form suitable for oral, topical
or parenteral use. For example, tablets, capsules, creams,
syrups, reconstituable powders and sterile forms suitable
for injection or infusion may be used. Such compositions
may contain conventional pharmaceu~i:cally acceptable
materials such as diluents, binders, colours, flavours,
preservatives, disintegrants and the like in accordance with
conventional pharmaceutical practice in a manner well known
¦ to those skilled in the formulation of antibiotics such as
penicillins and cephalosporins.
.. ,.j .
1 The MM 13902 or its salts may be present in the composition
¦ of the invention as sole therapeu~i-c agent or it may be
present together with a ~-lactam antibiotic. Suitable
, 20 ~-lactam antibiotics include those known to be susceptible
lactamases and also having some intrinsic resistance to
~ lactamases. Such ~-lactam antibiotics include ampicillin,
;~ amoxycillin~ benzylpenicillin, phenoxymethylpenicillin,
l~ propicillin, cephaloridine, cefoxitin, cephalothin, cephalexin,
j carbenicillin, ticarcillin and in vivo hydrolysable esters of
- 6 -
'1,` `: ~.
1 . .
lOS0~60
such compounds such as the phenyl, tolyl or indanyl esters of
carbenicillin or ticarcillin or the acetoxymethyl, pivaloyl-
oxymethyl or phthalidyl esters of ampicillin, benzylpenicillin,
amoxycillin, cephaloridine, cephalogylcin and the like.
The ratio of MM 13902 or salt thereof to ~ lactam antibiotic
is normally between 10:1 and 1:10, for example, between 3:1
and 1:3.
The total quantity of antibacterial agents present in any
unit dosage form will normally be between 50 and 1500 mgs
10 and will usually be between 100 and 1000 mgs.
Preferred unit dosage compositions according to this invention
may be administered one or more times a day, for example,
2 to 4 times a day, in the treatment of diseases of the
urinary tract, respiratory tract, soft tissues and the like.
Thus the compositions may be used in the treatment of such
diseases as bronchitis, gonorrhea, otitus media, mastitis
, and the like.
. ' .
In one of its aspects, this invention provides a process for
the preparation of MM 13902 or a salt thereof which comprises
20 chromatographically separating a solution of MM 4550 (Complex)
as hereinafter described into fractions consisting essentially
of a solution of MM 13902 or a salt thereof and other
fractions and isolating the MM 13902 or a salt thereof from
solution.
~ 7
.. . . . . . . ..
~050~60
By those fractions "consisting essentially of a solution
of MM 13902 or a slat thereof" is meant that either the only
antibiotic material present in that solution is MM 13gO2 or
a salt thereof or that if any other antibiotic material i5
present then it is there to a lesser extent than the MM 13902
or salt thereof. More sultably MM 13902 or a salt thereof
represents 70%, most suitably 80% and preferably 90-100%,
of the antibiotic material present in the fraction. We
have ~ound that an acceptable method of determining which
fractions consist essentially of solution of MM 13902
or a 8alt thereof is to moniter the uv absorption spectrum
of each sample. Thoseefractions showing a uv spectrum
similar to that of Fig. 1 normally contain MM 13902 or a
salt thereof substantially free from other antibiotic material
such as MM 4550 as herein~fter described. In general those
fractions showing a distinct uv absorption maximum at about
305 nm are of the desired purity.
.
Normally the preceeding process is used for the isolation of
, MM 13902 as a dibasic salt. If a monobasic salt of MM 13902
~ 20 or the free acid MM 13902 per se is required, they may be
-~ prepared by the acidification of a dibasic salt of MM 13902
¦ because in general the monobasic salts of MM 13902 and the
free acid M~ 13902 are not sufficiently stable to isolate
from mixtures such as MM 4550 (Complex) as hereinafter
, described. These monobasic salts and free acid are not
i readily obtainable owing to their low stability.
,` -:
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. ..
lOS046V
A~ previously stated, we believe that the chromato-
graphic isolation of MM 13902 is best carried out
using a ,alt of MM 13902 such as the di-sodium salt.
Salts of'MM 13902 are more soluble in aqueous solvent
systems than in highly lipophilic solvents and con-
sequently it is preferred to use aqueous solvent systems
in the chromatographic purifications used in this
invention.
In our hands aqueous solutions o~ electrolytes
buffered to approximately neutrality have proved
suitable for use in conjunction with polar support
~aterials such as basic ion-exchange resins. Thus
an aqueous solu-tion of sodium chloride buffered to
about pH 7 with a conventional buffer such as a
phosphate buffer may be used in conjunction with support
materials which may contain tertiary amino gro~lps or
~uaternary ammonium groups. We have found that basic
ion-exchange celluloses and basic ion-exchange cross
linked dextrans are suitable support materials and that
' 20 QAE Sephadex A25 in particular is a highly suitable
support material especially when the solvent system
comprises C.7m sodium chloride in pH7 phosphate buffer.
~ ,Sephadex' is a Registered Trade Mark).
Alternatively, in our hands solvent syste~s comprising
mixtures of water and water miscible organic solvents
~- such as a lower alkanol have proved suitable for vse
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~ S0460
in conjunction with inert support materials such as
silica gel or cellulose. A particularly suitable
solvent system for use with cellulose is a mixture of
water an~ isopropanol, especially a 7:3~isopropanol :
water mixture.
However, the product of the above procedure using ion-exchange
resins frequently contai~s a very high proportion of sodium chloride
- so that it is beneficial to de-salt the pooled solutions.
De-salting may b~ effected by passing the solution
down a column containing a lipophilic material onto which
the I~M 13902 is adsorbed bu-t w~lich does not adsorb the
sodiwn chloride. Suitabie column materials include
polystyrenes such as Amberlite XAD4. Alternatively gel ~iltration
may be utilised using filtration agents such as cross linked
: 15 dextrons such as Sephadex G 25 and polyacrylamide gels such as
Biogel P2 ('Amberlite', 'Sephadex' and 'Biogell are
Trade Marks). The an~ikiotic tnay be eluted from the
column using ~ater, aqueous methanol or the l~ke.
:
When the desired solu~ions are ob-tained by the above process
~0 ~e di-basic salt of MM 13902 may ~e obtained in solid form
by the removal of the solvent under m ld conditions. We have
found that an acceptable method of obtaining such solid material
iS to evaporate under reduced pressure and freeze-dry the pooled fractions
containing MM 13902.
If desired the preceeding process may be carried out in t~ro
or more steps. For example, a solution of MM 4550 (Complex)
-- 10 --
.. - . . . . . .
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~0504~iO
may be separated into fractions comprising MM 13902 or salt .
thereof contaminated with up to its own weight of other anti-
bacterial agents, the fractions may be freeze dried to yield
a ma~erial containing, for example, about 50-60t of MM 13902
or a ~alt thereof and this material may bhen be re-
chromatographed to yield a material which contains, for
example, 90-100% of MM 13902 or a salt thereof.
r,
For the purpose of this specification the term "MM 4550
(Complex)" is used to describe the material originally
designated as MM 4550 in ~ritish Patent No. 1,363,075.
.l MM 4550 (Complex) as produced on repetition of the Examples
of British Patent No. 1,363,075 is an impure material which
, contains in various proportions salts of MM 4550 (as
described hereinafter) and salts of MM 13902 and considerable
. quantities of other materials. MM 4550 (Complex) does not
: have the characteristic ultra-violet spectrum of MM 13902.
The I50(as described hereinafter) of MM 4550 (Complex) :
produced by repetition of the Examples of British Patent No.
1,3631075 is not normally below 0.0004 ug/ml. The ratio
of salts of MM 4550 and salts of MM 13902 present in MM 4550
~ (Complex) i8 believed to be highly variable and is thought
91 to d~p*n~ on such factors as the strain of organism ~sed :
I and/or the exact isolation techniques used but it has been
i generally found that the complex contains more MM 4550 then
- MM 13902. The preparation of MM 4550 (Complex) is described ~`
hereinafter in the section relating to "Descriptions".
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~0~0~60
For the purpose of this specification the term "MM 4550" i5
used to describe a ~ubstantially pure compound which is a
potent B-lactamase inhibitor and which also possesses a
certain degree of antibacterial activity. The properties
of MM 4550 are described hereinafteri~ the section relating
to "Descrip$ions".
In an alternative view, this invention provides a process
for the preparation of MM 13902 and its salts which process
comprises the cultivation of a MM 13902 producing strain of
Streptomyces and thereafter recovering the MM 13902 or salt
thereof from the culture.
For this Process we have found that MM 13902 producing
strains are strains of Streptomyces olivaceus and related
organisms as described hereinafter.
Most suitably the organism used is a strain of Streptomyces
olivaceus suoh as ATCC 21379, 21380, 21381, 21382~ 31126 or
mutants thereof.
A particularly preferred organism for use in this process is
Streptomyce~ olivaceus ATCC 31126.
When~r~ herein, the term "cultivation" means the deliberate
aerobic growth of an organism in the presence of assimilable
sources of carbon, nitrogen, sulphur and mineral sa lts.
Such aerobic growth may take place in a solid or semi-solid
(
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iO50460
nutritive medium, or in a liquid medium in which the nutrients
are dissolved or suspended. The cultivation may take place
on an aerobic surface or by submerged culture. The
nutritive medium may be composed of complex nutrients or may
be chemically defined. In our hands we have found media
containing complex nutrients such as yeast extract, soya bean
flour and the like to be particularly suitable. We have also
; found that the addition of cobalt ions, sulphate ions and
calcium carbonate to be beneficial.
We have found that cultivation at a temperature of 28 ~2C
gives acceptable yields of antibiotic and that a good time
to harvest the broth is 2-3 days after the initiation of
fermentation.
.~ :
When used herein the term "mutant" includes any mutant strain
whioh arises spontaneously or through the effect of an
external agent whether that agent is applied deliberately or
~, otherwise. Suitable methods of producing mutant ~trains
include those outlined by H.I. Adler in Techniques for the
Development of Micro-organisms in "Radiation and Radioisotope~
for Industrial Micro-organisms", Proceedings of a Symposium,
~ Vienna, 1972, page 241, International Atomic Energy Authority
i and these include:
i. Ionising radiation (such as X- and ~- rays), uv light,
~' uv light plus a photosensitizing agent (such as
-! 8-methoxypsoralen~ nitrous acid, hydroxylamine,
~ pyrimidine base analogues (such as 5-bromouracil),
.
- 13 -
.,
:. ~
~ os~o
acridines, alkylating agents (such as mustard gas,
ethyl-methane sulphonate), hydrogen peroxide, phenols,
formaldehyde, heat, and
ii. Genetic techniques such as recombination, transformation,
transduction, lysogenisation, lysogenic conversion and
selective techniques for spontaneous mu-tants.
We have found that use ofa mutation promoting agent can lead
to the production of organisms which have the ability to
produce enhanced qunatities of the desired antibiotics.
For example, irradiation of cultures of Streptomyces olivaceus
ATCC 21379 followed by isolation of the resulting strain which
appeared to produce the largest zone of activity on the KAG
assay as hereinafter described lead to the isolation of
Streptom~ces olivaceus ATCC 31126 which is a preferred
strain for use in this invention. ATCC 31126 has also been
depo~ited in the Netherlands as CBS 155.75.
In general, all isolation and purification procedures used
in obtaining the desired anti~iotic should take place at
non-elevated temperatures, for example, below 20C and more
~uitably not above 12C.
~he desired product is normally obtained predominantely from
the culture filtrate so that the preferred initial step in
the isolation process is the removal of solid material from
the fermentation, for example by filtration.
An impure preparation of MM 13902 or a slat thereof may be
.
;
1050460
obtained from the clariied culture filtrate by absorbing the
MM 13902 or a salt thereof onto an active material such as
active carbon or the like and thereafter eluting the desired
substance from the active material using a solvent such as
aqueous acetone. Normally this process is carried out on a
di-basic salt of MM 13902.
Alternatively an impure preparation of MM 1390~ or a salt thereof
may be obtained from the culture filtrate by extraction using
a lipophilic ammonium salt and a water immiscible solvent.
This is frequently more effective than the process described
above. (The NM 13902 may be obtained as the substituted ammonium
salt by evaporation of the organic solvent under reduced pressure.)
Preferably the initial solution of the MM 13902 substituted
ammonium salt is then back extracted into an aqueous phase by using
'~ a solution of an alkali metal iodide such as sodium iodide.
T~is last process variant generally leads to preparation of an
aqueous solution of an impure di-basic salt of MM 13902.
The impure forms of MM 13902 or its salts as described above
are normally subjected to the chromatographic procedures
hereinbefore described in order to produce material of
, acceptable purity.
The following Descriptions elucidate general information
useful in the isolation of antibacterial compounds. The
¦ following Examples are illustrative of aspects of the
invention.
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Descr iption 1
I50 Determination
The I50 value is the amount of material required to give
50% inhibition of hydrolysis of ampicillin by the ~-lactamase
enzyme of Escherichia coli B11, an orqanism containing an R
factor controlled ~-lactamase. This ~-lactamase is classified
as a type IIIa enzyme according to the classification of Richmond
and Sykes ~Adv. in Microbiol. Physiol. 9 31 (1973)]
The rate of ~-lactamase hydrolysis of ampicillin to its
penicilloic acid can be followed by a starch-iodometric assay
in which one measures the rate of formation of penicilloic acid
by following the decolorization of a starch-iodine complex.
This method and the preparation of the ~-lactamase are
described in a paper by Cole M., Elson S., and Fullbrook P.D.
(Biochemical Journal 1972, 127, 295-308). A slight
modification of the above method was used in that the sample of
inhibitor was preincubated with the enzyme for 15 minutes at
37C prior to adding the substrate ampicillin. The procedure
was as follows:-
~ :,
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- 16 - ~
.
~050460
Reagents - `
suffer: 0.05M pH7 potassium phosphate buf~er
Starch/iodine Prepared as described by Novick,
solution: siochemical J. (1962) 83, 236
Substrate: Ampicillin 40 ~g/ml in buffer
-lactamase Enzyme: Prepared from E.coli B11 as described by
Cole et al, siochem. J (1972) 127,295
The dilution of the enzyme preparation in
buffer was such as to give a fall of about
0.3 optical density units per 100 secs. in
the uninhibited reaction. Other ~-lacta-
mase producing strains of E.coli may be
used, in particular those carrying an R
factor for example R TEM.
Conditions -
The reactions were carried out in 1 cm cuvettes at 37C in an SP 800
Pye Unicam spectrophotometer. This instrument can carry four sample
-, cuvettes and their corresponding blanks. The first cuvette was
, 20 used for the control reaction and contained no inhibitor. The 2nd,
, 3rd and 4th cuvettes were used for various dilutions of the inhibi~
, tor.
Thus:
Sample Blank
Reagents Cuvette Cuvette
Starch/iodine reagent 1.0 ml 1.0 ml
E.coli ~-lactamase 0.1 ml
Buffer 0.3 ml 0.4 ml
Inhibitor of buffer 0.1 ml 0.1 ml
30 Substrate (added after incubation1.0 ml 1.0 ml
of above mixtures for 15 mins.
at 37C)
~i~ The reactions were followed by recording optical density change at
1~ 590 nm. and measuring the velocity of the reaction as optical
. . .
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1050460
densîty change per 100 secs. during the 3 - 6 minute time interval.
The inhibitor sample was diluted until a dilution was reached which
gave 50% of the rate of reaction seen in the no-inhibitor control.
. . :
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~OSQ4~;0
Description 2
The KAG As s ay
me KAG assay is a method for determining the presence of a
~-lactamase inhibitor in fermentation broths or during stages in
the isolation. Molten nutrient agar at 45C is seeded with a
suitable ~-lactamase producing strain of Klebsiella aerogenes and
then mixed with a sufficient quantity of a sterile solution of
penicillin G to give a concentration of 6 ~g/ml of penicillin G in
the agar. The agar is then poured into a petri dish and after solid-
ification equally spaced cylindrical wells are made in the layer of
agar by using a sterile metal cutter. The solutions to be tested
are introduced into the wells. The dish is then incubated at a
constant temperature between 27C and 37C. During the period of
incubation, any inhibitor in the test solution diffuses out from
the well into the agar and there inhibits the action of ~-lactamase
produced by the Klebsiella cells. The penicillin G is thus protected
from destruction by ~-lactamase and is present in sufficient concentration
to prevent the growth of the Klebsiella. In those parts of the agar
to which the inhibitor has not diffused in sufficient concentration,
the penicillin G is destroyed by the ~-lactamase, allowing dense growth
of the Klebsiella to develop. Clear circular zones of inhibition
of Klebsiella growth are thus formed around the wells containing
inhibitor the size of each zone depending on the concentration
of inhibitor in the solution under test. The potency (arbitrary
units/ml) of test solutions is obtained by reference to a standard
line of log.conc. inhibitor against zone diameter. This assay
system may also be used for bioautography.
`', ' '
.
~' .
.,
- 18 -
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~050460
n6~scription 3
Preearation of MM 4550 (Complex) comparable to that disclosed in
British Patent No. 1,363,075
Streptomyces olivaceus ATCC 2137~, was grown for 7 days at 28C
on a solid agar slant in a Roux bottle. The agar medium had
the following composition:-
Constituent Amount (g/l)
Yeast Extract 10.0
Glucose Monohydrate 10.0
Agar 15.0
Tap Water to1 1
(The "Yeast extract" was "Yeatex"~; as supplied by Bovril FoodIngredients, P.O. Box 18, Wellington Road, Burton-on-Trent, U.K.,
and the "Agar" was supplied by Oxoid Limited, Southwark Bridge Road,
London, S.E.l., ~.K.)
The medium was adjusted to pH 6.8 before sterilisation. 50 ml.
of sterile deionised water containing 0.02% Tween 80 (Registered
Trade Nark: Tween 80 is a polyoxyethylene sorbitan mono-oleate)
was added to a Roux bottle culture and the spores suspended by
shaking. This spore suspension was then added as inoculum to 75 1
of sterilised seed stage medium in a 100 1 stainless steel
i fermenter. The composition of the seed stage medium was as
follows:-
*Trade Mark
.
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105~)460
Consti-tuent Am_unt (g/l)
Soya-bean Flour lO.0
Glucose Monohydrate 20.0
Tap Water to 1 1
(The "Soya-bean Flour" was Arkasoy* 50 as supplied by
the British Arkady Co.Ltd., Old Trafford, Manchester).
To control foaming 50 ml. of 10% v/v Pluronic L81 (Registered
Trade Mark) in soya-bean oil was added to the fermentation medium
before sterilisation. (Pluronic L81 was supplied by Jacobsen van
den Berg U.K. Ltd., 231 The Vale, London, W.3.~ U.K.~ and is a
block polymer of ethylene oxide and propylene oxide).
The medium was steam sterilised in the fermenter for 20 mins at
120C. The seed stage culture was stirred at 340 r.p.m. with a
8.5 inch diameter vaned disc agitator and supplied with 150 l/min
sterile air through an open ended sparger. The culture vessel was
fitted with baffles. The temperature was controlled at 28C and
after incubation under these conditions for 45 hours, 7.5 l of this
seed culture was added as inoculum to 150 l sterile fermentation
medium in a 300 l stainless steel fermenter. The fermentation
medium had the following compositions:-
Constituent Amount (g/l)
Soya-bean Flour (Arkasoy lO.0
S O )
Glucose Monohydrate20.0
Chalk ~Precipitated Calcium
Carbonate) 0.2
' Cobalt Chloride (CoCl2,6H20) 0.001
Tap Water to l l
*Trade Mark
- 20 -
(
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1050460
300 mi. of 10% Pluronic L81 in soya-bean oil was added to prevent
foaming. The fermentation was harvested after 48 hours and clari-
fied by centrifugation. The clari~ied brew gave 50% inhibition in
the enzyme assay at a dilution of 1 in 100,000. 100 1 of the clari-
fied brew was stirred with 12 kg. wet weight of Whatman DE32
(Registered Trade Mark) ion exchange cellulose in the acetate form,
(as supplied by H. Reeve Angel & Co., 14 New Bridge Street, London
E.~.4, U.K; the material is a microerystaline cellulose substituted
by diethylaminoethyl groups).
The slurry was filtered and the MN 4550 (Complex) was eluted from
the cellulose with 12 1 of 0.5M potassium sulphate. The extract
was concentrated to 6 1 in a climbing film evaporator under vacuum
and below 30C. Much of the potassium sulphate was precipitated
by the addition of 12 1 of acetone. The solution was filtered and
concentrated to 200 ml. by evaporation under vacuum below 30C.
The concentrate was loaded onto a 76 mm x 2 m column of Amberlite
XAD-4 resin (Registered Trade Mark) (as manufactured by Rhom &
Hass Co., Philadelphia, U.S.A.; the material is a non-ionic
polystyrene resin), eluted with deionised water and the elutate
was collected in 140 ml. fractions. Active fractions, as detected
by the KAG assay were bulked (2.2 1) and concentrated to 275 ml.
by ultrafiltration using an Amicon UM-05 membrane (150 mm diameter)
(Registered Trade Mark) (as supplied by Amicon Ltd., 57 Queen's
Road, High Wycombe) under nitrogen pressure of 60 p.s.i. The
concentrate was freeze dried to yield 2.2g. of brown powder
(I50 0.004~ug/ml.).
: ~ .
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. .. , ... . : . . . .: .
~o5Q460
1 g. of the powder was dissolved in 1 l of 0.2M sodium sulphate and
mixed with l l of 2~ w/v tetra-n-butylammonium hydrogen sulphate
(as supplied by AB Astra, 5Odertalje, Sweden) in dichloro~ethane.
The dichloromethane phase was separated by gravity, cooled to -70~C,
filtered to remove ice and concentrated by evaporation to 20 ml.
400 ml. of 40-60C petroleum spirit was added to the concentrate
and the precipitate collected by centrifugation. The precipitate
was redissolved in dichloromethane (tO ml.) and extracted with
water (10 ml.) containing barium iodide (80 mg.) and barium
carbonate (70 mg.). The phases were separated, the aqueous phase
filtered and adjusted to pH 6.5. The solution was freeze dried to
yield a yellow powder. The solid was washed with acetone to
dissolve out excess barium iodide and the pale yellow solid recovered
by centrifugation and dried in vacuo. The yield was 13 mg.
15 (I50 0-0004 ~g/ml-)-
,
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- 22 -
~050460
Descrlption 4
Demonstration of Carbon Adsorption of Culture Filtrate useful
in obtaining material as described in British Patent No. 1,361,075
Culture filtrate obtained as in Description 3 gave a zone diameter
of 17 mm in a hole-in-plate agar diffusion antibacterial assay
against Klebsiella ~ , a æone diameter of 38mm in the
KAG assay and I50 at a dilution of 1 in 100,000. The clarified
culture filtrate (170 1) at 5C was percolated by upward flow
through a 9" diameter column packed to a height of 16" with active
charcoal (Farnell*B0, as supplied by Dearborn Chemicals Ltd.,
Dilton, Widnes, Lancs., 60-80 mesh pre-washed with N HCl and
buffered at pH 6 with phosphate) at a flow rate of 800-1000 ml/min.
Brew was washed from the carbon with deionised water (10 1) and
the column eluted with 20% acetone at 20 C. The active fractions
amounting to 10 1 were concentrated by evaporation in vacuo, below
30C to 6 1 and freeze dried to yield 282 g. of crude MM 4550
(Complex) preparation giving an I50 of 0.05/ug/ml. The recovery
of enzyme inhibitory activity was 22~.
.
An alternative activated charcoal which gives similar results is
Darcogranular car~on (as supplied by Honeywill-Atlas Ltd., Mill
Lane, Carshalton, Surrey).
,,.
*Trade Mark
- 23 -
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Description 5
Demonstration of Ion Exchange Resin Chromatography of Culture
Filtrate useful in obtaining material as described in British
Patent No.1~363,075
A 1 cm. internal diameter column was packed to a height of 6 cm.
(bed volume 4.7 ml.) with Dowex 21K ion-exchange resin (20-50 U.S.)
mesh, chloride form)(Dowex~:21K was supplied by B.D.H. Chemicals Ltd.,
Poole, Dorset, ~.K. and is a polystyrene-divinylbenzene resin
containing basic groups). The bed of resin was then washed with
approximately 4 x 4.7 ml. of 5% aqueous methanol, followed by
1 x 4.7 ml. of distilled water. Two litres of culture filtrate was
obtained essentially as described in Description 3 and applied to
the column. The resin was then washed with 50% aqueous methanol to
remove impurities.
- The MM 4550 (Complex) was eluted from the resin with 5% NaCl in 50%
aqueous methanol. Table 1 below shows the results obtained in terms
of units of activity. The MM 4550 (Complex) content of culture
filtrate was arbitrarily set at 8 units/ml. The eluted fractions
from the column were assayed using an antibacterial diffusion method
against Klebsiella aerogenes and the activity units were calculated
by reference to a standard line prepared by plotting ~one diameter
~ ,~
against units/ml. for various dilutions of culture filtrate (i.e.
the culture filtrate was used as a standard).
It can be seen that the column was an effective way of concentrating
- the MM 4550 (Complex). Fractions 2 - 5 inclusive contained 60% of
the activity in a total volume of only 37 ml. The total dry weight
- 24 -
,. . . .
;.. . - ~ . , : . :
,. : . .
1050460
of these fractions was about 210 mg. whereas 35 g. of solids had
been added to the column.
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1050460
T A B L E
RESULTS OF CHROMATOGRAPHY ON DOWEX 21K
.: :
Sample pHVolume Units/mlTotal Units
.~ _
Culture filtrate 6.5 1970 15760
Percolate 16.9550 C1 ~100
Percolate 27.0525 C1 ~100
Percolate 37.0595 C1 ~100
Percolate 47.0300 L~ C100
Eluate 1 7.8 7.0 R4 588
Eluate 2 7.8 7.0 328 2296
Eluate 3 7.7 8.5 440 3740
Eluate 4 7.7 10.0 320 2200
Eluate 5 7.6 11.5 160 1840
Eluaté 6 7.5 11.0 80 880
Eluate 7 7.6 14.2 66 937
Eluate 8 7.6 20.0 33 660
Total:13141
__ _ _
. ~
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Description 6
Demonstration of Ion-Pair Extraction of Culture Filtrate useful
in obtaining material as described in British Patent No. 1,363,075
Sodium sulphate (248 g.) was added to clarified culture filtrate
(10 l) prepared as described in Description 3 and the solution
extracted with 2~ tetra n-butyl-ammonium hydrogen sulphate in
dichloromethane (10 l) by stirring for 30 minutes. The phases
were separated and the dichloromethane phase cooled to 2C. A
small quantity of suspended water was removed by filtration through
Whatman No. 1 PS paper. The dichloromethane solution was con-
centrated to 20 ml. by evaporation ln vacuo below 30C. Theaddition of 400 ml. of petroleum ether (40 - 60) precipated a
gum which contained the MM 4550 (Complex).
The gum was collected by centrifugation, redissolved in 50 ml. of
dichloromethane and extracted with 50 ml. of water containing 1 9.
barium iodide and 1 g. barium carbonate by shaking for 2 minutes.
The solids present were filtered off and the two phases separated.
The aqueous phase containing the MM 4550 (Complex) as the barium
~` salt was adjusted to pH 6.5 and freeze dried to yield 317 mg. of
a crude preparation of MM 4550 (Complex) with an I50 f 0.001 ~g/ml.
... .
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- 26 -
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105~0
Description 7
Preparation of Partially Purified Antibiotic Complex containing
MM 4550 and MM 13902 using Tetra n-butyl Ammonium Hydrogen Sulphate
A crude preparation of MM 4550 (Complex) prepared as in Description 4
was redissolved in water (12.5 g. in 125 ml.) and extracted with
125 ml. of 10~ w/v tetra n-butyl ammonium hydrogen sulphate in
dichloromethane. The two phases were separated and the dichloro-
methane back extracted with 100 ml. water at 2C containing 190 mg.
sodium iodide by stirring slowly and adjusting the aqueous phase
to p8 6.4 with 2% NaHCO3. The solution was freeze dried and the
dry solid washed with acetone to yield 88 mg. of a preparation
mixed sodium salts of MM 4550 and MM 13902 having an I50 f
0.0002 ~9 against Escherichia coli ~-lactamase. The recover
of antibiotics was 70~.
The antibacterial activity of mixtures of ampicillin and material
prepared as described in Description 7 was determined by the serial
dilution method in nutrient agar. The minimum inhibitory concentrations
which weee obtained are ~iven in T~ble ~
'
- 27 -
..
1~504~0
T A_B L E 2
SYNERGISM BETWEEN AMPICILLIN AND MATERIAL OF DESCRIPTION 7
.... ____ . :- .'
Ampicillin + MM 4550 (Complex) at
Ampicillin _ _
O r g a n i s mAlone _ _
0.1 ~g/ml 1 ~g/ml10 ~g/ml
. . . _ . _
E. coli B11 >500 ~ 500 500 50
E. coli JT417 250 250 250 50
E. coli JT39 > 500 >500 500 25 *
Shigelia sonnei S239 125 125 125 25 *
Klebsiella aerogenes A 125 25 5 * ~ 0.1 *
_
Klebsiella aerogenes -
IP282 50 50 12.5 0.5 *
Proteus mirabilis 889 ~ 500 >500 50 0.5
Proteus mirabilis 247 >500 125 5.0 0.5
_
` Proteus morganii F50 50 25 0.5
Proteus rettgeri R110 250 125 25 * 0.25 *
, ,. ~ ,
Staph.aur _s tRussell~ 250 125 C0.1 0.25
Staph.aureus ~Russell H) >500 ~0.1 ~0.1 ~ 0.1
.,
. . , _. .. _ ' ''
* Partial inhibition by MM 4550 (Complex) alone at these
concentrations.
Similar synergistic effects were observed for mixtures
of A~oxycillin and MM 4550 (Co;plex).
'
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.
- - 28 -
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lOS()~tiV
Description 8
Preparation of Partially Purified Antibiotic Complex containing
MM 4550 and MM 13902 using cetyldimethylbenzylammonium chloride
Freeze dried product (I50 = 0.02 ~g/ml) from a Farnell carbon
column eluted with acetone/water as in Description 4 was dissolved in
distilled water at a concentration of 13 mg/ml. The solution was
adjusted to pH 6.5.
;
100 ml. of the solution was extracted with an equal volume of 0.1
cetyldimethylbenzylammonium chloride in dichloromethane. The
phases were separated by centrifugation and the organic phase back
extracted with 100 ml. of 0.05~ sodium iodide solution. The phases
were separated and any dichloromethane in the aqueous phase removed
by maintaining the solution under reduced pressure for 10 minutes.
The aqueous solution was freeze dried and the product of freeze
drying washed three times with 50 ml. portions of acetone. The
acetone washed product was dried in a vacuum desiccator to yield a
light brown powder (4.3 mg I50 = 0.00~ ~g/ml~.
;
'-
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- 29 -
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l(~S~?460
Deseription 9
Preparation of Partially Purified Antibiotic Complex contain~ng
MM 4550 and MM 13902 using Gel Filtration
.
1 g. of crude MM 4550 (Complex), I50 0.2 ~g/ml, prepared by the
method of Description 4 was chromatographed on Sephadex G25 (fine
grade), using acetone/water 4:6 v/v as eluant. The eolumn
dimensions were 2.5em x 32 em and elution was carried out at
1.5 ml/min. The aetive fraetions were combined, eoneentrated
ln vaeuo below 30C and freeze dried to yield 22 mg. amorphous
buff eoloured solid having an I50 of 0.005 ~g/ml. The reeovery
was 88% and purification 40-fold.
'
.,
- 30 -
:
1()50460
Description 10
Preparation of Purified Antibiotic Complex containing MM 4550
and MM 13902 using Cellulose Chromatography
MM 4550 (Complex) (610 mg.) prepared as in Procedure 7 was
chromatogr~phed on a 2.5cm. x 32cm. column of cellulose (Whatman~
CC 31) and eluted with a mixture of isopropanol/water, 7/3 (v/v)
at 1.5 ml/min. The antibacterially active fractions were
combined, concentrated in vacuo, below 30C and freeze dried to
yield 40 mg. of brown amorphous solid preparation of antibiotic
complex I50 of 0.00007~g/ml. The very small value of the
I50 indicates that the active material is of improved purity.
On a different occasion the above procedure yielded a materlal
having an I50 of 0.001 ~-g/ml. This material had the antibacterial
activity shown in Table 3 when determined by a standard microtitre
method in Oxoid sensitivity broth using light inocula tl% of
overnight broth).
~tTrade Mark
- 31 -
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T A B L E 3
ANTIBACTERIAL ACTIVITY OF MIXTURE OF MM 4550 and MM 13902
HAVING AN I5~ OF 0.001 ~g/ml
_
. Organism MIC
. . .___ . . -.,
Staphylococcus aureus (Oxford) 7.5
Staphylococcus aureus (Russell) 7.5
Streptococcus faecalis 125
Bacillus subtilis 0.9
Escherichia coli (10418 3.7 ~ :
Escherichia coli (B11) 15
Klebsiella aerogenes 1.8
Enterobacter cloacae 62 .
.
Proteus mirabilis 7.5
. .
Providentia stuartii 7 5
,, . ' ~:
Aclnetobacter anitratus 0.9 ~:
Pseudomonas aeruginosa 250
Serratia marcescens 7.5
Salmonella typhimurium 15
Shigella sonnei 7.5
.. j _
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- 32 -
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~050460
Description 11
MM 4550
MM 4550 may be recognised by its properties which are as follows:
MM 4550 is an acidic solid which in the form of a sodium salt
has the following characteristics:-
; 5 i. It is highly soluble in water, soluble in methanol and
substantially insoluble in chloroform, diethylether and
hydrocarbons.
ii. In aqueous solution, it has a characteristic ultra-violet
spectrum with absorption maxima at about 238 nm. and at
about 287 nm.
iii. When present at 0.4% w/w in a freshly prepared KBr disc, it
has a characteristic infra-red spectrum which has absorption
maxima at inter alia about 3450, 2950, 1765, 1695, 1510,
1390 and 1260 cm 1. If a further spectrum is taken about
one week after the preparation of the KBr. disc, the
spectrum shows considerable changes, for example, the large
peak previously at about 1765 cm-1 is considerably reduced
in size or is absent.
iv. It has a characteristic n.m.r. spectrum when taken in
D20 which spectrum posses inter alia (a) a pair of low
field doublets centered approximately at 2.45~ and 3.65
with coupling constants of approximately 15 Hz;
(b) a doublet centered at approximately 8.55t and
.:
(c~ a sharp singlet at approximately 7.95~,
:
. :
,
_ 33 -
~504~iO
v. When run over cellulose in a thin layer chromatography
system, it has the following very approximate RE values:
(a) butanol/isopropanol/water - 7:7:6 v/v; Rf = 0~6
(b) isopropanol/water - 7:3 v/v; Rf = 0.7
(c) n-butanol/ethanol/water - 7:7:6 v/v; Rf = 0.7
(d~ n-propanol/water 4:1 v/v; Rf = 0.6
vi. It possesses antibacterial activity against various
species including inter alia, strains of Staphylococcus
aureus, Bacillus subtilis, Escherichia coli, Klebsiella
aerogenes, Proteus mirabills, Acinetobacter anitratus,
Serratia marcescens and Shigella sonnei.
.:
vii. It possesses enzyme inhibitory activity against the
0-lactamases produced by various species including,
inter alia, Escherichia coli, Klebsiella aerogenes
and Staphylococcus aureus. It has an I50 (as herein-
before defined) of less than 0.0001 ~g/ml. against the
~-lactamase of Escherichia coli B11.
viii. When mixed with ampicillin, it synergyses its activity
against organisms including strains of Escherichia coli,
Rlebsiella aerogenes, Proteus mirabilis, Proteus morganii
and Staphylocoac=s aureas Rasse11.
:
~ - 34 -
1(~50460 :
ix. Amino acid analysis indicates that the material is
not a poly peptide or protein. No ~-aminoadipic acid
is found after acid hydrolysis.
x. It reacts with Ehrlich's reagent (300 mg. of 4-dimethyl-
aminobenzaldehyde dissolved in a mixture of 54 ml.
n-butanol, 9 ml. ethanol and 9 ml. concentrated
hydrochloric acid~ to produce a blue colour on paper
chromatograms and tlc sheets.
ix. It is not a general enzyme poison and does not inhibit
the following enzymes at concentrations in excess of
those required to inhibit the ~-lactamase of Escherichi
coli; monoamine oxidase, carbonic anhydrase, dopa
decarboxyla~e, trypsin, chytotryp~lin or urease.
': ~
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1~50460
Example 1.
Preparation of MM 4550 (Complex) and Separation into MM 4550 and
MM 13902
The isolation procedures detailed in the Description section may be
utilized in a variety of sequences. A particularly suitable
sequence is carbon adsorption, ion pair extraction and cellulose
chromatography as described below:
Culture filtrate (340 l) (obtained as in Description 3) was percolated
by upward flow at 800 - 1200 ml/min. through a column (9" diameter
x 21" high) packed with Farnell B0 carbon. The carbon had been used
before for adsorption of MM 4550 (Complex) and was regenerated by
~ washing with the following reagents:-
,,
0.5N NaOH; 0.2N NaOH/acetone (3:2); water; N HCl;
water; phosphate buffer pH 6; water.
The column was washed with water (20 l) to displace the culture
filtrate and eluted by downward flow with acetone/water 2:8 v/v '
; at 200 - 250 ml/min. One litre fractions were collected.
Fractions containing the MM 4550 (Complex) as determined by an
antibacterial diffusion assay using Klebsiella aerogenes were
combined l11 1) and concentrated by evaporation in vacuo below
30C to 5.5 1. The recovery of MM 4550 (Complex) at this staye
was 20~.
, ~" ~
:' :
.
- 36 -
1050460
The concentrate was cooled to 2C, 5.5 l of 2% w/v tetra n-butyl-
ammonium hydrogen sulphate in dichloromethane at -5C was added and
the two phases mixed together for 2 minutes. The dichloromethane
phase was separated and added to one litre of sodium iodide solution
(0.6%) at 0C. The two phases were stirred gently and the aqueous
phase gradually adjusted to between pH 6.5 - 7.0 with 2% aqueous
solution of NaHCO3. The aqueous phase was separated and freeze
dried. The dried solid was extracted with dry acetone to dissolve
; out excess sodium iodide and the acetone removed from the insoluble
residue in vacuo, to yield a buff coloured powder (1.1 g.). Overall
recovery of MM 4550 ~Complex) at this stage was 10%. The purification
calculated on total dissolved solids in the culture filtrate was
i 125-fold.
A 2.5cm. diameter column was packed with cellulose powder (Whatman
CC 31) in isopropanol/water, ~:3 v/v, to a height of 32cm. 500 mg-
` of the buff powder was dissolved in 2ml. isopropanol/water 7:3 v/v
and chromatographed using the same solvent at a flow rate o 1.5 ml/min.
Fractions (3 ml.) were collected from the column and those showing W
.
absorption maxima at about 285 nm. were combined, concentrated and
freeze dried to yield MM 4550 (Complex). Previous experiments have ~ -
;:j . : ,
established that fractions absorbing at about 285 nm. contained enzyme
inhibiting, antibacterially active material.
~,~ ~ ,,,;;.,.: :'
~ The yield of freeze dried MM 4550 (Complex) was 35 mg. It had a brown
,~ amorphous appearance and an I50 of0.0001~g/ml. The recovery of
active material at this stage was 3% overall and the purification
~,~ 600-fold overall.
i ~ '
~ ~ 37 ~
'; .
1050460
The antibacterial activity of this preparation was determined by
the standard Microtitre method of Oxoid sensitivity test broth
using light inocula. The resulting minimum inhibitory concentra-
tions were similar to those shown in Table 3 hereinbefore.
Analysis of the preparation by thin layer chromatography on cellu-
lose (Eastman Kodak "Chromagram"* sheets) developed with n-butanol;
isopropanol:water t7:7:6) separated two active substances, one with
an approximate Rfof 0.58 which was designated MM 4550 and one with
an approximate Rf of 0.72 which was de~ignated MM 13902. Both
these materials may be detected by bioautography on a variety of
organisms including B. subtilis, Staphylococcus aureus (Oxford),
Staphylococcus aureus (penicillin resistant), Escherichia coli
(penicillin sensitive and resistant), Salmonella typhimurium~
Proteus mirabilis, Proteus mor~anii and Klebsiella aerogenes. Some
.. ..
of the results obtained are summarised in Table 4.
In a further experiment, the two substances were separated on a
cellulose thin layer developed with isopropanol:water (8:2) and
extracted from the cellulose with phosphate buffer. Each extract
was assayed for ~-lactamase inhibition and both substances were
shown to be inhibitors of Escherichia coli ~-lactamase. Both
substances were also shown to be synergistic with benzylpenicillin
against Kiebsiella aerogenes.
*Trade Mark
.
: , , , . : :
..
. : . . . - , . .
~:)50460
T A B L E 4
ANTIBACTERIAL ACTIVITY OF MM 4550 AND MM 13902 AS DETERMINED BY
BIOAUTOGRAPHY (TLC WITH CELLULOSE AND BUTANOL/ISOPROPYL
ALCOHOL/WATER 7:7:6 v/v)
. .,
Zone Diameter on Bioautograph
O r g a n i s m . .
MM 4550 zone in MM 13902 zone in
mm at Rf 0.58 mm at Rf 0.70 ~:
. -
Klebsiella aerogenes20.0 mm 20.0 mm
Proteus mirabilis 17.5 mm 13.5 mm
Proteus morganii 16.0 mm 9.S mm
Salmonella typhi 19.5 mm 21.0 mm
Escherichia coli 10418 20.0 mm 13.5 mm
Escherichia coli B1117.5 mm 10.5 mm
. ~ ,:
.; Enterobacter aerogenes 6.5 mm No Zone .. ~ .
. .
Staphylococcus aureus(Oxford)13.0 mm 4.0 mm
l' Staphylococcus aureus(Russell)12.0 mm 4.0 mm :
: ac;llus subtilis 24.0 mm . _ ~:
~.i, ~' .
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10504~;0
Example 2.
Preparation of MM 4550 (Complex) and Separation into MM 4550
and MM 13902
Culture filtrate (1100 l) from the fermentation of streptomyces
olivaceus ATCC 31126 at pH 6.5 and 5C were percolated at 3.2 l/min.
through a column (0.3m x 1m) packed with Darco granular carbon.
[The carbon had already been used for adsorption of MM 4550 (Complex)
and was regenerated before use by washing with the following reagents:-
0.5N NaOH; 0.2N NaOH/acetone ~3:2); water; ~ HCl;
water; phosphate buffer, pH 6; water].
The column was washed with water (60 1) to displace the culture
filtrate and eluted with acetone/water (2:8 v/v) at 30C at 1.1 l/min.
60 1 was collected followed by 5 x 10 l fractions. Those fractions
containing the MM 4550 (Complex), as determined by the antibacterial
diffusion assay using Klebsiella aerogenes were pooled (40 1) and
concentrated in vacuo below 30C to 32 1. The recovery of MM 4550
(Complex) at this stage was 15%.
Ths concentrate was cooled to 5C, 16 1 of 0.2% cetyldimethyl-
benzylammonium chloride in dichloromethane at 5C was added and the
two phases mixed for 5 mins. The dichloromethane phase was separated
added to 3.75 1 sodium iodide solution (0.4%) at 5C. The two
phases were mixed for 5 mins., the aqueous phase was separated and
freeze dried. The dry solid was extracted with dry acetone to
dissolve out excess sodium iodide and the residual solid was dried
in vacuo to yield a buff coloured powder ~2.87 g). Overall recovery
,
oP MM 4550 (Complex) at this stage was 6%. The purification
calculated on total dissolved solids in the culture filtrate was
160-fold.
. , .
- 40 -
~0~04~)
A 63 mm diameter column was packed with cellulose powder tWhatman
CC 31) in isopropanol-water (7:3 v/v) to a height of 300 nm.
2.0 g. of the buff coloured powder was dissolved in 5 ml. isopropanol/
water (7:3 v/v) and chromatographed in the same solvent at 3 ml/min.
S Fractions containing MM 4550 (Complex), as determined by assay
against Klebsiella aerogenes were pooled, concentrated by evaporation
in vacuo below 30C and freeze dried to yield a brown solid (207 mg.).
The recovery of MM 4550 (Complex) for this stage was 51% and the
purification was 5-fold.
A 16 mm. diameter column was packed with cellulose powder (~hatman
CC 31) in n-propanol/water (4:1 v/v) to a height of 300 mm.
198 mg. of the brown solid was dissolved in water/n-propanol (1:1)
and chromatographed in n-propanol/water (4:1 v/v) at a flow rate of
0.5 ml/min. Franctions (6 ml.) were collected, bioassayed against
Klebsiella aerogenes and the uv spectrum of each fraction measured.
; Fractions 23 - 28, having a uv maximum at about 305 nm, contained
the di-sodium salt of MM 13902. These fractions we~e pooled, con-
centrated under vacuum and freeze dried to give 30 mg. of solid.
This preparation showed only one zone of antibiotic activity at
Rf 0.77 on thin layer chromatography using Eastman Kodak cellulose
plates with n-propanol/water (4:1 v/v). The zone was detected by
bioautography on Bacillus subtilis. (Fractions 29 - 40, having a
uv absorption maximum at about 285 nm, contained MM 4550. They were
combined, concentrated under vacuum and freeze dried to give 53 mg. of
solid which contained a salt of MM 4550 contaminated with a small
quantity of a salt of MM 13902).
' ~"""''
'
- 41 -
.: . . .. .
~0504~0
Description 12
Organisms
The property of producing MM ~550 ~Complex) was first discovered
in the cultures ATCC 21379, ATCC 21380, ATCC 21381 and ATCC 21382
which had been isolated from soil samples obtained from Spain,
New Zealand, South Africa and Israel respectively. In the
laboratory, these cultures appeared identical and all were
identified as Streptomyces olivaceus, by Dr. Bousfield, the
actinomycete expert at the National Collection of Industrial
Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, using
; the widely accepted 1967 classification of Hutter [Systematic der
Streptomyceten, S. Karger, Basel, 382 pp]. MM 4550 production
has since been found in other streptomyces species as may be
seen from Table 4.
S. olivaceus was first described by Waksman in 1919 [Cultural
Studies of Species of Actinomyces, Soil. Sci., 8,71 - 215].
Subsequently, in 1960, a group of Russian workers described a
species with very similar characteristics but which they named
Actinomycés (Streptomyces) fulvoviridis. [Kuchaeva et al.,
Trud. Inst. Mikrobiol., Akad Nauk. SSSR., 8, 226 - 253 (1960)].
Micro-organisms of the genus Streptomyces are extremely
variable in their morphological and physiological characteristics
depending on the conditions under which they are grown.
Descriptions of many species had been published before the
existence of this extreme variability had been recognised, with
consequent duplication and synonymy. H~tter (1967) lists 25
species as being synonymous with S. olivaceus including
fulvov1ridis. To resolve the confusion is nomenclature and
.,
- 42 -
. , .
lOSq~460
classification, the Lnternational Streptomyces Pro~ect was begun
in 1962, Shirling et al., Int.J.Syst.Bacteriol, 18, 69 - 189
(1968). Collaborators in this Project have carried out a series
of studies aimed at producing accurate descriptions of species
of Streptomyces under standard conditions. In this scheme,
standard descriptions have been produced of type strains of some
400 named species including Streptomyces olivaceus and
Streptomyces fulvoviridis.
The I.S.P. description of S. olivaceus and S. fulvoviridis
differ in two characters only: (1) the form of the sporophore
and (ii) ability to utilise inositol as sole carbon source.
S. olivaceus is described as follows:
Spore chain morphology: Section Spirales, with open spirals
intergrading through flexuous spore chains suggestive of Section
Rectiflexibiles. Mature spore chains generally long, often ~ith
more than 50 spores per chain.
Carbon utilisation: D-Glucose, L-arabinose, D-xylose~
I i-inositol, D-mannitol, D-fructose and rhamnose are utilised for
- growth. No growth or only trace of growth on sucrose and
raffinose.
S. fulvoviridis is described as follows:-
Spore chain morphology: Section Rectiflexibiles. Mature spore
chains moderately short with 10-50 spores per chain. Carbon
utilisation: D-Glucose, L-arabinose, D-Xylose, D-mannitol,
D-fructose and rhamnose are utilised for growth; utilisation of
sucrose is doubtful. No growth or only trace of growth on
i-inositol or raffinose.
.
" ,
,~ '.
- 43 -
. ~':,
1050460
Characterisation of a number of cultures named, as, or synony-
mous with S. olivaceus or S. fulvovirldis and other related species
was determined using the standard methods and media (Shirling et al.,
Int. J. Syst. Bacteriol. 16, 313 - 340, t1966) reco~mended in the
International Streptomyces Project (ISP).
The cultures were derived from different sources. ATCC 21379,
21380, 21381, 21382 were isolated from soil on the basis of pro-
ducing MM 4550 (Complex). The other strains were obtained from
various culture collections for comparison purposes.
Results of the tests for production of MM 4550 (Complex),
colour of aerial mycelium, sporophore shape, colour of substrate
myselium, soluble pigment production, melanin production and carbon
source utilisation are given in Tables 4 - 8. For all strains
the spore surface, as seen by electron microscopy, is smooth.
From the ISP description it is clear that the spiral growth
of the sporophore in S. olivaceus is a variable character. True
spirals were observed with a variable frequency in the t~rpe
species of S. olivaceus, i.e. ATCC 3335. The majority of the
, sporophores were long. No true spirals were observed from the
cultures ATCC 21379, 21380, 21381 or 21382 although the sporophores
were long and showed a tendency to spiral among a background of
Rectiflexibiles types. However, in two S. olivaceus strains,
NCIB 8238 and NCIB 8509, the majority of sporophores were of the
Rectiflexibiles type. In NCIB 8238 they were of medium length
while tho~e of NCIB 8509 were long.
The sporophores observed from ATCC 15863, the type species
of S. fulvoviridis, were shorter than those seen from isolates
- 44 -
:, :
' .:',,.' -': :::' : ., . ' . :.- . . , ~ .
~0S0460
ATCC 21379, 21380, 21381, 21382 and 31126 and were only of the
Rectiflexibiles types. In respect of this character therefore,
the isolates ATCC 21379, 21380, 21381, 21382 and 31126 correspond
fairly well but not exactly with the S. olivaceus description.
Isolates ATCC 21379, 21380, 21381, 21382 and 31126 show some
variability with regard to inositol utilisation which is the
other differentîating character according to the I.S.P. type
species descriptions between the species S. olivaceus and
S. fulvoviridis (Table 8). ATCC 31126 and ATCC 21380 utilise
_ _
inositol, which is characteristic of S. olivaceus, while the
other strains show doubtful or negative utilisation. However, it
is not generally considered satisfactory to separate off a species
on the basis of the ability to utilise a single carbohydrate. Such
a difference might arise by a single gene mutation and is often
considered to be of strain significance only. The difference in
sugar utilisatioD by the S. olivaceus strains NCIB 8138, NCIB 8509,
ATCC 21549, ATCC 12019 and ATCC 3335 suggest that many authorities
do not consider them of species signifioance. Thus ATCC 21379,
21380, 21381, 21382 and 31126 are properly designated S. oliva_eus.
Unless further studies uncover more important differences
between those cultures presently named S. olivaceus and
S. fulvoviridis, it is possible that they will eventually be inter-
nationally recognised as a single species. In this case the correct
name, by the priority rule, will be S. olivaceus, and this will
also include those cultures listed by Hutter as synonymous with
S. olivaceus.
. . .
' '.
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1 ,
i, .:
.,
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~0504~0
Examination of culture filtrates of the strains of
S. olivaceus and related species for the production of MM 4550
(Complex) may be carried out as follows:-
Culture filtrates of the listed strains were spotted onWhatman No.l paper strips at 20~u 1 per origin and chromatographed
in n-butanol/isopropanol/water (7:7:6) overnight in the cold.
Another set of strips were run in n-butanol/glacial acetic acid/
water (1~,3,5) also in the cold overnight. A partially purified
sample of MM 4550 (Complex) was also run in the two systems at
the same time as a market.
Alternatively, it is possible to extract 25 ml of clarified
brew with 12.5 ml of 0.2% cetylbenzylammonium chloride in
dichloromethane, separate the phases, retain the organic phase,
add 2.5 ml of sodium iodide solution (0.5%), shake, separate the
phases, retain the aqueous phase and use this chromatographically,
for example, by spotting 5~ onto thin layer chromotography strips
and run in n-butanol/isopropanol/water, 7:7:6.
Streptomyces olivaceus and related organisms have the follow-
ing prime characteristics:
(a) They possesssporulating aerial mycelia in either the grey or
yellow series.
(b) They possess a sporophone morphology of either the rextiflexibiles
or spirales type.
(c) They possess smooth surfaced spores.
(d) They do not produce melanin.
Species possessing the preceding characteristics include those
referred to in Tables S to 9.
- 46 -
.. . . .. .
:, . . :: ., : , - . : . :
. : : : , .:: . . ., : , . . , - : . . ,. :
... . : . . ~ - , ~ : . ., :
.' ' ~ . ~' , - . .:
' .-: -, , , . ' ' : '
1050460
T A B L E 5
ABILITY OF CULTURES STREPTOMYCES OLIVACEUS STREPTOMYCES FULVOVIRIDIS,
.. ~ . .
AND RELATED SPECIES TO PRODUCE MM 4550 (COMPLEX~
. ~
Culture MM 4550 (complex~
Production
..... . .
Streptomyces olivaceus ATCC 21379 f
" ATCC 31126 f
" ATCC 21380 f
' ATCC 21381 f
" ATCC 21382 f .--.
" NCIB 8238 f
" NCIB 8509 f
Streptomyces flavovirens ATCC 3320 f
Streptomyces flavus ATCC 3369 f
Streptomyces fulvoviridis ATCC 15863 f
Streptomyces argenteolus ATCC 11009 f
Streptomyces sioyaensis ATCC 13989 f
Streptomyces lipmanii NRRL 3584
(Streptomyces olivaceus ATCC 21549, ATCC 12019 and ATCC 3335
di'd not produce MM 4550 (comple~), ATCC 15863 has also been
deposited as RIA 660)
' "'
'.
, - 47 -
.
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- . . .- . .
- .. . . ~ , ,
', . ' ' . , ,. " ~ '
~, 10S0460
T A B L E 6
STREPTOMYCES OLIVACE~S, STPEPTOMYCES FULVOVIRIDIS AND RELATED SPECI~S
- COLOUR AND MORPHOLOGY OF MATURE SPORULATING AERIAL MYCELIUM
ON ISP MEDIA AFTER 14 DAYS GROWTH .
- .
_ _ . Sporophore
.~Culture SS YM GA OM Morphology
. mid size
ATCC 21379 Grey Grey Grey/White Grey Long RF .
ATCC 31126Grey/~rown Grey/Brown Grey Grey Long RF ,
ATCC 21380 ; Grey Grey Grey Grey Long RF
ATCC 21381 Grey Grey Pale Grey Grey/White Long RF .
ATCC 21382 ~ Grey/~hite Grey Grey Grey Long RF .
NCI~ 8238 Grey/White Grey Grey Grey Medium RF
NCI9 8509 Grey Grey Grey Grey Long RF
ATCC 21549 Grey Grey Grey Grey Long S
ATCC 12019 Grey Grey Grey Grey Long RR/S.
ATCC 3335 Gr0y Grey Grey Grey Long RF/S
ATCC 15863 Grey/Nhite Grey Grey/White Grey Medium RF i
ATCC 3320 Grey/Blue Grey Grey/Green Grey Long RF ;
ATCC 3369 Grey Grey Grey Pale grey~ . i
. . PrownMedium RF/RA
.. ATCC 11009 Pale Grey Grey~brown Grey/White Grey/Green Medium RF/RA,. . ATCC 13898White/Grey White Whi.te/ Grey/White Short S ;
. _ _ yellow .
SS ~ Inorganic salts - starch agar (ISP Medium 4)
YN = Yeast extract - malt extract agar (ISP Medium 2) '
GA ~ Glycerol - asparagine agar (ISP Medium 5) ~
OM ~ Oatmeal agar (ISP Medium 3) !
RF ~ Rectiflexibiles j
RA ~ R~tinaculiaperti ~
S ~ Spirales
:: :
.' ':
. . _ 48 -
. ~ .
.
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'; : ~ ' - ` i ~
.: ' '
s
~
-
: - :. , . , .
-: :, .
: : ~ , . . : . .,: . .: '.
'` 1050460
T A 8 L E 7 :
STREPTOMYCES OLIVACEUS, STREPTOMYCES FULVOVIRIDIS AND RELATED SPECI~S ..
- COLOUR O~ SUBSTRATE ~YCELIU~I AS VIE~IED FROM THE REVERSE SIDE . .
_ _ _
Culture SS YM GA OM
ATCC 21379 olive olive brown grey/brown olive green
ATCC 31126 olive brown olive brown brown olive brown
ATCC 21380 olive olive green olive brown olive yellow
ATCC 21381 dark brown dark brown olive green olive green .
ATCC 21382 olive olive brown olive green
NCIB 8238 brown olive/brown brown olive yellow
NCIB 8509 brown olive olive brown olive yellow .
ATCC 21549 buff/black brown/black brown/black buff/grey .
ATCC 12019 buff buff buff buff -
ATCC 3335 brown brown brown grey .
ATCC 15863 brown/grey dark brown olive brown olive yellow
ATCC 3320 yellow/brown olive/brown olive/brown orange/brown .
ATCC 33eg buff!grey yellow hrown buff/green yellow
ATCC 11009 black black/yellow black/yellow grey/green .
ATCC 13989 orange yellow yellow colourless
_ , _ ''. :'' ' .
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:
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()50460
T A B L E 8
. .
STREPTOMYCES OLIVhCEUS, STREPTOMYC~S_FULVOVIRIDIS AND RELATED SP~CIES
- PRODUCTION OF PIGMENTS IN CULTURE MEDIA
.
:
r - ~ - _ _ ~ ruble Non-Melanoid Pigments ~Melanin
Culture ' _ _. _ Production .
.~ SS YM GA OM
, _ __ _ _ _ ~
ATCC 21379 _ _ _ slight _ .
. yellow
ATCC 31126 _ _ . _ _ i
ATCC 21380 _ _ _ _ .. ,
~TCC 21381 _ _ _ _ _ ,
ATCC 21382 _ _ _ _ _ .
NCI~ 8238 f _ f f _ i
slight slight slight ,
'brown brown 'brown
NCIB 8509 _ _ _ Syefgllhtow _
ATCC 21549 _ . f f f _
. slight slight red/ slight
brown brown brown
ATCC 12019 _ _ _ _ _
ATCC 3335 _ . _. _ _ ~
: ., ATCC 15863 _ _ . _ _ .
ATCC 3320 f _ _ _ _
sliinhkt
ATCC 3369 _ _ _ _ _
. . ATCC 11009 _ f _ _ _
slight .
. . 'brown
: ATCC 13989 _ f _ _ _
: : slight
orange
_ _ _ _ _ ,
f = Pigment produced .
~ = Pigment not produced .
. * ' Tested on peptone-yeast-iron agar (ISP6), tyrosine
.~ agar (ISPl7? and tryptone-yeast broth (ISP 1) :~
- 50 - '
: ' '
' ` ' -
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. ' '" ' ' , .
,, '~` --'' ; - : ,
- :
., . .:. ' . , ~ ::
~ . 1 460 1 ~
~r A B L E 9
., . ,,..
STREPTOMYCES OLIVACEUS, STREPTOMYCES FULVOVIRIDIS AND RELATED SPECIES
.. ..
- CARBON UTILISATION TESTS
.
. ' ' ~ .,
.; . ATCC 21379 f _ _ f f f f f f :
.i ATCC 31126 f _ _ f f f f f f .
ATCC 21380 f _ _ f f f f f f
ATCC 21381 f _ _ _ f f f f f
ATCC 21382 f f _ _ f f f f f . ,.
~ NCIB 8238 f _ _ _ f f f f f ;
; NCIB 3509 , f f _ _ f f f f f
.' ATCC 21549 _ _ ._ f f f f f f
., . ATCC 12019 f f f f f f f f f
, . ATCC 3335 f _ f f f f f f f .. ::
.~ ATCC 15863 .f _ _ _ f f f f f i .
. ATCC 3320 . f _ ~ _ f .f f f f . : . .
ATCC 3369 f f f f f f f f . .
. ~ ATCC 11009 f _ .. f f f f f f .
ATCC 13989 _ i f f f _ f f _
. f denotes the compouhd is utilised ' :
denotes the compound is not utilised
, ~-f d-notes.utilis-tion is doubtful or very poor
~.. : ~
51- ' 'i.
`~i, '~ '~ . ' . .
J~ ;
`: 1050460
Exam.~le 3
A stock of spores of S.olivaceus ATCC 31126 was maintained by
storage in tubes of dry soil in a closed container with a dessicant
at a temperature of 20 C. A small quantity of soil stock
(approximately 20 mg) was transferred aseptically to a 500 ml
Erlenmeyer flask containing 100 ml of the following medium:
Constituent Amount (g/litre)
Glucose monohydrate 20.0
Soya-bean flour 10.0
Deionised water to 1 litre
The pH was adjusted to 6.5 before sterilisation. The soya
bean flour was 'Arkasoy 50' as supplied by British Arkady Co. Ltd.,
Old Strafford, Manchester, England.
The flask was incubated on a rotary shaker (240 r.p.m.) for
about 30 hours at 28C. 2 ml of the resulting vegetative growth
was then used to inoculate a solid agar slant in a Roux bottle.
The agar medium had the following composition:-
Constituent Amount
V-8* vegetable juice 20.0 ml
Agar 20.0 g
Deionised water to 1 litre
The pH was adjusted to 6.0 before sterilisation. (V-8 juice
is obtainable from Campbell's Soups Ltd., Kings Lynn, Norfolk,
England).
I The inoculation was spread on the agar surface by roc~ing
the bottle which was then incubated upright at 30C. After two
l~ *Trade Mark
,~ - 52 -
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ij
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.. . . , : . . .. , : ::: :.
. . . : . . . .
~5()460
days incubation, surplus liquid in the bottle was removed by
pipette and incubation continued for a further 4 days.
It had been previously found that the development of
actinophage in the slant culture is suppressed if this method
of preparation of the Roux bottle culture is adopted.
50 ml of sterilised deionised water containing 0.02% Tween
80 was added to a Roux bottle culture and the spores suspended
by shaking. This spore suspension was then added as inoculum
to 75 litres of sterilised seed stage medium in a 100 litre
10 staînless steel fermenter. The composition of the seed stage
medium was as follows:-
Constituent Amount (g/l)
Soya-bean flour ('Arkasoy 50') 10.0
Glucose monohydrate 20.0
Tap water to 1 litre
To control foaming, 50 ml of 10 % v/v 'Pluronic L81' in
soyal-bean oil was added to the fermentation medium before
sterilisation.
The medium was steam sterilised in the fermenter for
20 minutes at 120C. The seed stage culture was stirred at
140 rpm with a 7.5 inch diameter vaned disc agitator and
supplied with 75 litres/min sterile air through an open ended
sparger.
The temperature was controlled at 28C and after incubation
under these conditions for 48 hours the contents of the vessel
were added as inoculum to 1500 litres of sterile fermentation
medium in a 2000 litre stainless steel fully baffled fermenter.
The fermentation medium had the following composition:
~..
- 53 -
'.
.
,. .
.
10~04~0
Constituent Amount (g/litre)
soya-bean flo~r ('Arkasoy 50') 10.0
Glucose monohydrate 20.0
Chalk (precipitated calcium carbonate) 0.2
Cobalt chloride (COC12.6H2O) 0.001
Sodium sulphate (anhydrous) l.o
Tap water to 1500 litres
The pH was adjusted to 6.0 with sodium hydroxide before
sterilisation. 3 litres of 10% 'Pluronic L81' in soya bean oil
was added before sterilisation to prevent foaming. After
sterilisation the pH was again adjusted, to 7.0, with sterile
sodium hydroxide solution. The fermentation was stirred at
106 r.p.mO, the stirrer shaft being fitted with two 19 inch
diameter vaned disc impellers. Temperature was controlled
at 30C and airflow at 1200 litres/min. The fermentation was
harvested after 60 hours and clarified by centrifugation.
The material produced above was arbitrarily assigned an
activity of 340 units/ml. Assays were determined as described
in Description 2.
Culture filtrate (1050 1; 340 units/ml.) at 10C and
pH8 was extracted with dichloromethane (310 1.) at 10C
containing cetyldimethylbenzylammonium chloride (1200 g.) by
p~lping the two liquids at predetermined flow rates through
an in-line mixer. The phases were separated in a
Sharples continuous centrifuge having been admixed
- 54 - ,~
.
~ l~)S04~
for about two minutes. The dichloromethane phase (300 1)
was back extracted with aqueous sodium iodide. The back
extraction was performed in four batches using a total of 7 1
water containing 210 g. sodium iodide. The phases were
separated by gravity. The aqueous phase was adjusted from
pH 7.7 to pH 7.0 with hydrochloric acid and filtered.
The sodium iodide extract (7 1) contained 21,900 units/ml.
An ion exchange column was prepared by packing QAE
Sephadex A25 (obtained from Pharmacia Ltd.) in pE17 phosphate
10 buffer (0.05M) containing sodium chloride (0.3M) into a
10 cm diameter glass column to a height of 40 cm. The sodium
iodide extract (7 1) at 5C was percolated through the QAE
Sephadex at 50 ml./min. The column was eluted with
0.7M NaCl in 0.05M phosphate bufer, Pb 7 a1so
."' ..
. . ~
1' ~
' , :;'
.. . . .
~05(~4~0
at 5C at a flow rate of 25 ml./min. 2 l eluate was discarded
and 90 fractions (100 ml.) were collected. The fractions were
scanned in a u.v. spectrophotometer and those fractions showing an
absorption maximum at about 305 nm were pooled and adjusted to pH 7
(fractions 50 - 62, pooled volume 1440 ml. 71,000 units/ml. It
had previously been shown that fractions having a maximum in this
region contained MM 13902.
Sodium chloride (5 g./100 ml.~ was added to the pooled fractions
which were then percolated at 5C through a 6.3 cm diameter column
packed with A~berlite XAD 4 resin (as supplied by Rohm & Haas Ltd.)
to a height of 30 cm. at a flow rate of 20 ml./min. MM13902
was adsorbed to the resin under these conditions whereas the
inorganic impurities were not. MM 13902 was eluted at
room temperature with distilled water (200 ml.) followed by 50%
aqueous methanol. The eluate (1 l) was evaporated below 30C under
reduced pressure to 70 ml., adjusted to pH 7 and freeze dried to a
brown solid (1.62 g.) which was a partially purified salt of MM 13902
with an activity of 37,000 units/mg.
.'.
Partially purified MM 13902 di-sodium salt (1.0 g., 37,000 units/mg.)
was chromatographed on a cellulose column (3.8 cm. x 30 cm; Whatman
, :
CC31 cellose) eluted with n-propanol/water ~4:1 v/v) at a flow rate
!
of 2.5 ml./min. 170 ml. eluate was discarded and 100 x 15 ml.
! fractions collected. The fractions containing MM 13902 di-sodiumsalt (Nos. 37 - 43) as determined by uv absorption were pooled ~89 ml.),
evaporated below 30C under reduced pressure to remove n-propanol and
freeze dried to yield a yellowish powder ~219 mg.) with an activity of
; ~ 73,000 units/mg.
~: .
'~
- 56 -
~ 04~ :
The above material showed a uv absorption maximum at about 308 nm
with an E 1 of about 343. This material had the i.r. and n.m.r.
spectra shown in Figs.2 and 3 respectively. The antibacterial
activity of the material is shown in Table 10. Elemental analysis
of this material indicated that it contained inter alia nitrogen,
sulphur and sodium probably in the ratio N:S:Na - 2:2:2. The
positive and negative maxima of the circular dichroism curves
for di sodium MM 13902 were determined on a Cary-61 recording
spectropolarimeter at concentrations of 0.37 mg/ml at a path
length of 1 cm; the results were as follows:
(nm) ~ /m
186 + 67.24 x 10 4
' 221 - 67.24 x 10
286 - 3.3 x 10
323 - 3.2 x ~
:
,' ~ .
.
;'
- 57 -
... . . ,, , , , - .: :. :, . :
: , : : . . , : , . , . . . . :
~05~4~0
T A B L E 10
ANTIBACTERIAL ACTIVITY OF THE DI-SODIUM SALT OF MM 13902
(MICROTITRE METHOD - HEAVY INOCULUM, 1/100 OVERNIGHT BROTH)
.
Organism MIC (ug/ml)
, ' 7-
Bacillus subtilis A 0.15
Staph. aureus Oxford 0.3
Staph. aureus Russell 0.6
Strep. Faecalis 3.1
Enterbacter cloacae N1 25
E. coli 10418 0.15
E. coli JT410 5
Kleb. aerogenes A 0.03
Proteus mirabilis 13 0.6
Proteus vulgaris WO 90 0.6
Prov. stuartii 0.07
Ps. aerug nosa A 50
Salmonella typhimurium CT 10 0.3
Serratia marcescens US 39 2.5
Shigella sonnei 0.6-0.3
Haemophilus influenzae P6 0.08
Neisseria gonorrhoeae 0.08
. " . _ . _ ~
-,
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- 58 -
' .
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,
