Note: Descriptions are shown in the official language in which they were submitted.
13~863~
PREPARATION OF ANTIBIOTIC CHLOROPOLYS~'ORIN C
This invention relates to a process for the preparation
of the antibiotic chloropolysporin C from the related
compound chloropolysporin B. Chloropolysporins B and C are
glycopeptide antibiotics which exhibit antibacterial activity
and are useful in the treatment and prophylaxis of
infections, and as growth-promoting agents for animals.
As resistance to conventional antibiotics becomes
increasingly established in common strains of pathogenic
bacteria, the need for a wider variety of antibiotics for use
in the fight against such bacteria becomes ever more crucial.
~oreover, various antibiotics, for example chloramphenicol,
aureomycin, vancomycin and avoparcin, have been administered,
or have been proposed for administration, to poultry and
other farm animals, including the ruminants and pigs, for the
prophylaxis of disease or to promote growth or milk
production. However, an inherent disadvantage of the use of
antibiotics in this way is that there is some risk that
traces of the antibiotics or of metabolic products thereof
may be found in animal products intended for human
consumption (such as eggs, milk or meat); the alleged dangers
of such residues are increasingly criticized by some sections
of the community. There is, accordingly, a considerable
desire amongst farmers for an antibiotic substance which will
have the desired growth-promoting effect but which will leave
no, or no significant, residues in animal products.
In European Patent Application No. 84304765.5 (published
as EP-A-132349), assigned to the present assignees, there is
disclosed an antibiotic, there referred to as
"chloropolysporin", which was isolated from
2 ~31863~
~he culture medium of a microorgani6m identified as
MicroPolV6~0ra 6p. SANX 60983. It was sub~equently
discovered that the same microorganism, and hence others
of the genu~ MicroPolY6pora, produce6 a further two new
antibiotic 6ubstances that are highly effective against
Gram-po6itive bacteria ~nd that show considerable promise
for use as growth-promoting agents in animals, TheRe tWo
antibiotic~, named chloropoly6porin B and chloropoly6porin
C, their production by cultivation of microorganism~ of
the genus MicroDolYsDora, and their therapeutic and
veterinary use form the 6ubject of our European Patent
Application No. 86300176.4 (publi6hed as ~P-A-la7722),
When chloropoly6porin6 B and C are produced by
microbial culture. the yield of chloropolysporin C in the
resulting culture broth iB generally lower than that of
chloropolysporin B. On the other hand, it has been
establi6hed that chloropolysporin C exhibit6 higher
antimicrobial activity against many organisms than doe6
chloropolysporin B. Accordingly, chloropolysporin C iB
potentially the more valuable component, for instance as a
food additive, and there i8 a need for a procQ6s which can
be used selectively to produce chloropolysporin C.
It has now been discovered, in accordance with the
present invention, that chloropolysporin C can be produced
by selective hydrolysis of chloropolysporin B, u~ing
rhamno~ida~e.
It iS currently believed that chloropolysporins B and
C may be represented by the planar 6tructural formula (I):
~31863~
Cl oR3 ~`1
R10 ,br ~ oa2
N ~ CH3
~ OH
HO OH
in which:
for chloropolysporin B,
Rl represents a l-L-ri6tosamine residue;
R2 represents a l-D-mannose residue:
R3 reprefien~s a l-D-glucoge residue: and
R4 repre6ents a l-L-rhamno6e residue:
for chloropoly~porin C,
R represents a l-L-ri6tosamine re6idue:
R2 represent6 a l-D-mannose residue:
R3 represents a l-D-glucose residue: and
R repre6ents a hydrogen atom.
(Rigto6amine iB 3-amino-2,3,6-trideoxy-L-ribohexopyranose).
Accordingly, chloropolysporins B and C diPfer only in
the substituent represented by -oR4 in the above formula.
It is probable that the various assymetric carbon
atoms shown in the above formula (I) adopt, in
chloropolysporins B and C, specific configurations, but
these have not, to date, been elucidated.
`' 1318630
The proces6 of the invention involves an enzymatic
hydroly~i6 and can be carried out under conditions such as
reaction temperature, reaction time and pH which are
generally well known Der se for this type of reaction.
For instance, it can be carried out in solution, with or
without stirring, at a temperature of from 20 to 50OC,
preferably 30O to ~0C, and mo6t preferably at about
37OC. A conventional aqueous solvent may be employed for
thi6, for instance a 0.01 to o.lN phosphate buffer
solution or a TRIS-HCl buffer 601ution. The pH of the
reaction sslution will usually be wit~in the range of from
4 to 8, and preferably from 5 to 6. The reaction time
will depend upon the other reaction conditions employed,
such as the te~perature, pH, and concentration of the
enzyme and of the chloropolysporin B 6ubstrate, but it
will generally be within the range of from 5 hours to 3
days, and preferably from 15 to 24 hours.
The enzyme preparation used in the proce6s of the
invention will usually have a rhamnosida6e activity of
from 100 to 1,000 units/mg, and more preferably from 200
to 500 units/mg. The enzy~e i~ generally used iD an
amount wich corre6ponds to a weight ratio of rhamnosîdase
to chloropolysporin B within the range of from 1:0.01 to
1:50, and more preferably from 1:0.05 to 1:20.
There i~ no particular limitation on the type of
rhamnosidase preparation used in the process of the
invention, provided that it posse66e6 adequate
rhamno6idase activity. In practice, an a-L-rhamno~idase
(Enzyme Commis6ion No. ~.2.1.40) will normally be
employed. The enzyme may be used in pure form, or in the
form of a crude rhamno~idase preparation such as those
produced commercially ~rom various microorganism6, and
which may add~tionally contain some different enzymes
and/or other materials besides the rhamnosidase itself,
provided that such contaminant6 do not adversely affect
the proce6s of the reaction to an unacceptable degree.
5 ~3186~0
Examples of 6uch rhamno~idase preparation6, which may
be used in the proces6 of the inYention, include those
produced by Sankyo Co Ltd , Japan, under the ~ ~
IlNaringinase'' and ~Sclase", and that produced by Tanabe
Seiyaku Co Ltd,, aapan, under the trade name
~Kumitana6e~ Naringinase i6 an enzyme preparation in
which the major enzyme component i~ a-rhamnosidase, with
a minor amount of ~-gluco~idase, and i~ produced by the
cultivation of the microorganism A6Derqillus usamii or of
certain Penicillium species. Sclase is a pectinase-rich
preparation containing naringinase, and i8 produced by
cultivation of Coniothvrium diPlodiella~ A6Der~ ua
usamii or AsDeraillu~ nicer. Kumitanase is
a-rhamnosidase derived from AsDer~illus niaer.
AB an alternative to using a rhamnosidase preparation
in soluble form, an immobilisQd enzyme preparation may
also be used, either one in which the enzyme itself has
been immobili~ed in a suitable carrier, or one containing
immobili6ed cells of a rhamnosidase-producing
microorganism - for example a naringinase-producing
microorganism 6uch as AsDeraillus usamii, or a
sclase-producing microorganism such as AsPeraillu6 niqer.
If such an immobilised form is used, the support material
and techniques used for immobilisation of the en~yme or
enzyme-producing microorganism may be cho~en from tho~e
conventionally employed for this purpose in the enzyme art.
The process of the invention can be carried out by
using as the 6tarting material a ~olution of previously
i601ated chloropoly6porin B in either crude or purified
form, and the concantration of chloropoly6porin B in the
reaction solution i6 not particularly critical. However,
in accordance with a preferred embodiment of the
invention, the starting material may be a crude culture
6 i3~863~
broth containing chloropolysporin B, or a mixtuee of
chloropolysporins B and C, a~ obtained by cul~ivation of a
chloropolysporin-producing MicroPol~6Pora strain, by a
proce6s such a6 already mentioned. In thi~ way, it is not
nece6sary to isolate or purify the chloropoly~porin B
starting material before subjecting it to the proces6 of
the invention, and this i6 advantageou6 especially for
large-6cale commercial production. For example, the
concentration of chloropoly~porin B starting material in
6uch a crude culture broth may be in the range of from
1,000 tO 10,000 y/ml. More generally, the concentration
of chloropolysporin B in the reaction solution will
usually be at least 1 to 5 y/ml,
At the end of the reaction, the chloropolysporin C
formed by means of the enzymatic hydrolysis can be
eecovered from the reaction mixture and purified by mean6
of variou~ techniques which are Der se well known in the
production of biochemicals. For instance, it can be
adsorbed onto an adsorbing resin such as I'Diaion"
(Registered Trade Mark) HP 20 (product of Mitsubi~hi
Chemical Industries Ltd.) or "Amberlitel~ (Registered Trade
Mark) XAD-2 or XAD-4 ~products of Rohm S Haas Co.), and
eluted from the resin with a ~uitable eluent such as
aqueous acetone or aqueou~ methanol. Another purification
method which may be employed is rever6e phase
chromatography, for example u~ing a silanizing 6ilica gel
with a uniform particle size, preferably with a particle
diameter within the range o from 0.06 to 0.2 mm. The
eluent employed for thi~ technique may suitably be a
mixture of aqueous ammonium formate and acetonitrile, a
mixture of tri1uoroacetic acid and acetonitrile, or a
mixture of phosphata buffer and acetonitrile; and the mo6t
preferred eluent is a mixture containing about 85 par~s by
volume sf 0.2% trifluoroacetic acid and about 15 parts by
volume of acetonitrile.
7 131~63~
Thin layer chromatography and high performance liquid
chromatography may be used to monitor the concen~ration of
starting material and reaction product throughou~ the
reaction, including the purification QtageS.
Since the ~tructure~ of chloropolysporin6 B and C have
not been completely elucidated, they may be characterized
by the properties 6et out below, in conjunction with the
accompanying drawings. In ~he drawings:-
Figures 1, 2 and 3 re~pectively show the ultra~ioletabsorption 6pectrum, the infrared abQorption spectrum
and the nuclear magnetic re60nance spectrum of
chloropoly6porin B;
Figures 4, 5 and 6 respectively 6how the ultraviolet
ab60rption spectrum, the infrared absorption spectrum
and the nuclear magnetic resonance spectrum of
chloropolysporin C.
Chloropolysporin B, as its sulfate, is characterized
by the properties:
(a) it takes the form of an amphoteric white powder,
~oluble in water:
(b) specific rotation: ~a]25-64.5 (C-1.04, O.lN
aqueous hydrochloric acid, ~odiu~ D-line);
(c) elemental analysi~:
C, 48.33%; H, 5.05%; N, 5.48%: Cl, 5.11%: S, 1.00~;
(d) on acid hydroly~i~ it yield6:
neutral ~accharides: glucose, mannose and rhamno~e;
amino acids: 3-chloro-s-hydroxyphenylglycine and
N-methyl-~-hydroxyphenylglycine;
8 13~ 8~30
(e) ultraviolet absorption 6pectrum:
a~ illu~trated in Figure 1 of the accompanying
drawings, having an ab~orption maximum ~max at
Z80nm (Elcm=51) in a 0.1 N solution of hydrochloric
acid, the absorbence, E, being measured at a
concentration of 1% w~v;
(f) infrared absorption spectrum:
the infrared absorption spectrum (v cm 1) measured
on a KBr disc is as shown in Figure 2 o~ the
accompanying drawing6;
(g) nuclear magnetic resonance spectrum:
the nuclear magnetic resonance 6pectrum (~ ppm),
measured at 270 MHz in deuterated dimethyl sulfoxide
u6ing tetramethylsilane as the internal standard, is
as illustrated in Figure 3 of the accompanying
drawing~;
(h) 501ubility:
soluble in water, sparingly soluble in methanol and
acetone, and in601uble in ethyl acetate, chloroform
and benzene:
(i) color reactions:
positive in ninhydrin and Rydon-Smith reaction~;
(j) thin layer chromatography:
Rf value=0.65, using a cellulose sheet (Ea~tman~ as
adsorbent and a 15:10:3:12 by volume mixture of
9 1318630
butanol, pyridine, acetic acid and water as the
developing solvent:
(k) high voltage paper electrophore~i6:
using Toyo~s filter paper No. 51A in a O.lM
TRIS-hydrochloric acid buffer 601ution of pH 7.5 (3300
~olt/60cm, 1 hour), the migration distancè (detected
by bioautography with Bacillus subtilis PCI 219) from
the origin to the cathode wa6 4cm;
(1) molecular formula:
C83H8934N8~13 o.5H2S04. lOH20;
(m) molecular weight:
the molecular weight, measured by FAB-MS, was 1846
(~+, 18g7 ) .
"FAB-MS" is Fast Atom Bombardment Mas6 SpQctroscopy.
Chloropolysporin C, as its sulfate, may be
characterized by the following propertie6:
(a) it takes the form of an amphoteric white powder,
solubls in water;
(b) specific rotation: ~a] -64.4 (C=1.08, O.lN
aqueous hydrochloric acid, codium D-line);
lc) elemental analysis:
C, 50.53%; H, 4.69~; N, 6.14%; Cl, 5.62~; S, 1.12%;
10 13186~0
(d) on acid hydrolysis it yield6:
neutral saccharides: gluco6e and mannose:
amino acids: 3-chloro-4-hydro~yphenylglycine and
~-methyl-p-hydroxyphenylglycine:
(e) ultraviolet absorption 6pectrum:
as illustrated in Figure 4 of the accompanying
drawings, having an absorption maximum ~max at
280nm (ElC~=57) in a 0.1 N solution of hydrochloric
acid, the ab60rbence, E, being measured at a
concentration of 1% wJv;
(f) infrared absorption 6pectrum:
the infrared absorption spectrum (~ cm 1) measured
on a XBr di~c i~ as shown in Figure 5 of the
accompanying drawings:
(g) nuclear magnetic resonance spectrum:
the nuclear magnetic resonance spectrum (~ ppm),
measured at 400 MHz in deuterated dimethyl sulfoxide
using tetramethyl6ilane as the internal standard, is
aa illu~trated in Figure 6 of the accompanying
drawings:
(h) solubility:
601uble in water, 6paringly soluble in methanol and
acetone, and insoluble in ethyl acetate, chloroform
and benzene:
(i) color reactions:
positive in ninhydrin and Rydon-Smith reactions:
11 1318630
(j) thin layer chromatography:
Rf value=0.65, u~ing a cellulo6e 6heet (Eastman) a~
ad60rbent and a 15:10:3:12 by volume mixture of
butanol, pyridine, acetic acid and water a~ the
developing solvent;
(k) molecular formula:
C77H7930N8C13. O.SH2S04. SH20;
(1) molecular weight:
the molecular weight, measured by FAB-MS, was
1700 (MH+, 1701).
The acute toxicity (LD50) value, a6 determined by
intravenous admini6tration in mice (ICR, male, 5 weeks
old) was 215 mgtkg for chloropolysporin B and 250 mg/kg
for chloropolysporin C.
The minimal inhibitory concentrations (MICj of
chloropolyspolins B and C against variou6 Gram-positive
and Gram-negative bacteria were determined by the two-fold
agar dilution method, using a Mueller-Hinton agar medium
(protuced by Difco); the MIC against anaerobic bacteria
wa6 determined using a GAM agar medium (produced by
Nis6ui). The results are shown in Tables 1 and 2.
12 ~3~86~
TABLE 1
Test strain MIC (~g/ml)
Chloeopolysporin
B C
_ _ . .
Staphylococcus aureus FDA Z09P JC-l 1.56 1.56
StaphYlococcu6 aureu~ SANK 70175 3.13 1.56
Staphylococcus aureus Smith 12.5 6.25
StaPhylococcu6
ePidermidis SANX 71575 3.13 3.13
Enterococcu6 faecalis SANK 71778 1.56 1.56
Bacillu8 subtili6 PCI 219 0.78 0.78
.
MYcobacterium ~meqmatis ATCC 607 25.0 12.5
Escherichia coli NIHJ JC-2 >100 >100
Klebsiella pneumonlae PCI 602 >100 >100
Pseudomonas aeruaino6a NCTC 10490 >100 >100
Serratia marcescens SANK 73060 >100 >100
Proteus mirabili_ SANK 70g61 >100 >100
. . _ . . _ . _
TABLE 2
~est strain MIC (~g/ml)
Chloropoly6porin
B C
Bacteroides fra~ilis >100 >100
Eubacterium cvlindroides 6.25 3.13
Fusobacterium necroDhorum >100 >100
PeDtococcus a6accharolvticu6 6.25 3.13
PeDtostreptocQccus Parvulus 0.78 0.39
Propionibacterium acnes 0.78 0.39
Clo~tridium ~Ymbiosum 1.56 0.39
Clostridium ramosum 1.56 1.56
Clos~ridium perfrinqens 0.20 0.10
Clostridium difficile 0.78 0.39
. _ . _ _ ~, ., __ ....... _
13 ~318~
From the results given in the above Table6, it can be
seen that chloropoly~porins B and C are effective against
aerobic Gram-po6itive bacteria, such as Sta~hvlococcu~
aureus, StaphYlococcu6 eDidermidis, Enterococcus faecali6,
Bacillu~ 6ubtili6 and MYcobacterium &meamatis, and against
anaerobic Gram-positive bacteria, such as Eubacterium
cYlindroides, PePtococcus asaccharolvticus,
ProPionibacterium acnes, Clostridium 6vmbio6um,
Cl06tridium Perfrinaen6 and Clostridium difficile.
CompariEon of the propertie6, chemical, physical and
biological, given above of chloropoly6porin6 B and C with
those of known antibiotic6 leads to the conclusion that
they belong to the class of glycopeptide antibiotics
containing chlorine, such a6 vancomycin, avoparcin a and
~, actinoidins A and B or A-35512 B. However,
chloropolysporin6 B and C can be clearly di6tingui6hed
from these ~nown antibiotics on the basis of the
differences shown in the following Table 3. Specifically,
they have different neutral saccharide components and
different amino acids are produced on acid hydrolysis.
Moreover, they move a different distance on high voltage
paper electrophoresi6 (HVPE 3300 volts~60 cm, 1 hour, pH
7.5, in 0.1 M TRIS-hydrochloric acid buffer solution), and
they have different chlorine contents.
1318630
14
TABLE 3
Antibiotic Neutral Amino acid Distance Chlorine
saccha- content
ride . (%)
Vancomycin Glucose A~partic acid 4.89
N-methylleucine
Avoparcin a Glucose, 4-Hydroxyphenyl- 9.4cm 1.85
Mannose, glycine, N-methyl-
Rhamnose P-hydroxyphen
qlYcine
Avoparcin ~ Gluco6e, 3-Chloro-4-hydroxy- 9.4cm 3.65
Manno6e, phenylglycine, N-
Rhamno~e methyl-~-hydroxy-
_ phenYl~lvcine
Actinoidin A Glucose, 4-Hydroxyphenyl- 2.02
Manno6e glycine, Phenyl-
alanine
~ .
Actinoidin B Glucose, 2-Chloro-3-hydroxy- 3.96
Manno~e, phenylglycine,
Phenvlalanine
A-35512B Gluco8e, 1.82
Manno6e,
Rhamnose,
Fuco~e
Actaplanin Glucose, 1.96
Mannose
Rhamno6e
Ri~tocetin A Gluco6e, 0
Manno6e
Rhamnose
Arabinose _ _ _ _
Ristocetin B Gluco~e,
Mannose,
Rhamnose _
1 31~;3~
TABLE ~ (Cont'd)
Chloro~oly- Glucose, 3-Chloro-4- 4 cm 5.11
spoein B Mannose, hydroxyphenyl-
Rhamno~e glycine, N-methyl-
~-hydroxyphenyl-
qlvcine
Chloropoly- Glucose, 3-chloro-s- 5.62
sporin C Mannose hydroxyphenyl-
glycine, N-
methyl-~-hydroxy-
Dhenvl~lYcine
The value reported above a6 ~'Di6tance~' is the di6tance
of movement on high voltage paper electrophorasis,
measured u6ing bioautography with Bacillus subtilis PCI
219 as the te6t organi6m.
From the above finding6, it can be seen that
chloropolysporins B and C can be used as antibiotic6
against various diseases caused by bacterial infections.
The route of administration can vary widely and may be
parenteral (e.g. by ~ubcutaneous, intravenou~ or
intramu~cular in~ection or by suppository) or oral (in
which case it may be in the form of a tablet, capsule,
powder or granule). The do6e will, of course, vary with
the nature of the disea6e to be treated, the age,
condition and body weight of the patient and the route and
time of administration; however, for an adult human
patient, a daily dose of from 0.1 to 1.0 grams is
preferred and thi6 may be administered in a single do~e or
in divided dose6.
Moreover, in view of the strong activity of
chloropolysporins B and C against infectious bacteria of
the genus Cl06tridium, they can be expec~ed to be valuable
growth-promoting agent~ for veterinary use. Bacteria of
16 13~
the genu~ Cl06tridium, particularly Clostridium
perfrin~en6 and CloGtridium difficile, are often present
in the intestine6 of farm animal6 and are the cause of
diarrhoea. Since chloropolysporins B and C have a strong
activity against 6uch microorganisms, they would 6uppre66
the growth of such microorgani6m6 in the intestines and
thu6 impro~e the microbial balance of the intestines.
This, in turn, would improve feed efficiency, thus
contributing to weight gain and improved milk production
in variou6 farm animal6, including ruminants, pigs and
poultry. Moreover, chloropoly6porins B and C, in common
with the other glycopeptide antibiotics, are likely to
have a low rate of absorption through the digastive organs
and, as a result, where the chloropolysporin B or C i6
administered in the feed, little will remain in the animal
body and hence in animal product6, such meat, milk or
eggs. When the chloropolysporin B or C is used as a
growth-promoting agent for animals, it is preferably
administered orally. ~lthough it may be formulated into
an edible compo6ition with any suitable carrier or
diluent, it is particularly convenient to administer it in
admixture with an animal feed or with drinking water.
When the chloropolysporin B or C i6 u6ed a6 a feed
additive, it may be mixed alone with the feed or it may be
mixed in combination with other non-toxic edible
excipient6, nutrient 6upplement6 (e.g. vitamins, mineral6
or amino-acids), other antibiotic6, anticoccidial agents
or enzyme6. For admini6tration to animal6 as a
growth-promoting agent, the chloropoly6porin B or C need
not nece66arily be in a completely purified form and it
may be used in a crude or partially purified form, a6
obtained at any de6ired stage during the extraction and
purification de6cribed above. For use as a
growth-promoting agent, chloropoly6porin ~ or C is
preferably employ~d in an amount of from 1 to 200, more
preferably from 5 to 60, ppm by weight on the basis of the
feed, drinking water or other carrier to which it is
1 3 ~
17
added; where an impure ~orm of chloropolysporin B or C is
employed, a concentration having equivalent activity is
used.
~ nimal~ to which chloropolyspo~in B or C can be
administered include farm mammals (e.g. cattle, horse6,
swine, ~heep and goat6), poultry (e.g. chickens, turkey~
and ducks) and pet animals (e.g. dogs, cats and birds).
Mo6t 6ignificantly, when chloropolysporin B or C i~
admini6tered orally to animal~, their growth is
effectively promoted, but it i6 little ab~orbed from the
gastro-inte6~inal tract and it exhibits low retention in
animal tis6ues: thus, there iB an almos~ complete ab6ence
of chloropoly6porin B or C residue6 in the products (e.g.
milk, meat or eggs) of animal6 to which it ha6 been
administered, which is a great advan~age from the view
point of food hygiene.
Chloropoly6porins B and C are produced by the
cultivation of a MicroPolyspora strain herein identified
as MicroDolvsPora 6p. SANK 60983, which was i~olated from
a ~oil sample collected in Tochigi Prefecture, Japan.
The microorganism, MicroPolvspora sp. SANK 60983, has
the characteristics de6cribed hereafter and i6 as
described in Europsan Patent Application No. 8~304765.5
(publication ~P-A-132349), referred to above. These
characteristics were determined by cultivation on various
media prescribed by the ISP (International StreptomyceE
Project) or with the media recommended by S.A. Waksman in
Volume 2 of ~The Actinomycete6", in all caRes at a
temperature of 28C.
l. MorPholoaical Charac~eristics
Strain SANK ~0983 grows relatively well on variou6
media. The aerial mycelium i6 hardly visible on almost
18 ~318~3~
all media but may occasionally be vi6ible on
glycerol-asparagine agar or cn potato extract-carrot
extract agar. The aerial and vege~ative mycelia bear, at
the top and the middle, short chains of spore6, normallY
from 1 to 20, al~hough occasionally more than 20, ~pore&.
No distinct fragmentation of the hyphae i6 observed with
the strain, although it may be ob6erved during later
stages of the cul~ure.
2. Culture Characteri6tic~
Strain SANK 60983 can produce pale yellow,
yellowish-brown or yellowish-gray colors. Aerial hyphae
are not ob6erved on mo~t media, although white aerial
hyphae are produced on ~ome media. No 601uble pigment iB
produced. Table 4 shows the resul~6 obtained after
cultivation for 1~ day~ at Z8C on variou6 6tandard
culture media. The color name6 and number6 u6ed were
as6igned according to the ~Guide to Color Standard~, a
manual published by Nippon Shiki6ai Kenkyusho, Tokyo,
Japan.
lg 13~8630
TA8LE 4
_ .,
~edium Growth Aerial ~ever~e Soluble
MYcelium Piqment
Yea6t Abundant, None Yellowi~h- None
extract- raised, brown
malt wrinkled, (8-8-8
extract yellowish-
agar brown
(ISP 2) (8-8-8)
Oatmeal Good, None Dull None
agar smooth, yellow
(ISP 3) dull (8-8-9)
yellow
(8-8-9)
.
Inorganic Abundant, ~one Yellowish- None
salt- smooth, gray (2-9-10)
starch yellowi6h- to pale
agar gray yellowi6h-
(ISP g) (2-9-10) brown
to pale . (6-8-9)
yellowi6h-
brown
~6-8-9)
Glycerol- Good, Poor, Yellowi6h- Nohe
a~paragine wrinkled, white brown
agar yellowish- (2-9-10)
(ISP 5) brown
(2-9-10)
~31~63~
TABLE 4 (Cont~d)
Medium Growth Aerial Reverse Soluble
Mvcelium Piament
Peptone- Moderate, None Pale None
yeast smooth, yellowish-
extract- pale brown
iron agar yellowish-
(ISP ~) brown (4-8-9)
(2-8-9)
, _ _
Tyrosine Abundant, None Dull None
agar raised, yellow
(ISP 7) wrinkled, (10-8-~)
pale
yellowi6h-
brown
(14-8-9)
Sucrose Abundant, None Pale None
nitrate raised, yellowi6h-
agar wrinkled, brown
pale (4-8-8)
yellow
(12-8-10)
Glucose- Moderate None Yellowi6h- None
asparagine smooth, gray
agar yellowish- (2-9-10)
gray
(2-9-10)
6 3 ~
21
TABLE ~ ~Cont'd)
_ _ _ _ .
Medium Growth Aerial Rever~e Soluble
Mvcelium P ament
Nutrient Modera~e None Pale None
agar 6mooth, yellowi~h-
(Difco) ~ale brown
yellowi~h- (6-8-9)
brown
(6-8-9)
Water Poor, None Yellowi~h- None
agar 6mooth, gray
yellowi6h- (1-9-10)
gray
(1-9-10)
Potato Moderate Poor, Yellowish- None
extract- gmooth, white gray
carrot yellowi6h- (2-9-l0)
extract gray
agar (2-9-l0)
._
3._ PHYSIOLOGICAL PROP~RTIES.
The ehy6iological eropertie~ of ~train SANK
60983 are shown in Table 5.
TABLE 5
Decompo~ition: ~denine
Ca6ein
Xanthine
Hypoxanthine +
Urea +
22 ~318630
TABLE S (Cont'd)
Hydrolyfiis of starch +
Liquefac~ion of gelatin +
Coagulation of milk
Peptonization of milk
Reduc~ion of nitrate +
Secretion of
deoxyribonuclea6e +
Melanin formation: ISP 1
ISP 6
ISP 7
Acid production from:
Sodium Acetate
Sodium Succinate
Sodium Citrate
Sodium Pyruvate
Sodium Tartarate
D-Gluco6Q +
L-Arabinose +
D-Xylose +
Ino6itol +
D-Mannitol +
D-Fructo6e +
L-Rhamno6e +
Sucro6e
Raffino6e +
23 13 1 8~3 0
TABLE 5 (Cont_~d)
. ~
Utilization of D-Glucose +
carbon 60urce6: L-Ar~binose
~-~ylo~e +
Inositol +
D-Manrlitol +
D-Fructose
L-Rhamnose +
Sucrose
Raffinose +
Growth in NaCl: 3% w/v
5~ w/v +
7% w/~ +
10% w/v
Range of growth 10C
temperature: 20C +
28C +
37C +
45C
In the above Table, "+" means po6itive, ~'-" means
negative and "+" means B lightly po 6 itive.
Although coagulation and peptonization of milk are
both reported as negative, they may occasionally turn
positive after long-term culture.
4. Whole Cell ComPOnent6
Acid hydrolyzates of bac~erial cells were as&ayed by
paper chromatography, u~ing the me~hod of M.P. Lechevalier
et al. ~I~The Actinomycetes Taxonomy~!, page 225 (1980)].
meso-Diaminopimelic acid, arabinose and galacto~e were
24 ~3 i 863 a
found to be present in ~he cell walls, which are thus of
Type IV, whilst the whole cell 6ugar pattern i6 of Type
A The characteristic acyl group of the cell wall was
also investigated by the method of Uchida et al. ~J. Gen.
Appl. Microbiol, 23,2~9 (1977)] and found to be ~he acetyl
group.
None of the known genera of actinomycete~ has been
reported to be capable of forming 6pores in the middla of
the hyphae. However, from a comparison of other
characteri6tics, the new 6train i~ clearly related to the
genera ActinoDolv6Dora, SaccharoPolys~ora, Pseudonocardia
and MicroDolv6~0ra. However, both ActinoPolY6Pora and
Saccharop_lyspora allow spores to grow only on the tips of
aerial hyphae, and the for~er is a highly halophilic
genus, whilst the characteri6tic acyl group of the cell
wall of the latter i6 the glycolyl group. For the6e
reason6, the new strain SANK 60983 cannot be assigned to
either of the6e genera. Although strains of the genu6
Pseudonocardia can grow spores on the aerial hyphae and on
the vegetative mycelium, a6 does 6train SANK 60983, the
6ite of it6 growth takes place only at the tip of the
hyphae and, moreover, lt6 hyphae characteri6tically grow
by budding thus, strain SANK 60983 cannot be as6igned to
the genus P6eudonocardia.
The only difference between the genus MicroDol~sPora
and 6train SANK 60983 is that the site of growth of spores
of MicroDoly~ora iB limited to the tips of the hyphae,
whereas that of SANK 60983 iB at both the tip and the
middle of the byphae.
At the present time, when there ha~ been virtu~lly no
discu6sion in learned circles as to the implication~ for
taxonomy of differences of thi6 type, it would seem
inappropriate to differentiata between genera solely on
the basis of difference6 in the si~e of growth of their
13~
spores. Accordingly, it seems most sati6factory to regard
the strain SANK 60983 as repre6entative of a new species
of the genu6 MicropolYs~ora and it h~s, accordingly, been
named MicropolYspora 6p. SANK 60983. It should, however,
be remembered that a~ignment of a strain of microorgani6m
to any particular species, genus or even family i8 largely
a matter of consen~us amongst tho~e experienced in the
study of ~he particular class of microorgani~m involved
and ~he original a6signment of a microorganism can be, and
not infrequently i8, changed after wider discus6ion.
The strain SANK 60983 ha~ been depo~ited with the
Fermentation Re6earch Institute, Agency of Industrial
Science and Technology, Ministry of International Trade
and Industry, Japan, on 10th March 1983 under the
acces6ion No. FER~ P-6985 and was re-deposited in
accordance with the conditions stipulated by the Budapest
Treaty with said Fermentation Research In6titute on 4th
June 1984 under the acces6ion No. FER~ BP-538.
I~ has been established that strain SANK 60983
produces chloropoly6porin6 B and C. However, as i6 well
known, the properties of microorganisms falling within the
general category of the actinomycete6 can vary
considerably and ~uch microorganisms can readily undergo
mutation, both through natural cause6 and as the re~ult of
induction by artificial means. Accordingly, other
microorqanisms which can be clas~ified within the genus
Micropolvspora and which 6hare with the 6train SANK 60983
the characteristic ability to produce chloropoly6porins B
and C can al60 be used to produce the 6tarting material
for the process of the present invention.
The cultivation of microorganisms of the genus
MicroPoly6pora to produce chloropoly6porin B and C can be
performed under conditions conventionally employed for the
cultivation of actinomycetes ~pecies, preferably in a
1318~3~
26
liquid culture, and de6irably with 6hakin~ or stirring and
aeration. The nutrient medium u6ed for the cultivation i6
completely conventional and contain6 such confitituents as
are commonly u6ed in the cultivation of the
actinomyce~es. Specifically, the medium should contain an
assimilable carbon source, sui~able example of which
include gluco~e, maltose, sucro6e, mannitol, molasses,
glycerol, dextein, starch, soybean oil and cottonseed oil;
an assimilable nitrogen ource, suitable examples of which
include soybean meal, peanut meal, cottonseed meal, fish
meal, corn steep liquor, peptone, meat extract, pres6ed
yeast, yeas~ extract, sodium nitrate, ammonium ni~rate or
ammonium sulfate: and one or more inorganic salts, ~uch a6
sodium chloride, pho6phates, calcium carbonate and ~race
metal 6alt6. Where cultivation is effected in a liquid
medium, it i8 generally desirable to incorporate an
anti-foaming agen~ (for example silicone oil, vegetable
oil or a suitable surfactant) in the medium,
The cultivation is suitably performed a~ a
sub~tantially neutral pH value and at a temperature of
from 2~ to 30C, more preferably at about 28C.
The production of chloropoly6porin6 B and C as
cultivation proceed6 may be monitored by a variety of
conventional microbiological assay techniques for
monitoring the production of antibiotic~ (when they are
produced by microbial culture) and which require little or
no elaboration here. A suitable technique might be the
paper disc-agar diffusion a66ay (using, for example, a
paper di6c of diameter about 8mm produced by Toyo Kagaku
Sangyo Co.f Ltd) and using, for example, Bacillu~ subtili~
PCI 219 or StaPhylococcus aureu6 FDA 209P JC-l as tbe ~e~t
organism.
The amount of chloropolysporins B and C produced
normally reaches a maximum after cultivation has proceeded
27 ~ ~1 8~ ~Q
for 55-70 hour6 and i~ is clearly desirable to separate
the chloropolysporin~ from the culture medium no later
than the time when this maximum ha6 been reached.
However, this period may vary, depending upon the
cultivation conditiong and technique~, and a shorter or
longer period may be appropriate, depending upon the
circum6tance~. The correct cultivation time may readily
be a~6es6ed for every ca6e by routine experiment, u6ing
suitable monitoring techniques, e.g. as described above.
Chloropolysporin6 B and C are mainly relea6ed into the
liquid portion of the cultured broth and can thu6 be
eecovered by removing solid matter, including the
mycelium, for example by filtration (preferably u6ing a
filter aid such a6 diatomaceou6 earth) or by
centrifugation. It can then be recovered from the
separated liquid portion by conventional technique6 and,
if desired, then purified.
Chloropolysporin6 B and C are preferably 6eparated
from other products in said liquid portion by mean6 of an
ad60rbent, either by ad60rbing the impuritie6 or by
adsorbing the chloropoly6porins or by ad60rbing both
separately or together and then eluting the
chloropolycporins. A wide range of ad60rbQnt6 may be
u6ed; examples which we have found to be particularly
sati6factory include: activated carbon; and adsorbing
re6ins such a~ Amberlite (registered trade mark) XAD-2,
XAD-4 or XAD-7 (products of Rohm and Haa6), Diaion
(regi~tered trade mark) ~P 10, HP 20, CHP 20P or HP 50
(products of Mitsubishi Chemical Industries Co., Ltd.) and
Polyamide gels (a product of Woelm Pharma, ~est Germany~.
The impuritie~ pre6ent in the liquid por~ion may be
removed by pa6~ir.g the solution containing the
chloropolysporin~ through a layer or column of one or more
of the aforemen~ioned adsorbent6 or by ad~or~ing the
chloropolysporin~ on one or more of the adsorbents and
2~ 13~8~30
then eluting the chloropolysporin6, either separately or
together, with a 6uitable eluent. Suitable eluents
include mixture6 of methanol, acetone or bu~anol with
water.
The chloropoly6porins B and C thu~ obtained may be
further purified by variou~ means. Suitable method~
include: partition column chromatography u~ing a cellulo~e
product, such a6 Avicel (a regi6tered trade mark for a
~roduct of A~ahi Chemical Industry Co., Ltd.) or Sephadex
LH-20 (a regi6tered trade mark for a product of Pharmacia,
Sweden); reverse pha~e column chroma~o~raphy u~ing a
carrier for the rever6e pha~e; extraction based on the
di~ferences in di~tribution in solvents between
chloropolysporins B and C and their contaminating
impurities; or the counter-current distribution method.
These purification techniques may be used singly or in
combination and may, if needed, be repeated one or more
times.
If de~ired, chloropolysporin~ B and C can be separated
from each other by chromatography. A preferred 6y6tem for
this purpoBe i8 Sy6tem 500 (a product of Waters Co.),
using the Preppack C18~cartri~ é. A suitable eluent is
a buffered mixture containing acetonitrile and maintained
at a slightly acidic pH value.
Depending upon the culture condition~,
chloropolysporins B and C can exist in the mycelium from
the cul~ure broth and can be extracted therefrom by
conventional techniques. For example, ~hey can be
ex~racted with a hydrophilic organic solvent (such a6 an
alcohol or acetone), and then the solvent removed from the
exteact to leave a re6idue, which i6 dis601ved in an
aqueous medium. The chloropQlysporins can be extracted
from ~he resulting 601ution and purifie~ as de6cribed
above.
29 1318630
Chloropoly~porin~ B and C thu6 obtained have, a~ ~heir
sulfates, the phy~ical and chemical propertie6 described
above. They are normally and preferably 6eparated from
the culture bro~h in the form of a water-601uble sal~ and
are most conveniently charac~eri~ed in the form of such a
salt, i.e., as herein, in the form of the sulfate, since
chloropolysporin~ B and C them6elves (i.e. the free ba6es)
are insoluble in water.
Where the chloropolysporin B or C i6 isolated in the
f~rm of a salt, it may be converted to the free base by
conventional mean~, such a6 the use of ion-exchange resins
or of adsorbents for reverse phase chromatography. An
aqueous solution of a salt will normally have an acidic pH
value: adjustment of this pH value to approximate
neutrality will result in mainly preciei~ation of the free
ba~e, which may then be collected by suitable means, e.g.
filtration or centrifugation. This product will, however,
normally be contaminated by impurities, including minor
proportion6 of the relevant salt , and will, therefore,
normally require further purification. Accordingly, a
more preferred method is by using, for example, a suitable
ion-exchange resin or an adsorbent for reverse phase
chromatography. These compounds, however, ~hare wi~h
known glycopeptide antibiotics, such as avoparcin, the
property of being very difficult to isolate in the form of
the ~ree base [see e.g. W.~. McGahren et al., Journal of
Antibiotics, XXXVI, 12, 1671 (1983)] and they are,
accordingly, preferably isolated and employed in salt form.
The invention is further illustrated by the following
non-limiting Examples, which describe the production of
chloropolysporin C by enzymatic hydrolysi6 of
chloropolysporin B, and by the following Preparation which
describes ~he production of the starting material by
microbial culture.
~3~86~
EXAMPLE 1
3.8 g of ~he enzyme naringina6e (product of Sankyo Co.
Ltd.) were added to a solution of 10.5 g of
chloropolysporin B in 2 litres of 0.06M aqueous pho~phate
buffer (pH 5.8), and the mixture was allowed to react for
21 hours at 37C under gentle stirring, until the
chloropolyspor}n B ~tar~ing material could no longer be
detected by HPLC.
After completion of the reacton, the reaction mixture
wa~ pas6ed through a column containing 370 ml of Diaion
HP20 ad~orbent resin (product of Mitsubi~hi Chemical
Indu6trie6 Ltd.), and the column was then wa6hed with
water and eluted with 700 ml of 50~ v/v aqueous acetone.
The solvent was evaporated off from the eluate, under
reduced pre6sure, and the residue was lyophilized giving
7.8 q of crude product, which was then purified by the
following procedure.
The whole of the crude product was dissolved in 30 ml
of 50% v/v aqueous methanol, and the solution was ad60rbed
r c,~;/e".~, rfc ,~ " GI
on a 3 x 50 cm column of Toyopearl HW-~OF resln ta~product
of Toyo Soda Co. Ltd.~. The column was developed and
eluted with 50% v/v aqueous methanol. The eluate was
collected in fractions of 20 ml each, and the fraction~ in
which chloropolysporin C was detected by HPLC were
selected. These fractions were combined and the solvent
was distilled off from them under reduced pres6ure. The
re6idue was acidified to pH 4.0 with hydrochloric acid and
lyophilized, yielding 5.6 g of chloropolysporin C
hydrochloride.
EXAMPLE 2
200 mg of the enzyme 6clase (product of Sankyo Co.
Ltd.) were added to a solution of 10 mg of
31 ~318~3~
chlo~opoly~po~in B in 5 ml of o.lM ~queou~ phosphate
buffer, and the mixture was allowed to react for about 24
hour~ at room t~mperature, under gentle stirring After
completion of the reaction, the mixtu~e wa6 ad60rbed on~o
a column containing 5 ml of Diaion HP 20 re6in ~product of
Mit6ubishi Chemical Indu6trie6 Ltd.), and the column wa6
then wa6hed with water and eluted with 50~ v/v aqueou6
acetone. The fractions of eluate containing
chloropoly6porin C were conden~ed under reduced pres6ure
and the re~idue was lyophilized, giving 5 mg of crude
product. The crude product may be purified by the
following procedure.
8 g of crude product containing chloropoly6porin C was
dis601ved in 40 ml of water and the 601ution adju6ted to
pH 3.0 with dilute hydrochloric acid. Thi6 solution was
ad60rbed onto a 4 x 40 cm column of Polyamide gel (product
of Woelm GmbH ~ Co.), which wa6 developed with water, and
fractions of 20 ml each were collected. The fraction6
containing chloropoly6porin C (as detected by HPLC) were
combined and conden6ed under reduced pre66ure. The
re6idue wa6 acidified to pH 4.0 with hydrochloric acid and
then lyophilized, giving 4.6 g of chloropoly6porin C
hydrochloride.
PRE~PARAT I ON
one loopful growth of MicroDolY~Dora 6p. SANK 60983
wa6 inoculated into a 500 ml Erlenmeyer flask containiag
80 ml of medium A, which ha6 the following compo6ition
(percen~age6 are by weight):
MEDIUM A
Glucose 3%
Pressed yeast 1%
Soybean meal 3~
Calcium carbonate 0.4%
Magnesium sulfate 0.2%
Anti-foaming agent (supplied under
the trademark Nissan CB-442) 0.01%
Water the balance
(adjusted to pH 7.0~
The microorganism was then cultured for 84 hours at
28C, using a rotary shaker at 220 r.p.m.
25 ml of the resulting seed culture were inoculated into
each of four 2 litre Erlenmeyer flasks, each containing 500
ml of medium B, which has the following composition
(percentages are by weight):
MEDIUM B
Glucose 5%
Yeast extract 0.1%
Soybean meal 1%
Polypepton (a trademark for a product of Daigo
Eiyo Co. Ltd., Japan) 0.4%
Beef extract 0.4%
Sodium chloride 0.25%
Calcium carbonate 0.5%
Anti-foaming agent (supplied under the
trademark Nissan CB-442) 0.01%
Water the balance
(adjusted to pH 7.2)
The microorganism was then cultured at 28C for 24
hours, using a rotary shaker at 220 r.p.m.
32
~1863~
33
The resulting cultured broth~ were combined. 750 ml
of thiS broth were then inoculated into each o~ two 30
litre jar fermentors, each containing 15 litres of
medium B, and the microorganism was then cultured at
28C for 69 hours, whilst aerating at the ra~e of 15
li~res per minute and stirring.
At the and of this time, batches of cultured broth
separately cultivated as described above were combined
to give a total of 30 li~res of cul~ured broth. Celite
5g5 (a registered trade mark for a product of
Johns-Manville Products Corp, New Jersey, U.S.A.) filter
aid was added to the cultured broth and the mix~ure wa6
filtered, to give 30 litres o~ a filtrate. This
filtrate was adsorbed on 3 litre~ of Diaion HP 20 (a
product of Mitsubishi Chemical Industrie~ Co., Ltd.),
and the ad60rbent was wa~hed with water and then eluted
with 50~ ~/v aqueous acetone. Acetone wa6 evaporated
from the combined active fraction6 by evaporation under
reduced pre6~ure; and the concentrate thus obtained was
lyophilized, giving 44 g of a crude powder.
41 g of this powder were dissolved in water and
adsorbed onto 1.8 litres of Diaion HP 20, wa~hed with 5
litre6 of water and 2 litres of 10~ v/v aqueous acetone,
and ehen eluted with 4 litres of 50~ v/v aqueou6
acetone. The active fraction~ from the elution were
collected and condensed to a volume of 1 litre by
evaporation under reduced pressure. The condensate was
centrifuged at 5000 r.p.m. and the resulting precipitate
was dried, to give 9.6 g of crude powder containing
chloropolysporin~ B and C.
This crude powder was dis601ved in 1 litre of 50
v/v aqueous methanol and then adsorbed on~o Z00 ml of
acidic alumina (a product of Woelm Pharma, West
Germany), which had previously been equilibrated with
34 ~ 63 ~
50% v/v aqueous methanol. The ad~orbed product was then
eluted with the same solvent, and ~:he active fraction6,
a total of 1.1 litres, were collected. The combined
active fractions were pas6ed through 60 ml of Dowex 21 K
(OH ~, and eluted with water. The active fractions
from this elution, a ~o~al ~olume of 1.2 li~res, were
collected and ~hen condensed by evaporation under
reduced pressure to a volume of 30 ml, This condensa~e
was lyophilized, to give 1.23 g of powder. The powder
wa~ dissolved in aqueous hydrochloric acid of pH 4.0 and
then adsorbed onto 56 g of Polyamide filled wi~h water
(a product of Woelm Pharma, ~est Germany). This was
subjected to gradient elution with 400 ~1 of water and
1.2 litres of methanol, in 20 ml fractions, up to
raction 80. Fractions 30-60 were collected and
combined. The methanol was distilled off under reduced
pres6ure and the resulting concentrate was lyophilized,
to give 738 mg of a white powder containing
chloropoly6porins B and C.
4.4 g of this crude white powder containing
chloropolysporins B and C were dissolved in 80 ml of a
mixed solvent consi~tinq of 15 parts of acetonitrile and
85 parts of a buffer solution (containing 0.2~ wtv
sodium heptanesulfonate, 2.5~ w/v acetic aeid and 0.5%
w/v concentrated aqueous ammonia): the solution was then
ad60rbed on a Syste~ 500 chromatography system (a
product of Waters Co), using a Preppack C18
cartridge. This was developed and eluted with the same
mixed solvent as mentioned above at a flow rate of
100-150 ml per minu~e. Chloropolysporin ~ was elu~ed in
the solvent after between 800 ml and 1700 ml of the
eluant had passed ~hrough the cartridge, whils~
chloropolysporin C was eluted after between 1700 and
4700 ml of the eluant had passed.
~3~3~
The active fractions containing chloropoly6porin B
were collected and adju6ted to a pH value o~ 7Ø They
were then concentrated by evaporation under reduced
pres6ure, to di6till o~f the acetonitrile. The
re~ulting concentrated 601ution was ad~orbed on a Diaion
HP 20 column (100 ml), washed with water. and then
eluted with 500 ml of 70~ v/v aqueous ace~one. The
eluate wa~ condensed by evaporation under reduced
pre6sure, and the residue wag lyophilized to afford
chloeopolysporin B heptane6ulfonate as a powder.
200 mg of this powder were di~olved in S ml of
water, and then 1 ml of a 10~ w/v aqueou6 601ution of
sodium dodecyl~ulfate was dropped into the re6ulting
solution. The precipitate which formed was collected by
centrifugation at 3000 rpm for 10 minute6. Thi6
precipitate was su~pended in water and the su6pen6ion
was again centri~uged at 3000 rpm for 10 minute6 to wash
the precipitate. Thi6 operation was reeeated a further
three times to wash the precipitate. The precipitate
was then dissolved in 3 ml of methanol and the insoluble
residue was filtered off. 2 ml of a 0.5M methanolic
solution of teiethylamine 6ulfate were added dropwise
and the re6ulting precipitate wa6 collected by
centrifugation at 3000 rpm for 10 minute~. This
precipitate was su6pended in a small amount of methanol
and again centrifuged at 3000 rpm for 10 minute6. This
wa6 repeated a further three times to wa6h the
precipitate. The precipitate wa6 then dis601ved in 1.5
ml of water and the in601uble residue wa~ filtered off.
Lyophilization of the fil~rate gave 65 mg of
chloropolysporin B ~ulfate.
