Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a process for preparing and
purifying thymidine phosphorylase of bacterial origin and to a
preparation of purified thymidine phosphorylase.
This application is a division of Canadian Patent
Application S.N. 244,671, filed January 27, 1976.
The invention has particular application in the field
of culture media for microbes and in particular culture media
used in testing for the susceptibility of bacterial to anti-folate
anti-microbial agents such as sulphamethoxazole (SMX) and/or
trimethoprim (TMP).
It has been known for a number of years that culture
J`. media in common use are often unsuitable for determining
sensitivity of bacteria to sulphonamides or trimethoprim, i.e.
agents interfering with the synthesis of folates in these
organisms. This unsuitability manifests itself by giving long
tailing end-points when the serial dilution method is used and
~ by partial growth within the inhibition zones when the diffusion
5/" method is employed, It has been shown by Bushby (Med. J, Aust.Special Supplement (1973) 1: 10) and Koch and Burchall (Applied
Microbiology (1971) 22: 812) that thymidine is a very potent
' reversing agent of the inhibiting activities of sulphonamides
and trimethoprim.
In 1945 Harper and Cawston (J. Path. Bact., 57: 59)
, showed that when lysed horse blood was added to a poor sus-
ceptibility test medium, it could convert this into a satis-
factory one. Since this early work, and that of several other
workers, it has become common practice to include lysed horse
blood in antibacterial susceptibility test media, in order to
reduce the partial growth often observed within the inhibition
zones produced by sulphonamides. More recently this method has
also been shown to be similarly effective in testing with respec~
to trimethoprim (Bushby, Postqraduate Med. J. (1969) 45: 10,
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Harper and Cawston established that a) lysed horse
blood was more effective than whole blood in neutralizing
sulphonamide-antagonizing substance(s), b) blood of several
other species was inactive, c) the activity of the lysate
increased with incubation time and temperature (up to 30) and
d) the lysate did not affect the reversal of sulphonamide-
inhibition by p-aminobenzoic acid. They concluded that the
lysed blood contained a factor which neutralizes sulphonamide-
antagonizing substance(s).
This so-called Harper-Cawston Factor is effective only
with media which contain a moderate level of thymidine, i.e.
from about 0.1 to 15 ~g/ml. Below 0.1 ~g/ml the activity of
; the drugs is not antagonized, thus, removal of such a small
amount of thymidine has no effect on the drug inhibition observed.
At very high levels of thymidine, i.e. greater than 15 ~g/ml,
the activity of Harper-Cawston Factor is not sufficient to
overcome the reversal of the activities of the sulphonamides and
trimethoprim, possibly because the high concentration of thymine,
produced as a result of the cleavage of thymidine, can replace
the much more active thymidine in the reversal.
The Harper-Cawston Factor has been reported to be
thymidine phosphorylase Bushby in Trimethoprim Sulfamethoxazole
in Bacterial Infections: A Wellcome Foundation SYmposium. Ed ;~
Bernstein & Salter, Churchill Livingston, Edinburgh & London,
1973, p. 31-38, and(Bushby, Med. J. Aust. Special Sup~lement,
1973, 1: 10-18). It has been pointed out in the latter
reference that "although thymidine interferes with the in vitro
activity of TMP/SMX, it is not usually present in animals in
sufficiently high concentrations to affect the in vivo activity."
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One disadvantage of including lysed horse blood in a
culture medium is that it imparts a reddish brown color to the
medium; the greater the amount of horse blood, the deeper the
color. This coloration is undesirable since it greatly inter-
; feres with the assessment of bacterial growth after incubation.
For example, in the case of fluid media the increased color
decreases the transparency of the media making optical density
measurements far less accurate. With solid media there is a
decrease in contrast of the agar making it more difficult to
precisely measure inhibition zone sizes and to determine thepresence or absence of partial growth within the zones of
' inhibition. Furthermore the requirement of the addition of lysed
horse blood to bacterial culture media means that the media
are virtually impossible to define, a somewhat undesirable
` characteristic. A further disadvantage of using horse blood is
that it is commercially available in very limited supply and
from only a very few suppliers world-wide.
,~ It has recently been found that the addition of the
isolated and purified enzyme thymidine phosphorylase of bacterial
origin, to a wide variety of commonly used growth media, improves
those media for susceptibility testing of bacteria to anti-
folate drugs.
There is thus provided a composition for testing the
susceptibility of bacteria to anti-folate drugs which comprises
a bacterial growth medium in combination with purified thymidine
phosphorylase of bacterial origin.
Addition of the enzyme thymidine phosphorylase improves
many media for susceptibility testing of bacteria to anti-folate
drugs, such as sulphamethoxazole and trimethoprim, for example
Mueller-Hinton Broth and Agar, Oxoid Sensitivity Test Broth and
Agar, Wellcotest Sensitivity Test Agar, Brain Heart Infusion
Broth and Agar, and Typtone Soya Agar.
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Thymidine phosphorylase has previously been purified
from bacteria such as Salmonella tvphimurium, Bacillus cereus,
Bacillus stearothermophilus and Haemophilus influenzae and
particularly from a strain of Escherichia coli requiring thymine
and methionine for growth. This latter purification involved
an extremely lengthy process consisting of precipitation,
- fractionation, several chromotographic steps and dialysis
(Schwartz, M., 1971, Eur. J. Biochem. 21: 191-198). The enzyme
preparation recovered from this process, however, was only 25-
fold purer than the crude cell extract.
It has recently been found that certain strains of
bacteria, in particular a certain strain of E, coli, produce
inordinate amounts of thymidine phosphorylase under appropriate
growth conditiGns and that this enzyme may be isolated and
i~ purified by applying the cell extract to specific adsorbents
; and eluting it therefrom to give a much higher yield of a
much purer preparation than has heretofore been achieved.
Moreover, this new isolation/purification procedure is
adaptable to large scale production of the enzyme.
According to the invention there is provided a process
for preparing purified thymidine phosphorylase of bacterial
origin, which process comprises the steps of extracting the
crude enzyme from bacteria into an aqueous medium, and
cubjecting the extract to a fractionation procedure which
includes adsorption, chromatographic and dialysis steps. This
process is characterized in that 1) a strain of bacteria is
selected which has the capability of producing high concentrat-
ions of thymidine phosphorylase under appropriate growth
conditions and 2) the crude cell extract obtained therefrom
is applied to a calcium phosphate gel absorbent containing sub-
stantially equivalent amounts of Ca and P04_, preferably as
a first step in purification.
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In particular the process comprises the steps of
extracting the crude enzyme from a culture of Escherichia coli
into an aqueous medium and purifying the extract of the crude
enzyme by subjecting it to a fractionation procedure which
includes, as a first step, adsorption onto a calcium phosphate
gel containing substantially equivalent amounts of calcium and
phosphate ions in the ratio of 3 : 2 respectively, followed by
elution therefrom and the subsequent further purification by
column chromatography and dialysis.
' 10 Preferably, the eluant is subsequently contacted with
DEAE cellulose and/or cellulose-epichlorhydrin triethanolamine
(ECTEOLA-cellulose), with dialysis against water or a suitable
buffer being carried out after elution from DEAE-cellulose and
before adsorption to E~TEOLA-cellulose. -
Escherichia coli B-96 (ATCC 13473) is eminently
advanta~eous for the purposes of the present invention.
Salmonella typhimurium LT-2 (ATCC 15277) is an example of
another strain of bacteria which produces large amounts of
thymidine phosphorylase and thus i9 useful in the practice
of this invention. In certain cases, where a more thermo-
stable enzyme is desirable, the enzyme isolated from B. stearo-
thermophilus has proven effective.
The E. coli strain ATCC 13473 may be cultured in a
minimal saltæ medium containing a suitable carbon source and
additional purines. Alternatively the bacteria may be cultured
in a yeast extract medium. A crude extract of the enzyme may
then be made by sonication of the bacterial cells in phosphate
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buffer followed by centrifugation to remove the cell debris.
The crude extract is for instance admixed with a
small amount of calcium phosphate gel and then centrifuged
to remove unwanted protein. The supernatent so obtained
may then be admixed with a further aliquot of calcium
phosphate gel and the enzyme absorbed thereto. The enzyme
activity may be eluted from the gel by sequential washings
with phosphate buffer. The enzyme may then be adsorbed to
DEAE-cellulose, washed and eluted therefrom. After dialysis,
the preparation may be adsorbed to ECTEOLA-cellulose and
eluted therefrom.
; Monitoring of the elutions for enzyme activity, at
all stages of the purification, may conveniently be carried
out using a spectrophotometric assay at a selected wave-
length.
me enzyme so purified can be conveniently made
available as a suspension in aqueous ammonium sulphate.
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- The enzyme is also present in a number of vertebrate
.~ tissues and can be purified therefrom, but the levels in
mammalian tissues are generally much lower than in bacteria.
Furthermore purified microbial thymidine phosphorylase is
` many times more active than the purified mammalian counter-
part.
Indeed horse blood has been found to contain about
40 to 100 units of thymidine phosphorylase activity (as
~ defined herein) per ml. The E. coli sonicate of Example 1
, 10 contains greater than 1000 times this concentration. Thus,
` the advantages of an economical method of preparing the
purified enzyme from a bacterial source are many and
significant. Not only is there a more readily available,
inexpensive source of the enzyme, but the process for
- extracting and purifying it is much more economical.
~; The concentration of thymidine phosphorylase
incorporated into the media is preferably in the range of
about 2 to 200 units of enzyme activity/ml of medium, more
preferably between 5 and 100 units/ml and most preferably
between 7 and 50 units/ml. One unit of enzyme activity of
purified enzyme is that amount of the enzyme which catalyzes ~-
the formation of one nanonole of thymine/minute ~rom a one
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`-` iO774ZO
millimolar solution of thymidine at 25C in the presence of
200 mM potassium phosphate buffer at pH 7.4.
Media, to which purified thymidine phosphorylase has
; been added, may be suitable for testing the susceptibility
to anti-folate drugs of a variety of organisms, such as
r Streptococcus pyoqenes, Staphylococcus aureus, Vibri_ comma,
Erysipelothrix rhusicpathiae, Serratia marcescens, Klebsiella
pneumoniae, Kleb. aero~Lenes, Sal. typhosa, E. coli, Shiqella
flexnerl, Shiq. dysenterlae, Enterobacter aeroqenes, Entero.
cloacae, Citrobacter freundii, Proteus vulqaris, Pr. mirabiles,
Pr. rettgeri, and Pseudomonas aeruqinosa. Strep faecalis
is an outstanding exception, because with this organism,
thymine is as effective as is thymidine in virtually reversing
the activity of inhibitors of folate reductase, e.g., tri-
methoprim.
The purified enzyme may be added to the desired
- medium at any suitable stage of manufacture or preparation.
For example, it may be added aseptically as a sterile solution
after autoclaving of the medium, and when the temperature
has dropped to about 50-55C. After the enzyme has been
added, the medium should be processed as soon as possible so
that the enzyme-treated medium is not maintained at 50-55C.
for more than about 5-10 minutes in order to minimise inact-
ivation of the enzyme.
There is also provided a method of preparing a
composition suitable for testing the susceptibility of
bacteria to antifolate drugs which comprises the admixture of
a purified preparation of thymidine phosphorylase of bacterial
origin with a bacterial growth medium.
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One particular advantage of the composition so pro-
duced is that it is light colored and transparent, which
facilitates the accurate evaluation of bacterial growth in
the determination of bacterial sensitivity to antifolate
drugs.
According to another aspect of the invention there
7 i q provided a stabilized thymidine phosphorylase preparationcontaining ammonium sulfate. The formulation of increased
stability may be a suspension of the enzyme in an a~monium -
sulfate solution.
In particular there i~ provided in accordance with
the invention a preparation of purified thymidine phosphorylase
comprising a concentrated solution of the enzyme, containing
at least 5 mg protein/ml, in phosphate buffer containing a -
stabilizing amount of ammonium sulphate.
It is known in the prior art that thymidine phos- -
phorylase of bacterial origin iss~able at -20C. but that at
4C. activity decreases at a significant rate. It has now
been found that formulations of the purified enzyme can be
made remarkably stable to decomposition by adding ammonium
sulfate to the preparation provided that the protei~ content
of the preparation is at least 5 mg protein/ml. The protein
content need not necessarily all consist of the enzyme.
Concentrated solutions of the enzyme (Smg protein/ml or greater)
in phosphate buffer containing lC% ammonium sulfate, for
, example, may be stored for long periods of time with little
or no loss of activity. Stable suspensions of thymidine
, phosphorylase in aqueous ammonium sulfate may also be prepared.
.
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The following examples illustrate the invention but
do not limit it in any way.
EXAMPLE 1 - Thymidine Phosphorylase_Purification
E. Coli B-96 (ATCC 13473) was grown in aerated
vessels at 34C in a minimal salts medium containing Na2HP04
(18.9 g/1), KH2P04 (6.3 g/l), MgSO4. 7H20 (0.2 g/l), (~H4)2S04
(2.0 g/l), adenosine (0.5 g/l), and casamino acids (8.0 g/l).
The cells were harvested by centrifugation when the optical
density of the culture at 600 nm (without dilution) reached
2Ø The following operations were carried out at 3C unless
otherwise specified. The cell paste (25 g) was suspended in
two times its weight of 5 mM potassium phosphate buffer,
pH 8.0 (Buffer A). The cell suspension was sonicated in 5
ml aliquots, each for two 12 second periods with a 50 second
cooling interval. A Branson Model 5125 Sonic Oscillator was
used at a power setting of six. The sonicates were pooled and
centrifuged for 20 minutes at 48,000 x g. To the supernatant
(Stage I - see Table I below) was slowly added 25 ml of a
calcium phosphate gel suspension (31 mg dry solid per ml,
aged at 3C for 5 months). The resulting suspension was
stirred for 10 minutes and then centrifuged at 12,000 x g for
5 minutes. The supernatant was mixed with an additional 100
ml of the calcium phosphate gel suspension, and the mixture
was stirred for l0 minutes and centrifuged at 9,700 x g for
15 minutes. The resulting gel pellet was washed with Buffer A
(100 ml) by resuspension and centrifugation at 9,700 x g for
15 minutes.
The enzyme activity was eluted from the gel pellet
by two sequential washings; the first with 10 mM potassium
phosphate buffer, pH 8.0 (l00 ml) and the second with 20 mM
potassium phosphate buffer, pH 8.0 (100 ml). The two washes
were combined (Stage II).
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The remainder of the procedure was carried out at
25C. The combined washes were applied to DEAE-cellulose
column, 1. 8 cm in diameter by 6 cm high, which was previously
equilibrated with 20 mM potassium phosphate, pH ~.4 (Buffer B).
The loaded column was washed with Buffer B (100 ml), and the
enzyme was then eluted with a linear gradient of phosphate
buffer. This gradient was prepared by adding with thorough
mixing 200 mM potassium phosphate buffer, pH 6.4 (200 ml) to
: a mixing chamber, which was originally filled with Buffer B
(200 ml), at such a rate as to maintain constant volume within
the mixing chamber. The fractions containing the highest
enzyme activity w~re pooled (Stage III) and dialyzed using a
cellulose dialyzer tubing (1/4" diameter) against water (2 1)
for 1 hour, The dialysis was repeated, and then the dialysate
was applied to an ECTEOLA-cellulose column (1. 8 X 5 cm) pre-
viously equilibrated with Buffer A. The loaded column was
washed with Buffer A (100 ml). The enzyme was then eluted
with a linear gradient eluant prepared as above using a mixing
'~ reservoir initially filled with Buffer A (200 ml) into which
200 mM potassium phosphate buffer, p~ 8.0 (200 ml) was syphosed
by gravity as the column solution proceeded, The fractions
containing enzyme activity were pooled (Stage IV) and sufficient
ammonium sulfate added to give a suspension of the enzyme in 80%
ammonium sulfate solution. This overall procedure resulted in
a 100-fold increase in specific activity with respect to
protein and the virtually complete removal of nucleic acids.
Thymidine phosphorylase activity was assayed spectro-
photometrically by measuring the decrease in absorbance at 290
nm accompanying the phosphorolysis of thymidine to thymine.
At 290 nm and pH 7.4 thymidine has a higher extinction co-
efficient than thymine (~=-480M lcm 1), The assay mixture
contained 200 mM potassium phosphate buffer, pH 7.4 and 1 mM
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10774Z0
. thymidine in a total volume of 2.5 ml. One unit of enzyme
activity is that amount which catalyzes the formation of one
nanomole of thymine per minute from thymidine at 25C.
Table I summarizes the results of the purification
described in this example. This procedure has been scaled
. up 60 fold with similar results.
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10774Z0
EXAMPLE 2 - Solid Mediu_
- Mueller-Hinton (Difco) Agar was prepared in the
normal manner and autoclaved. After the medium had been removed
from the autoclave and allowed to cool to 50-55C, a sterile
solution of purified thymidine phosphorylase as prepared in
Example 1 containing sufficient enzyme to give a final
concentration of 40 ~nits of the purified enzyme per ml of
medium, was added aseptically. The medium was thoroughly
mixed, poured into sterile Petri dishes and allowed to cool
to room temperature.
EXAMPLE 3 - Sensitivity Testing
; Mueller-Hinton Agar (wilson) and Brain-Heart Infusion
Agar (Difco) were prepared in the normal manner and autoclaved
. . .
(three batches of each). After removal from the autoclave,
one batch of each medium was poured into sterile Petri
dishes. The other two batches of each medium were allowed
to cool to 55C. To one batch of each was then added
sufficient lysed horse blood to give a final concentration
of S%, and to the final batch of each medium was added a
sterile solution of thymidine phosphorylase sufficient to
give a final concentration of 40 units/ml. Each batch was
thoroughly mixed and then poured into sterile Petri dishes. -
After cooling to room temperature, each agar plate was
, . .
- seeded with a 2 ml of a diluted (10 ) overnight broth culture
(Mueller-Hinton broth) of E. coli. Excess fluid was removed,
and the plates were allowed to dry. Filter paper discs
containing 1.25 mg TMP, 1.25 mg TMP + 23.75 mg SMX, and 23.75
mg SMX were then placed on the surface of the plates, which
were then incubated at 37C for 16 hours giving a lawn of
not quite confluent growth. The zones of inhibition were
determined and expressed as the distance from the edge of the
disc to the edge of the growth of uninhibited colonies.
Table II summarizes the results.
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; Table II: Sensitivity Testing
_ _Size of Zone in mm
Reversing T~ S~ ~ 1.2S ~g
~lediuDI Agent +
, 1.25 ~g23.75 ~gS~ 23.75 ~g
.. .
Thymidine
Phosphorylase 22(~5) 17(20) 26(31)
40 Units/ml
Brain-Heart . Lysed
Infus on Agar Y7.orse B1ood ' 14(~ 0) ~1(29)
(Difco) 5%
Nil (22) (23) (31
~ Thymidin2
Phosphorylase 24(27) 24(26) 32(36)
40 Units/mi
~ Muelier-~in~on Lysed
;; A~ar horse Blood25(28) 24(2~) 31(35)
,j ~r~;Jil sok~ 5%
~ Nil (25) (263 (35)
.
( ) Incll~des zone of partial inhibition.
:
~ueller-l7.inton agar is i7Qproved for sensitivity testino to about
the same degree by the addition of bacterial thymidine phosphoryiase as
i~ .by the addition of lysed horse blood. Ho~7ever, the enzyme improves
Brain-Heart Infusion ~o~r significantly more effectively than does lysed
h.orse biood. In addition, the virtually colorless plates p.oduced
according to this invention ~7er~ read and evaluated mu~h mo.e easily and
qu~ckly than the colored hor~e b_ood plates.
C7~L~ 15
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