EP0000071B1 - Préparation stabilisée de thymidine-phosphorylase et bouillon de culture la contenant - Google Patents

Préparation stabilisée de thymidine-phosphorylase et bouillon de culture la contenant Download PDF

Info

Publication number
EP0000071B1
EP0000071B1 EP78100132A EP78100132A EP0000071B1 EP 0000071 B1 EP0000071 B1 EP 0000071B1 EP 78100132 A EP78100132 A EP 78100132A EP 78100132 A EP78100132 A EP 78100132A EP 0000071 B1 EP0000071 B1 EP 0000071B1
Authority
EP
European Patent Office
Prior art keywords
preparation
thymidine phosphorylase
enzyme
stabilised
thymidine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP78100132A
Other languages
German (de)
English (en)
Other versions
EP0000071A1 (fr
Inventor
Thomas Anthony Krenitsky
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wellcome Foundation Ltd
Original Assignee
Wellcome Foundation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wellcome Foundation Ltd filed Critical Wellcome Foundation Ltd
Publication of EP0000071A1 publication Critical patent/EP0000071A1/fr
Application granted granted Critical
Publication of EP0000071B1 publication Critical patent/EP0000071B1/fr
Expired legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)

Definitions

  • This invention relates to a preparation of thymidine phosphorylase for incorporation into culture media used for the testing of the susceptibility of bacteria to antifolate anti-microbial agents such as sulphamethoxazole (SMX) and/or trimethoprim (TMP), in particular it relates to an improved stabilised thymidine phosphorylase preparation and a culture medium containing it.
  • SMX sulphamethoxazole
  • TMP trimethoprim
  • thymidine At very high levels of thymidine, that is greater than about 15 ,ug/ml, the activity of the 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/Sulphamethoxazole in Bacterial Infections: A Wellcome Foundation Symposium Ed. Bernstein Et Salter, Churchill Livingston, Edinburgh Et London, 1973, 1, 10-18; Ferone et al, Antimicrobial Agents and Chemotherapy, (1975), 7, 91). It has been pointed out in the former reference that "although thymidine interferes with the in vitro activity of trimethoprim/sulphamethoxazole, it is not usually present in animals in sufficiently high concentrations to affect the in vivo activity.”
  • thymidine phosphorylase purified from Escherichia coli suggested that under certain conditions thymine, phosphate, and thymidine phosphorylase may form a dead-end complex, that is a complex which is itself not catalytically active but the formation of which must be reversed before the enzyme can form catalytically active complexes. This finding suggested that the dead-end complex might be more stable than the free enzyme. Since thymine is an undesirable additive to the media; as hereinabove explained, a substitute for this was looked for.
  • a stabilised thymidine phosphorylase preparation containing uracil and inorganic phosphate.
  • the thymidine phosphorylase for use in the present invention may be obtained by purification from a number of bacteria such as Salmonella typhimurium, Bacillus cereus, Bacillus stearothermophilus, Haemophilus influenzae and particularly from a strain of Escherichia coli requiring thymine and methionine for growth.
  • the purification may be carried out by the method described by Schwartz, Eur., J. Biochem., (1971), 21, 191-198, which method involves a somewhat lengthy process of precipitation, fractionation, chromatography and dialysis.
  • a more preferred process is that described in Belgian Patent No. 837 946 and published German Patent application No. P 26 02 996.9 which disclose that a certain strain of E.
  • coli. produces inordinate amounts of thymidine phosphorylase under appropriate growth conditions and that it may be isolated and purified by applying the cell extract to specific adsorbents and eluting it therefrom, to give a much higher yield and purity than the method of Schwartz.
  • Monitoring of the eluates at all stages of the purification process employed may be carried out using a spectrophotometric assay at a selected wavelength in order to ascertain enzyme activity which is expressed in International Units (l.U.), one International Unit being equivalent to that amount of enzyme that will phosphorylise one micromole of thymidine to thymine under the assay conditions used (see Example 1). The peaks that show the highest concentration and purity are selected.
  • the enzyme so purified as above is then made available, as previously stated, in a stable form by addition of a combination of uracil and inorganic phosphate.
  • a combination of uracil and inorganic phosphate can be used, potassium or ammonium phosphate are preferred.
  • the concentration of thymidine phosphorylase incorporated into the media is preferably in the range of about 0.01 to 1,000 International Units/ml and more preferably between 0.02 to 10 International Units/ml.
  • the useful concentration limits for uracil and phosphate to produce a stabilised thymidine phosphorylase preparation are 0.5 mM to saturation, preferably 1 to 20 mM, for uracil, and 0.1 mM to saturation, preferably 0.1 to 1.0 M for the inorganic phosphate.
  • the serum albumin is preferably added at a concentration of 0.2 to 5% w/v.
  • the sterility of the above described formulctions is of great importance in view of their applicaticn to the testing of the sensitivities of bacteria to antifolates. It is often desirable, therefore, to add an antimicrobial agent to the formulation in order to ensure sterility. It is important however that the antimicrobials employed are able to sterilise the formulation without affecting the enzyme stability. It has been found that alkali metal azides such as sodium azide or potassium azide are excellent antimicrobials for the purposes of the present invention since they do not interfere with enzyme activity and in the use of the formulations of the present invention are diluted out to ineffectiveness as antimicrobial agents.
  • the antimicrobial agent as hereinabove defined, may be incorporated into the preparation at a concentration of 0.001 to 0.4% w/v, preferably 0.002 to 0.2% w/v.
  • uracil alone or potassium phosphate alone are not as effective in stabilising the enzyme as is their combination. Furthermore, this combination is much more effective with low concentrations of enzyme than is the formulation used in Example 1.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (12)

1. Préparation de thymidine phosphorylase stabilisée, caractérisée en ce que la préparation comprend aussi de l'uracile et un phosphate inorganique.
2. Préparation suivant la revendication 1, caractérisée en ce que le phosphate inorganique est présent en concentration de l'intervalle de 0,1 mM à la saturation.
3. Préparation suivant l'une quelconque des revendications précédentes, caractérisée en ce que le phosphate inorganique est le phosphate de potassium ou le phosphate d'ammonium.
4. Préparation suivant l'une quelconque des revendications 1 à 3, caractérisée en ce que l'uracile est présent en concentration de l'intervalle de 0,5 mM à la saturation.
5. Préparation suivant l'une quelconque des revendications 1 à 4, caractérisée en ce que la préparation comprend aussi de l'albumine de sérum.
6. Préparation suivant la revendication 5, caractérisée en ce que l'albumine de sérum est présente en concentration de l'intervalle de 0,2 à 5% poids/volume.
7. Préparation suivant l'une quelconque des revendications précédentes, caractérisée en ce que la préparation comprend aussi un agent antimicrobien.
8. Préparation suivant la revendication 7, caractérisée en ce que l'agent antimicrobien est un azide de métal alcalin.
9. Préparation suivant la revendication 8, caractérisée en ce que l'azide de métal alcalin est présent en concentration de l'intervalle 0,001 à 0,4% poids/volume.
10. Préparation suivant l'une quelconque des revendications précédentes, caractérisée en ce que le pH de la préparation tombe dans l'intervalle de pH 6 à pH 8.
11. Milieu de culture pour l'épreuve de la sensibilité des bactéries à l'égard des agents antimicrobiens antifoliques, caractérisé en ce qu'une préparation de thymidine phosphorylase suivant l'une quelconque des revendications 1 à 10 est incorporée au milieu.
12. Milieu de culture suivant la revendication 11, caractérisé en ce que la thymidine phosphorylase est présente en concentration de l'intervalle de 0,01 à 1000 unités internationales par ml.
EP78100132A 1977-06-14 1978-06-09 Préparation stabilisée de thymidine-phosphorylase et bouillon de culture la contenant Expired EP0000071B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2466577 1977-06-14
GB2466577 1977-06-14

Publications (2)

Publication Number Publication Date
EP0000071A1 EP0000071A1 (fr) 1978-12-20
EP0000071B1 true EP0000071B1 (fr) 1981-08-05

Family

ID=10215321

Family Applications (1)

Application Number Title Priority Date Filing Date
EP78100132A Expired EP0000071B1 (fr) 1977-06-14 1978-06-09 Préparation stabilisée de thymidine-phosphorylase et bouillon de culture la contenant

Country Status (13)

Country Link
US (1) US4219621A (fr)
EP (1) EP0000071B1 (fr)
JP (1) JPS548791A (fr)
AU (1) AU516545B2 (fr)
CA (1) CA1112193A (fr)
DE (1) DE2860890D1 (fr)
DK (1) DK264878A (fr)
ES (1) ES470728A1 (fr)
FI (1) FI60031C (fr)
HU (1) HU179360B (fr)
IL (1) IL54898A (fr)
IT (1) IT1106103B (fr)
ZA (1) ZA783394B (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU94045954A (ru) * 1993-02-11 1996-10-20 Гист-Брокейдс Н.В. (NL) Способ обнаружения остатков противомикробных веществ в жидкостях и набор для его осуществления
JP2011136911A (ja) * 2009-12-25 2011-07-14 Tosoh Corp 甲状腺刺激ホルモンレセプターの安定化方法
CN103305487B (zh) * 2012-11-20 2014-11-05 上海理工大学 一种利用短乳杆菌发酵生产胸苷磷酸化酶的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1513461A (en) * 1975-01-27 1978-06-07 Wellcome Found Bacterial culture medium

Also Published As

Publication number Publication date
AU516545B2 (en) 1981-06-11
CA1112193A (fr) 1981-11-10
AU3706178A (en) 1979-12-20
IL54898A0 (en) 1978-08-31
IT7849852A0 (it) 1978-06-13
FI781887A (fi) 1978-12-15
EP0000071A1 (fr) 1978-12-20
ZA783394B (en) 1980-02-27
ES470728A1 (es) 1979-02-01
IL54898A (en) 1981-03-31
DK264878A (da) 1978-12-15
FI60031C (fi) 1981-11-10
IT1106103B (it) 1985-11-11
HU179360B (en) 1982-10-28
JPS548791A (en) 1979-01-23
JPS6156998B2 (fr) 1986-12-04
FI60031B (fi) 1981-07-31
US4219621A (en) 1980-08-26
DE2860890D1 (en) 1981-11-05

Similar Documents

Publication Publication Date Title
Takaku et al. In vivo anti‐tumor activity of arginine deiminase purified from Mycoplasma arginini
Andrews Purification of lactose synthetase a protein from human milk and demonstration of its interaction with α‐lactalbumin
US4244943A (en) Method for preparing urokinase injection
WO1992002133A1 (fr) Compositions diagnostiques et therapeutiques ameliorees
Ferone et al. Identification of Harper-Cawston factor as thymidine phosphorylase and removal from media of substances interfering with susceptibility testing to sulfonamides and diaminopyrimidines
Sarkar et al. Isolation and characterization of glutamine synthetase from chicken neural retina
US3855142A (en) Enzymatic denture cleanser
Lehrer et al. Increased content of microbicidal cationic peptides in rabbit alveolar macrophages elicited by complete Freund adjuvant
EP0000071B1 (fr) Préparation stabilisée de thymidine-phosphorylase et bouillon de culture la contenant
EP0726076B1 (fr) PROCEDE DE STABILISATION DE PROTéINE C ACTIVEE OU NON ACTIVEE ET COMPOSITION STABILISEE
Okuda et al. Inhibitory effects of ciprofloxacin and sparfloxacin on DNA gyrase purified from fluoroquinolone-resistant strains of methicillin-resistant Staphylococcus aureus
US4366249A (en) Storage stable cholesterol oxidase compositions
Wriston Jr 5 L-Asparaginase
CA1077379A (fr) Milieu de culture bacteriologique
US5658948A (en) Enhancement of benzalkonium chloride preservative activity in formulations containing an incompatible drug using amino acids having net positive charge
León et al. Fosfomycin resistance plasmids do not affect fosfomycin transport into Escherichia coli
Boxer et al. Leukocyte disorders: quantitative and qualitative disorders of the neutrophil, Part 2
Feder et al. Stabilization of proteolytic enzymes in solution
Formica et al. Production, isolation, and properties of azetomycins
EP0065800B1 (fr) Solutions d'enzymes et procédé pour leur préparation
US4579960A (en) Stable solutions containing thimerosal
US4178212A (en) Stabilized thymidine phosphorylase formulation
EP1735437B1 (fr) Proteinase k hautement purifiee
CA1077420A (fr) Thymidine phosphorylase dans une preparation stabilisee
US3004891A (en) Stable, aqueous solutions of sodium phosphite, sodium formaldehyde sulfoxylate, and dihydrostreptomycin sulfate

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): BE CH DE FR GB NL SE

17P Request for examination filed
GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): BE CH DE FR GB NL SE

Designated state(s): BE CH DE FR GB NL SE

REF Corresponds to:

Ref document number: 2860890

Country of ref document: DE

Date of ref document: 19811105

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 19840319

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 19840321

Year of fee payment: 7

Ref country code: DE

Payment date: 19840321

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 19840331

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 19840630

Year of fee payment: 7

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 19850630

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Effective date: 19860630

BERE Be: lapsed

Owner name: THE WELLCOME FOUNDATION LTD

Effective date: 19860630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Effective date: 19870101

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Effective date: 19880609

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Effective date: 19880610

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Effective date: 19880630

GBPC Gb: european patent ceased through non-payment of renewal fee
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19890228

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19890301

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

EUG Se: european patent has lapsed

Ref document number: 78100132.6

Effective date: 19890220

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT