EP0000071B1 - Stabilisierte Thymidinephosphorylase- Zusammensetzung und diese enthaltender Nährboden - Google Patents

Stabilisierte Thymidinephosphorylase- Zusammensetzung und diese enthaltender Nährboden Download PDF

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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
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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
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English (en)
French (fr)
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EP0000071A1 (de
Inventor
Thomas Anthony Krenitsky
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Wellcome Foundation Ltd
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Wellcome Foundation Ltd
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Publication date
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    • 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.

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  • 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. Stabilisierte Thymidinphosphorylase-Zubereitung, dadurch gekennzeichnet, daß sie auch Uracil und anorganisches Phosphat enthält.
2. Zubereitung nach Anspruch 1, dadurch gekennzeichnet, daß das anorganische Phosphat in einem Konzentrationsbereich von 0,1 mM bis zur Sättigung vorliegt.
3. Zubereitung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß das anorganische Phosphat Kalium- oder Ammoniumphosphat ist.
4. Zubereitung nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß das Uracil in einem Konzentrationsbereich von 0,5 mM bis zur Sättigung vorliegt.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß die Zubereitung auch Serum-Albumin enthält.
6. Zubereitung nach Anspruch 5, dadurch gekennzeichnet, daß das Serum-Albumin in einem Konzentrationsbereich von 0,2 bis 5% Gew./Vol. vorliegt.
7. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß die Zubereitung auch ein antimikrobielles Mittel enthält.
8. Zubereitung nach Anspruch 7, dadurch gekennzeichnet, daß das antimikrobielle Mittel ein Alkalimetallazid ist.
9. Zubereitung nach Anspruch 8, dadurch gekennzeichnet, daß das Alkalimetallazid in einem Konzentrationsbereich von 0,001 bis 0,4% Gew./Vol. vorliegt.
10. Zubereitung nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, daß der pH der Zubereitung im Bereich pH 6 bis pH 8 liegt.
11. Kulturmedium für die Untersuchung der Empfindlichkeit von Bakterien gegenüber folathemmenden und antimikrobiellen Mitteln, dadurch gekennzeichnet, daß das Medium eine Zubereitung aus Thymidinphosphorylase nach einem der Ansprüche 1 bis 10 enthält.
12. Kulturmedium nach Anspruch 11, dadurch gekennzeichnet, daß die Thymidinphosphorylase in einem Konzentrationsbereich von 0,01-1000 Internationale Einheiten/ml vorhanden ist.
EP78100132A 1977-06-14 1978-06-09 Stabilisierte Thymidinephosphorylase- Zusammensetzung und diese enthaltender Nährboden Expired EP0000071B1 (de)

Applications Claiming Priority (2)

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

Publications (2)

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EP0000071A1 EP0000071A1 (de) 1978-12-20
EP0000071B1 true EP0000071B1 (de) 1981-08-05

Family

ID=10215321

Family Applications (1)

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EP78100132A Expired EP0000071B1 (de) 1977-06-14 1978-06-09 Stabilisierte Thymidinephosphorylase- Zusammensetzung und diese enthaltender Nährboden

Country Status (13)

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

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT611001E (pt) * 1993-02-11 2002-10-31 Dsm Nv Unidade para a detencao de residuos de compostos antibacterianos em liquidos
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
FI60031C (fi) 1981-11-10
IT1106103B (it) 1985-11-11
EP0000071A1 (de) 1978-12-20
DE2860890D1 (en) 1981-11-05
DK264878A (da) 1978-12-15
FI60031B (fi) 1981-07-31
ZA783394B (en) 1980-02-27
FI781887A (fi) 1978-12-15
HU179360B (en) 1982-10-28
JPS6156998B2 (de) 1986-12-04
ES470728A1 (es) 1979-02-01
IL54898A0 (en) 1978-08-31
IT7849852A0 (it) 1978-06-13
US4219621A (en) 1980-08-26
IL54898A (en) 1981-03-31
AU516545B2 (en) 1981-06-11
JPS548791A (en) 1979-01-23
CA1112193A (en) 1981-11-10
AU3706178A (en) 1979-12-20

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