EP0000071B1 - Stabilised thymidine phosphorylase preparation and culture medium containing it - Google Patents
Stabilised thymidine phosphorylase preparation and culture medium containing it Download PDFInfo
- 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
Links
- 238000002360 preparation method Methods 0.000 title claims description 28
- 102000013537 Thymidine Phosphorylase Human genes 0.000 title claims description 25
- 101710132082 Pyrimidine/purine nucleoside phosphorylase Proteins 0.000 title claims description 24
- 239000001963 growth medium Substances 0.000 title claims description 9
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 24
- 229940035893 uracil Drugs 0.000 claims description 12
- 239000004599 antimicrobial Substances 0.000 claims description 11
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 8
- 229910052816 inorganic phosphate Inorganic materials 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 108010071390 Serum Albumin Proteins 0.000 claims description 5
- 102000007562 Serum Albumin Human genes 0.000 claims description 5
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 5
- 235000011009 potassium phosphates Nutrition 0.000 claims description 5
- -1 alkali metal azide Chemical class 0.000 claims description 4
- 230000003432 anti-folate effect Effects 0.000 claims description 4
- 229940127074 antifolate Drugs 0.000 claims description 4
- 239000004052 folic acid antagonist Substances 0.000 claims description 4
- 229910052783 alkali metal Inorganic materials 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 239000004254 Ammonium phosphate Substances 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical group [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 description 27
- 108090000790 Enzymes Proteins 0.000 description 27
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 18
- 230000000694 effects Effects 0.000 description 14
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 10
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 9
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 9
- 238000009472 formulation Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 229940104230 thymidine Drugs 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 229960001082 trimethoprim Drugs 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 239000001166 ammonium sulphate Substances 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 229940113082 thymine Drugs 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 229960005404 sulfamethoxazole Drugs 0.000 description 4
- 150000003456 sulfonamides Chemical class 0.000 description 4
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000193755 Bacillus cereus Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 241000606768 Haemophilus influenzae Species 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000009097 Phosphorylases Human genes 0.000 description 1
- 108010073135 Phosphorylases Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241000277284 Salvelinus fontinalis Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000002829 antibacterial sensitivity test Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- TZLVRPLSVNESQC-UHFFFAOYSA-N potassium azide Chemical compound [K+].[N-]=[N+]=[N-] TZLVRPLSVNESQC-UHFFFAOYSA-N 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000003019 stabilising effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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)
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 (en) | 1978-12-20 |
| EP0000071B1 true EP0000071B1 (en) | 1981-08-05 |
Family
ID=10215321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP78100132A Expired EP0000071B1 (en) | 1977-06-14 | 1978-06-09 | Stabilised thymidine phosphorylase preparation and culture medium containing it |
Country Status (13)
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2176220T3 (es) * | 1993-02-11 | 2002-12-01 | Dsm Nv | Procedimiento para la deteccion de compuestos antibacterianos en muestras de ensayo y unidad para su realizacion. |
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1513461A (en) * | 1975-01-27 | 1978-06-07 | Wellcome Found | Bacterial culture medium |
-
1978
- 1978-06-09 EP EP78100132A patent/EP0000071B1/en not_active Expired
- 1978-06-09 DE DE7878100132T patent/DE2860890D1/de not_active Expired
- 1978-06-13 US US05/915,153 patent/US4219621A/en not_active Expired - Lifetime
- 1978-06-13 DK DK264878A patent/DK264878A/da not_active Application Discontinuation
- 1978-06-13 JP JP7142278A patent/JPS548791A/ja active Granted
- 1978-06-13 ZA ZA783394A patent/ZA783394B/xx unknown
- 1978-06-13 HU HU78WE578A patent/HU179360B/hu unknown
- 1978-06-13 FI FI781887A patent/FI60031C/fi not_active IP Right Cessation
- 1978-06-13 IT IT49852/78A patent/IT1106103B/it active
- 1978-06-13 AU AU37061/78A patent/AU516545B2/en not_active Expired
- 1978-06-13 ES ES470728A patent/ES470728A1/es not_active Expired
- 1978-06-13 IL IL54898A patent/IL54898A/xx unknown
- 1978-06-13 CA CA305,302A patent/CA1112193A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| AU516545B2 (en) | 1981-06-11 |
| ES470728A1 (es) | 1979-02-01 |
| FI781887A7 (fi) | 1978-12-15 |
| FI60031C (fi) | 1981-11-10 |
| IL54898A0 (en) | 1978-08-31 |
| IT7849852A0 (it) | 1978-06-13 |
| JPS6156998B2 (enrdf_load_html_response) | 1986-12-04 |
| IT1106103B (it) | 1985-11-11 |
| US4219621A (en) | 1980-08-26 |
| EP0000071A1 (en) | 1978-12-20 |
| AU3706178A (en) | 1979-12-20 |
| FI60031B (fi) | 1981-07-31 |
| HU179360B (en) | 1982-10-28 |
| ZA783394B (en) | 1980-02-27 |
| DE2860890D1 (en) | 1981-11-05 |
| CA1112193A (en) | 1981-11-10 |
| JPS548791A (en) | 1979-01-23 |
| IL54898A (en) | 1981-03-31 |
| DK264878A (da) | 1978-12-15 |
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