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.

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• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
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• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
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• 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)

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• 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 ES ES470728A patent/ES470728A1/es not_active Expired
  • 1978-06-13 IT IT49852/78A patent/IT1106103B/it active
  • 1978-06-13 JP JP7142278A patent/JPS548791A/ja active Granted
  • 1978-06-13 AU AU37061/78A patent/AU516545B2/en not_active Expired
  • 1978-06-13 HU HU78WE578A patent/HU179360B/hu unknown
  • 1978-06-13 CA CA305,302A patent/CA1112193A/en not_active Expired
  • 1978-06-13 US US05/915,153 patent/US4219621A/en not_active Expired - Lifetime
  • 1978-06-13 FI FI781887A patent/FI60031C/fi not_active IP Right Cessation
  • 1978-06-13 IL IL54898A patent/IL54898A/xx unknown
  • 1978-06-13 DK DK264878A patent/DK264878A/da not_active Application Discontinuation
  • 1978-06-13 ZA ZA783394A patent/ZA783394B/xx unknown

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