Patent US4753323A successfully refreshed from Google Patents.
EP0102216A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Biosynthesis of unnatural cephalosporins
Assignee: Queen's University
•
Filing: 1983-08-12
•
Granted: —
•
Expires: 2003-08-12
Abstract
Abstract A peptide precursor of the ACV (aminoadipyl- cysteinylvaline) type, in which the valine moiety may be replaced by any readily available amino acid may be converted into a cephalosporin of the type where R, is H, lower alkyl or functionalized carboxylic group and R 2 is H or lower alkyl, by reacting the precursor with novel cyclase, epimerase and ring expansion enzymes isolated from a cell-free extract of a prokaryotic organism such as Streptomyces clavuligerus. The three novel enzymes may be immobilized separately or together on a suitable support medium and the conversion may be carried out in a continuous mode.
Inventors
- 👤 Saul Wolfe
- 👤 Donald Westlake
- 👤 Susan Jensen
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 05:11
CPC Classifications
C; C07; C07K; C07K5/00; C07K5/04; C07K5/06; C07K5/06139; C12; C12N; C12N11/00; C12N11/18; C12N9/00; C12N9/90; C12P; C12P35/00; C12P35/06; C12P37/00; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/815; Y10S435/816; Y10S435/8215; Y10S435/822; Y10S435/886
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
86 words
A process for producing unnatural cephalosporins of the formula where R 1 = H, lower alkyl, or functionalized carboxylic group and R 2 = H or lower alkyl and derivatives thereof, characterized in that a starting material comprising L-a-aminoadipyl-L-cysteinyl-D-valine and analogs thereof in which an amino acid is substituted for the valine moiety, is reacted with cyclase, epimerase. and a ring expansion enzyme isolated from a cell-free extract of a prokaryotic organism for sufficient time and in the presence of sufficient co-factors to produce said cephalosporins.
Claim 2
Dependent Claim
86 words
A process for producing unnatural cephalosporins of the formula where R 1 = H, lower alkyl, or functionalized carboxylic group and R 2 = H or lower alkyl and derivatives thereof, characterized in that a starting material comprising L-a-aminoadipyl-L-cysteinyl-D-valine and analogs thereof in which an amino acid is substituted for the valine moiety, is reacted with cyclase, epimerase. and a ring expansion enzyme isolated from a cell-free extract of a prokaryotic organism for sufficient time and in the presence of sufficient co-factors to produce said cephalosporins.
Claim 3
Dependent Claim
31 words
A process as claimed in claim 1, characterized in that said amino acid is selected from the group consisting of a-amino butyric acid, allo-isoleucine, isoleucine, glutamic acid, arginine, glutamine and lysine.
Claim 4
Dependent Claim
25 words
A process as claimed in claim 1, characterized in that said co-factors include at least one of ferrous sulfate, ascorbic acid, dithiothreitol and alpha- ketoglutarate.
Claim 5
Dependent Claim
29 words
A process as claimed in claim 1 characterized in that said cyclase, epimerase and ring expansion enzymes are immobilized on a column and said reaction is a continuous reaction.
Claim 6
Dependent Claim
27 words
5 . A process as claimed in claim 1 characterized in that said reaction is a batch process terminated by addition of methanol as to precipitate proteins.
Claim 7
Dependent Claim
74 words
A process for chromatographically isolating cyclase, epimerase and a ring expansion enzyme from each other and from a cell-free extract of a prokaryotic organism characterized by: (a) precipitating contaminating proteins from said cell-free extract by addition of ammonium sulfate to 40% saturation; (b) separating precipitated protein from a supernatant; (c) adding ammonium sulfate to 70% saturation to said supernatant thereby precipitating desired enzymes;. and (d) suspending said precipitated desired enzymes in pH 7 buffer.
Claim 8
Dependent Claim
74 words
A process for chromatographically isolating cyclase, epimerase and a ring expansion enzyme from each other and from a cell-free extract of a prokaryotic organism characterized by: (a) precipitating contaminating proteins from said cell-free extract by addition of ammonium sulfate to 40% saturation; (b) separating precipitated protein from a supernatant; (c) adding ammonium sulfate to 70% saturation to said supernatant thereby precipitating desired enzymes;. and (d) suspending said precipitated desired enzymes in pH 7 buffer.
Claim 9
Dependent Claim
27 words
A process as claimed in claim 1 or 6 characterized in that said prokaryotic organism is selected from the group comprising S. clavuligerus, S.cattleya and S. lipmanii.
Claim 10
Dependent Claim
22 words
A process as claimed in claim 6, characterized in that epimerase is separated from the suspension of (d) by gel filtration chromatography.
Claim 11
Dependent Claim
31 words
A process as claimed in claim 6, characterized in that after separation of epimerase from the suspension of (d), cyclase and said ring expansion enzyme are separated by ion exchange chromatography.
Claim 12
Dependent Claim
34 words
A stable epimerase reagent capable of epimerizing isopenicillin N and analogs thereof to penicillin N and the respective analogs thereof characterized by a molecular weight of about 60,000 and derived from a prokaryotic organism.
Claim 13
Dependent Claim
34 words
A stable epimerase reagent capable of epimerizing isopenicillin N and analogs thereof to penicillin N and the respective analogs thereof characterized by a molecular weight of about 60,000 and derived from a prokaryotic organism.
Claim 14
Dependent Claim
24 words
An epimerase reagent as claimed in claim 10 characterized in that said prokaryotic organism is selected from S. clavuligerus, S, cattleya and S. lipmanii.
Claim 15
Dependent Claim
33 words
A stable cyclase reagent capable of cyclizing ACV and analogs thereof to isopenicillin N and the respective analogs thereof characterized by a molecular weight of about 36,500 and derived from a prokaryotic organism.
Claim 16
Dependent Claim
33 words
A stable cyclase reagent capable of cyclizing ACV and analogs thereof to isopenicillin N and the respective analogs thereof characterized by a molecular weight of about 36,500 and derived from a prokaryotic organism.
Claim 17
Dependent Claim
24 words
A cyclase reagent as claimed in claim 12 characterized in that said prokaryotic organism is selected from S. clavuligerus, S. cattleya and S. lipmanii.
Claim 18
Dependent Claim
36 words
A stable ring expansion enzyme reagent capable of ring expanding penicillin N and analogs thereof to desacetoxycephalosporin and the respective analogs thereof characterized by a molecular weight of about 29,000 and derived from a prokaryotic organism.
Claim 19
Dependent Claim
36 words
A stable ring expansion enzyme reagent capable of ring expanding penicillin N and analogs thereof to desacetoxycephalosporin and the respective analogs thereof characterized by a molecular weight of about 29,000 and derived from a prokaryotic organism.
Claim 20
Dependent Claim
25 words
A ring expansion enzyme as claimed in claim 14 characterized in that said prokaryotic organism is selected from S. clavuligerus, S. cattleya and S. lipmanii.
Claim 21
Dependent Claim
71 words
An enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV and analogs thereof to desacetoxycephalosporin and the respective analogs thereof characterized by an epimerase having a molecular weight of about 60,000, a cyclase having an MW of about 36,500 and a ring expansion enzyme having a molecular weight of about 29,000, all of which are derived from a prokaryotic organism and are immobilized on a diethyl- aminoethytrisacryl chromatographic resin.
Claim 22
Dependent Claim
71 words
An enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV and analogs thereof to desacetoxycephalosporin and the respective analogs thereof characterized by an epimerase having a molecular weight of about 60,000, a cyclase having an MW of about 36,500 and a ring expansion enzyme having a molecular weight of about 29,000, all of which are derived from a prokaryotic organism and are immobilized on a diethyl- aminoethytrisacryl chromatographic resin.
Claim 23
Dependent Claim
104 words
A process for preparing an enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV and analogs thereof to deacetoxycephalosporin and the respective analogs thereof, characterized by: (a) precipitating contaminating proteins from a cell-free extract of a prokaryotic organism by addition of ammonium sulphate to 40% saturation; (b) separating said precipitated protein from a supernatant; (c) adding ammonium sulphate to 70% saturation to said supernatant thereby precipitating said cyclizing epimerizing and ring expanding enzymes; (d) suspending said precipitated enzymes in a pH 7 50mM buffer; and (e) loading said enzymes onto a chromatographic column comprising diethylaminoethyltrisacryl so as to immobilize said enzymes thereon.
Claim 24
Dependent Claim
104 words
A process for preparing an enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV and analogs thereof to deacetoxycephalosporin and the respective analogs thereof, characterized by: (a) precipitating contaminating proteins from a cell-free extract of a prokaryotic organism by addition of ammonium sulphate to 40% saturation; (b) separating said precipitated protein from a supernatant; (c) adding ammonium sulphate to 70% saturation to said supernatant thereby precipitating said cyclizing epimerizing and ring expanding enzymes; (d) suspending said precipitated enzymes in a pH 7 50mM buffer; and (e) loading said enzymes onto a chromatographic column comprising diethylaminoethyltrisacryl so as to immobilize said enzymes thereon.
Claim 25
Dependent Claim
16 words
A process as claimed in claim 17 characterized in that said buffer is a Tris-chloride buffer.
Claim 26
Dependent Claim
17 words
A process as claimed in claim 6 characterized in that said buffer is a 100mM Tris-chloride buffer.
Claim 27
Dependent Claim
30 words
A process as claimed in claim 19 characterized in that said cyclase and said ring expansion enzyme are eluted from a chromatographic column at 150mM Tris-chloride and 110mM Tris-chloride respectively.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.