Preparing Compound Having A 1-thia-5-aza-bicyclo (4.2.0) Octane Ring System (e.g., Cephalosporin, Etc.) Patents (Class 435/47)
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Patent number: 5629171Abstract: Important intermediates for preparing cephalosporin antibiotics, 7-amino-cephalosporanic acid (7-ACA) and 7-aminodeacetylcephalosporanic acid (7-ADAC), are prepared by a novel bioprocess in which a transformed Penicillium chrysogenum strain is cultured in the presence of an adipate feedstock to produce adipoyl-6-APA (6-amino penicillanic acid); followed by the in situ expression of the following genes with which the P. chrysogenum has been transformed:1) an expandase gene, whose expression product converts the adipoyl-6-APA by ring expansion to adipoyl-7-ADCA;2) an hydroxylase gene whose expression product converts the 3-methyl side chain of adipoyl-7-ADCA to 3-hydroxymethyl, to give the first product, 7-aminodeacetylcephalosporanic acid (7-ADAC); and3) an acetyltransferase gene whose expression product converts the 3-hydroxymethyl side chain to the 3-acetyloxymethyl side chain of 7-ACA. The final product, 7-ACA, is then prepared by cleavage of the adipoyl side chain using an adipoyl acylase.Type: GrantFiled: May 11, 1995Date of Patent: May 13, 1997Assignee: Gist-Brocades B.V.Inventors: Michael J. Conder, John A. Rambosek, Phyllis C. McAda, Christopher D. Reeves
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Patent number: 5618687Abstract: An enzymatic process for the production of 7-amino cephalosporanic acid from cephalosporine C is disclosed. The process is a two-stage enzymatic reaction which can be performed in a single reactor. The invention further includes a mutant strain of Trigonopsis variabilis which produces increased amounts of D-amino acid oxidase, and a mutant strain of Acinetobacter sp., which increased amounts of deacylase, which are enzymes necessary for the present process.Type: GrantFiled: November 2, 1994Date of Patent: April 8, 1997Assignee: Biopure CorporationInventors: Bing L. Wong, Yong Q. Shen
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Patent number: 5599702Abstract: D-amino acid oxidase (DAO) in purified, partially purified or crude form is immobilized on a porous bead-shaped support that is a copolymer of vinyl acetate and/or vinyl alcohol and a crosslinking agent. A preferred crosslinking agent is N,N'-divinylethyleneurea. The amount of crosslinking agent is 1 to 60% by weight of the copolymer, and acyloxy groups of vinyl acetate may be present on the support as such or may have been hydrolyzed to hydroxyl groups. The support preferably has a mean particle size of 20 to 800 .mu.m and a mean pore diameter of 2 to 10,000 nm. The D-amino acid oxidase is preferably obtained from Trigonopsis variabilis and purified by ion exchange chromatography. The D-amino acid oxidase may be covalently bonded to a spacer such as epichlorohydrin or its homolog that is bound to the support. Catalase may also be immobilized.Type: GrantFiled: May 17, 1995Date of Patent: February 4, 1997Assignee: Hoechst AktiengesellschaftInventor: Klaus Sauber
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Patent number: 5597704Abstract: A method of converting cephalosporin C to glutaryl-7-aminocephalosporanic acid. The method including obtaining a cell preparation from a microorganism which produces D-amino acid oxidase, the preparation being a cell-free extract or a suspension of permeated cells; adding a D-.alpha.-amino acid to the preparation; after the adding step, heating the preparation at 50.degree.-75.degree. C. for 5-60 minutes; and incubating cephalosporin C in the preparation. Also disclosed are a method of converting cephalosporin C to glutaryl-7-aminocephalosporanic acid in the absence of exogenous H.sub.2 O.sub.2, and a method of screening for a D-amino acid oxidase-producing microorganism.Type: GrantFiled: May 1, 1995Date of Patent: January 28, 1997Assignee: Food Industry Research and Development InstituteInventors: Yun-Huey Lee, Wen-Shen Chu, Wen-Hwei Hsu
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Patent number: 5589354Abstract: An enzyme preparation that exhibits cephalosporin haloperoxidase activity is isolatable from a microorganism species of the Rathayibacter genus. This enzyme preparation can convert cephalexin to a halogenated cephalosporin antibiotic in a single step. A particular, unique microorganism that can provide the cephalosporin haloperoxidase enzyme preparation is Rathayibacter biopuresis.Type: GrantFiled: December 20, 1994Date of Patent: December 31, 1996Assignee: Biopure CorporationInventors: Bing L. Wong, Yong-Qiang Shen, Yung-Pin Chen
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Patent number: 5559005Abstract: Important intermediates for preparing cephalosporin antibiotics, 7-amino-cephalosporanic acid (7-ACA) and 7-aminodeacetylcephalosporanic acid (7-ADAC), are prepared by a novel bioprocess in which a transformed Penicillium chrysogenum strain is cultured in the presence of an adipate feedstock to produce adipoyl-6-APA (6-amino penicillanic acid); followed by the in situ expression of the following genes with which the P. chrysogenum has been transformed:1) an expandase gene, e.g., from Cephalosporium acremonium, whose expression product converts the adipoyl-6-APA by ring expansion to adipoyl-7-ADCA;2) an hydroxylase gene whose expression product converts the 3-methyl side chain of adipoyl-7-ADCA to 3-hydroxymethyl, to give the first product, 7-aminodeacetylcephalosporanic acid (7-ADAC); and3) an acetyltransferase gene whose expression product converts the 3-hydroxymethyl side chain to the 3-acetyloxymethyl side chain of 7-ACA.Type: GrantFiled: May 27, 1994Date of Patent: September 24, 1996Assignee: Merck & Co., Inc.Inventors: Michael J. Conder, Phyllis C. McAda, John A. Rambosek, Christopher D. Reeves
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Patent number: 5559006Abstract: "A method for selectively deactivating catalase while retaining D-amino acid oxidase activity is disclosed. The catalase and oxidase are both present in whole cells or a cell-free extract. The method comprises combining the whole cells or the cell-free extract with a basic solution at a pH between about 11 and about 12. The catalase activity is eliminated and the oxidase activity is unaffected. This results in the production of a solution which contains oxidase activity but no catalase activity. The whole cells and cell-free extract are preferably from Triginopsis variabilis.Type: GrantFiled: February 22, 1995Date of Patent: September 24, 1996Assignee: Eli Lilly and CompanyInventor: Jeffrey T. Vicenzi
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Patent number: 5527697Abstract: An esterase with a molecular weight of 55,200 .+-.300 Da is suitable for the chemoselective conversion of a compound of the formula II into a compound of the formula I.Type: GrantFiled: July 12, 1994Date of Patent: June 18, 1996Assignee: Hoechst AktiengesellschaftInventors: Werner Aretz, Klaus Sauber
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Patent number: 5514561Abstract: Chromogenic and fluorogenic substrates for .beta.-lactamase, methods for synthesis thereof and methods for detecting .beta.-lactamase in a sample are provided. The substrates are substantially colorless or substantially nonfluorescent .beta.-lactam compounds which include an electronegative leaving group. The leaving group comprises a carbamate, carbonate, thiocarbamate or thiocarbonate linkage and a fluorescent moiety or a moiety capable of producing a visually detectable colored product. Upon cleavage of the lactam ring by .beta.-lactamase, the leaving group is liberated and fluorescence or a colored product is produced.Type: GrantFiled: April 14, 1994Date of Patent: May 7, 1996Assignee: Becton, Dickinson and CompanyInventors: J. Michael Quante, Randal A. Hoke, Patrick D. Mize, Daniel L. Woodard, O. Elmo Millner, deceased
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Patent number: 5512454Abstract: O-acylated cephalosporins are produced by reacting 3-hydroxymethylcephalosporins with an acyl donor containing at least three carbon atoms and an enzyme which is a Rodosporidium toruloides esterase, a wheat germ lipase, an Aspergillus niger lipase, an orange peel acetylesterase or a Bacillus subtilis esterase. A preferred enzyme is the esterase from Rodosporidium toruloides ATCC 10657. The enzyme can be used while in whole cells or in soluble or inmobilized form.Type: GrantFiled: February 3, 1994Date of Patent: April 30, 1996Assignee: Bristol-Myers Squibb CompanyInventors: John J. Usher, Guna Romancik, Michael Politino, David A. Lowe
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Patent number: 5474912Abstract: Targeted gene insertion methodology can increase the production of an antimicrobial metabolite and comprises the steps of:a) identifying the gene in unaltered chromosomal DNA which encodes an enzyme that catalyzes the rate controlling step in a biosynthetic pathway in the production of increased concentration of a precursor to a core molecule which leads to the antimicrobial metabolite; andb) inserting into unaltered chromasomal DNA a genetic delivery vehicle (such as a vector, phage, or virus) which carries a gene which encodes the enzyme that catalyzes the rate controlling step in the biosynthesis in the production of a core molecule which is a precursor to the antimicrobial metabolite into unaltered chromosomal DNA, said delivery vehicle being compatible with said unaltered chromosomal DNA, the delivery vehicle being generated byinserting at least one exact or modified copy of the gene determining the rate controlling step into the compatible delivery vehicle, andinserting the delivery vehicle containing tType: GrantFiled: March 3, 1994Date of Patent: December 12, 1995Inventors: David H. Sherman, Wei-Shou Hu, Li-Hong Malmberg
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Patent number: 5472853Abstract: N-Terminal .beta.,.gamma.-didehydrovaline di- and tri-peptides are reacted with isopenicillin N synthetase to form 3-exomethylenecepham-4-carboxylic acids, e.g., .delta.-(L-.alpha.-aminoadipoyl)-L-cysteinyl-D-.beta.,.gamma.-didehydroval ine is incubated with the purified enzyme to provide 7.beta.-(L-.alpha.-aminoadipoylamino)-3-exomethylenecepham-4-carboxylic acid. The 3-exocepham products are useful intermediates to 3-alkoxy-3-cephem and the 3-halo-3-cephem antibiotics.Type: GrantFiled: June 18, 1993Date of Patent: December 5, 1995Assignee: University of OxfordInventor: Jack E. Baldwin
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Patent number: 5470717Abstract: The present invention relates to an improved method for the preparation of certain .beta.-lactam antibiotics by enzymatic acylation or by deprotection of a protected intermediate.Type: GrantFiled: May 12, 1994Date of Patent: November 28, 1995Assignee: Gist-Brocades N.V.Inventor: Kim Clausen
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Patent number: 5441874Abstract: A method for the acylation of the 7-amino group of the cephalosporanic ring, according to which a 7-ACA amino thiazolyl protected adduct is prepared by acylating said amino group by an aminothiazolyl acetic acid whose amino group has been protected by a phenyl acetyl or a phenoxy acetyl group, the amino group being then deprotected by aqueous hydrolysis in the presence of penicillin G amidase or respectively penicillin V amidase.Type: GrantFiled: July 7, 1993Date of Patent: August 15, 1995Assignee: Finpael S.p.A.Inventors: Maurizio Zenoni, Claudio Fuganti
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Patent number: 5424203Abstract: A method for selectively deactivating catalase while retaining D-amino acid oxidase activity is disclosed. The catalase and the oxidase are both present in whole cells or a cell-free extract. The method comprises combining the whole cells or the cell-free extract with a basic solution at a pH between about 11 and about 12 for a time of about 1 hour and 45 minutes to about 2 hours. The catalase activity is eliminated and the oxidase activity is uneffected. This results in the production of a solution which contains the oxidase and which has no catalase activity. The pH of the solution containing the oxidase is then lowered to provide a D-amino acid oxidase capable of enzymatic oxidation of cephalosporin C to glutaryl-7-ACA in high yields. The whole cells and cell-free extract are preferrably from Triginopsis variablilis.Type: GrantFiled: March 11, 1994Date of Patent: June 13, 1995Assignee: Eli Lilly and CompanyInventor: Jeffrey T. Vicenzi
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Patent number: 5424196Abstract: Transformation of Cephalosporin C or its derivatives and salts into 7-aminocephalosporanic acid or its derivatives by an enzymatic two stage process with enzymes immobilized on a solid matrix.Type: GrantFiled: January 3, 1994Date of Patent: June 13, 1995Assignee: Antibioticos S.p.A.Inventors: Stefano Cambiaghi, Sergio Tomaselli, Roberto Verga
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Patent number: 5338667Abstract: Disclosed is a microbiological process which utilizes a biologically pure culture of Sphingomonas sp. DSM 7007 in order to produce a malonyl-7-aminocephalosporanic acid derivative product. Furthermore the process uses cephalosporin C derivative as a substrate for the conversion of the cephalosporin C into the product. The process takes place by one time or continuous addition of substrate so that the concentration of cephalosporin C does not exceed 20 percent by weight. Furthermore, the process is performed at a temperature range of 0-60 degrees celsius and a pH range of 5 to 9.Type: GrantFiled: April 27, 1993Date of Patent: August 16, 1994Assignee: Lonza Ltd.Inventors: Sabine Schurr, Andreas Tschech
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Patent number: 5332663Abstract: 7-amino-cephalosporanic acids having the formula ##STR1## are obtained by submitting compounds having the following formula to enzymatic conversion: ##STR2## wherein R represents H, OH, O--CO--R", R" being an alkyl radical with from 1 to 4 carbon atoms, R.sup.1 represents a carboxylic group. The reaction is carried out in the presence of a microorganism selected from the Pseudomonas, Bacillus, Achromobacter genera, or in the presence of an enzyme, either free or immobilized, derived therefrom.Type: GrantFiled: July 14, 1992Date of Patent: July 26, 1994Assignee: Ministero dell'Universita' e della Ricerca Scientifica e TecnologicaInventors: Ezio Battistel, Pietro Cesti, Giuliana Franzosi, Vilhelmus van der Goes
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Patent number: 5320948Abstract: A cephalosporin C acylase from Pseudomonas diminuta N-176 is characterized by its ability to catalyze the conversion of cephalosporin C, glutaryl 7-ACA, adipyl 7-ACA, succinyl 7-ACA, N-acetylcephalosporin, N-benzoylcephalosporin C and cephalothin into 7-aminocephalosporanic acid. The enzyme contains an .alpha.-subunit having a molecular weight of 26 kDA and a .beta.-subunit having a molecular weight of 58 kDa. A method for the recombinant production of the present cephalosporin C acylase in Escherichia coli is provided.Type: GrantFiled: August 26, 1992Date of Patent: June 14, 1994Assignee: Fujisawa Pharmaceutical Co., Ltd.Inventors: Morita Iwami, Ichiro Aramori, Masao Fukagawa, Takao Isogai, Hitoshi Kojo
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Patent number: 5318896Abstract: An important intermediate for preparing cephalosporin antibiotics, 7-aminodesacetoxy cephalosporanic acid (7-ADCA), is prepared by a novel bioprocess in which a transformed Penicillium chrysogenum strain is cultured in the presence of an adipate feedstock to produce adipoyl-6-APA (6-amino penicillanic acid); and the in situ expression of an expandase gene, e.g., from Streptomyces clavuligerus, with which the P. chrysogenum has been transformed, converts the adipoly-6-APA by ring expansion to adipoyl-7-ADCA. The final product, 7-ADCA, is then prepared by cleavage of the adipoyl side chain using an adipoyl acylase. The entire synthesis, accordingly, is carried out using bioprocesses, and is efficient and economical.Type: GrantFiled: August 28, 1992Date of Patent: June 7, 1994Assignee: Merck & Co., Inc.Inventors: Michael J. Conder, Phyllis C. McAda, John A. Rambosek
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Patent number: 5296358Abstract: The enzymatic preparation of cephalosporanic derivatives, or their salts, ving the formula: ##STR1## wherein R is --CO--COOH or --COOH, R.sub.1 is H, OH, or --O--CO--R" and R" is an alkyl group with 1 to 4 carbon atoms, is carried out by the oxidative deamination of compounds, or their salts, having the formula: ##STR2## with an oxidase D-Aminoacid enzyme derived from Rhodotorula glutinis NCIMB 40412. The enzyme can be in a free or immobilized form.Type: GrantFiled: June 3, 1992Date of Patent: March 22, 1994Assignee: Ministero Dell'Universita' e Della Ricerca Scientifica e TecnologicaInventors: Ezio A. Battistel, Pietro Cesti, Giuliana Franzosi, Vilhelmus van der Goes, Silvana Bonicelli, Mirella Pilone
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Patent number: 5284754Abstract: A process for the continuous conversion of cephalosporin derivatives into glutaryl-7-aminocephalosporanic acid derivativesA process for the continuous conversion of cephalosporin derivatives into the corresponding glutaryl-7-aminocephalosporanic acid derivatives in the presence of a catalyst containing D-amino-acid oxidase is described. The product yield can be increased, where appropriate, by addition of hydrogen peroxide.Type: GrantFiled: September 3, 1991Date of Patent: February 8, 1994Assignee: Hoechst AktiengesellschaftInventors: Thomas Bayer, Klaus Sauber
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Patent number: 5268271Abstract: Synthesis of semi-synthetic monobactamic or .beta.-Lactamic antibiotics by using derivatives stabilized by various methods of penicillin G acylase from various microbial sources according to a thermodynamically controlled strategy in monophase water/cosolvent organic apolar systems, wherein the concentration of the cosolvent varies between 30% and 90%, the temperature between -10.degree. C. and 50.degree. C., the pH between 4.5 and 8.5, with concentrations of the antibiotic nucleus between 0.5 an 875 mM and acyl donor between 0.2 mM and 1M, with a relationship antibiotic ring/activated or free acyl donor, using a buffer between 0 and 1M. Application to the pharmaceutical industry.Type: GrantFiled: August 2, 1991Date of Patent: December 7, 1993Assignee: Cosejo Superior de Invetigaciones CertificasInventors: Jose M. Guisan Seijas, Roberto Fernandez Lafuente, Gregorio Alvaro Campos, Rosa M. Blanco Martin, Cristina Molina Rosell
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Patent number: 5264346Abstract: A novel colorimetric method for beta-lactamase assay and its applications are disclosed. The novel assay method for beta-lactamase is based on the discovery described in this invention. The chromophore formed by oxidation of either the N-alkyl derivative of p-phenylenediamine or the 3,3',5,5'-tetraalkyl derivative of benzidine can be decolorized by the open beta-lactam ring end product resulting from the hydrolysis of a penicillin in the presence of a mercury-containing compound. The same chromophore can be decolorized by the open beta-lactam ring end product resulting from the hydrolysis of a cephalosporin in either the presence or the absence of the mercury-containing compound. None of the intact beta-lactam antibiotics can decolorize the chromophore in either the presence or the absence of the mercury-containing compound. The final concentration of the mercury-containing compound in the decolorization mixture ranges from approximately 0.Type: GrantFiled: June 3, 1991Date of Patent: November 23, 1993Inventor: Kirk C. S. Chen
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Patent number: 5162228Abstract: A promoter of a glyceraldehyde-3-phosphate dehydrogenase gene of Acremonium chrysogenum and a transformant which is obtained by using the expression plasmid containing the promoter and is capable of producing a cephalosporin are disclosed.Type: GrantFiled: December 13, 1989Date of Patent: November 10, 1992Assignee: Takeda Chemical Industries, Ltd.Inventors: Yasuhiro Sumino, Hiroyuki Kimura, Masaru Suzuki
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Patent number: 5114852Abstract: The present invention provides a chimeric enzyme gene which codes for a monooxygenase having both monooxygenase activity derived from cytochrome P-450 and reducing power supplying ability derived from NADPH-cytochrome P-450 reductase.The present invention further provides a yeast expression plasmid which contains said chimeric enzyme gene and expresses said monooxygenase gene; a transformed yeast strain which carries said yeast expression plasmid; a monooxygenase which has both the monooxygenase activity and the reducing power supplying ability as mentioned above; and a process for producing said monooxygenase.Type: GrantFiled: March 27, 1990Date of Patent: May 19, 1992Assignee: Director-General of Agency of Industrial Science & TechnologyInventors: Yoshiyasu Yabusaki, Hiroko Murakami, Toshiyuki Sakai, Megumi Shibata, Hideo Ohkawa
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Patent number: 5104800Abstract: A process for the one-step conversion of cephalosporin C and derivatives thereof to the corresponding 7-aminocephalosporanic acid and derivatives comprising treating said cephalosporin C and derivatives with a cephalosporin C amidase enzyme of a recited sequence, the DNA encoding said enzyme, and expression thereof in a suitable host, e.g., Bacillus species under the control of a suitable promoter.Type: GrantFiled: June 27, 1989Date of Patent: April 14, 1992Assignee: Merck & Co., Inc.Inventors: Mark S. Crawford, David B. Finkelstein, John A. Rambosek
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Patent number: 5084390Abstract: Recombinant Cu/Zn superoxide dismutase (SOD) polymers having an extended in vivo half-life composed of SOD monomers covalently coupled to each other, carboxy terminus to amino terminus, by a polypeptide spacer such as a fragment of the hinge region of an immunoglobulin.Type: GrantFiled: February 14, 1990Date of Patent: January 28, 1992Assignee: Chiron CorporationInventors: Robert A. Hallewell, Guy Mullenbach
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Patent number: 5079146Abstract: The present invention relates to a new method of protection of the carboxy group in the chemistry of the compounds of .beta.-lactam type. According to such a method, the carboxy group is protected by being transformed into its corresponding phenyl-acetoxy-methyl ester, which is then removed by an enzymatic route by means of penicillinacylase.In particular, if the .beta.-lactam compound also bears a phenyl-acetamidic substitutent, this latter is simultaneously hydrolysed during the same step of removal of the carboxy function protecting group.Type: GrantFiled: April 4, 1989Date of Patent: January 7, 1992Assignee: Sclavo S.p.A.Inventors: Claudio Fuganti, Eva Baldaro, Daniela Faiardi, Ameriga Lazzarini
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Patent number: 4847200Abstract: A process for producing unnatural penicillins and cephalosporin derivatives thereof in which peptide analogs of ACV in which the L-.alpha.-aminodipyl moiety is replaced by L-S-carboxymethyl cysteine or other obvious substituents, are reacted with cyclase, epimerase, ring expansion and hydroxylase enzymes isolated from a cell free extract of a prokaryotic organism such as S. clavuligerus. The product depends upon the presence or absence of co-factors such as ferrous ion, .alpha.-ketoglutarate, and ascorbate. In an alternative embodiment, a penicillin analog having the formula: ##STR1## may be reduced with L-cysteine to produce an analog of isopenicillin N which may be reacted with the enzyme reagent to produce the desired penicillin or cephalosporin.Type: GrantFiled: September 5, 1986Date of Patent: July 11, 1989Assignee: Queen's University at KingstonInventors: Saul Wolfe, Donald Westlake, Susan Jensen
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Patent number: 4753881Abstract: Deacetoxycephalosporin C synthetase is provided in purified form via chromatography of crude cell-free extracts over a weak anion exchange resin followed by gel filtration and hydroxylapatite chromatography, all carried out in the presence of glycerol, a C.sub.1 -C.sub.3 alkyl monohydric alcohol, e.g., ethanol, a sulfhydryl containing reducing agent, e.g., dithiothreitol, and ascorbate. The purified enzyme which possesses both expandase and hydroxylase activities can be further purified by chromatography over a strong anion exchange resin.Type: GrantFiled: September 10, 1986Date of Patent: June 28, 1988Assignee: Eli Lilly and CompanyInventors: Wu-Kuang Yeh, Joe E. Dotzlaf
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Patent number: 4745061Abstract: A D-aminoacid transaminase which converts CPC with .DELTA.-keto acids into .DELTA.-ketoadipinyl-7-ACA can be isolated from Bacillus Licheniformis ATCC 9945. This transamination can be applied to other D-amino acids and can also be used for the preparation of D-amino acids from .DELTA.-keto acids. The enzyme is also suitable for resolving racemates of D,L-amino acids and for detection of .DELTA.-keto acids alongside L-amino acids.Type: GrantFiled: December 20, 1985Date of Patent: May 17, 1988Assignee: Hoechst AktiengesellschaftInventors: Werner Aretz, Klaus Sauber
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Patent number: 4693977Abstract: Enzymes extracted from a prokaryotic .beta.-lactam producing microorganism are immobilized on a support for producing desacetoxycephalosporin and analogs thereof from L-.alpha.-aminoadipyl-L-cysteinyl-D-valine and analogs thereof. The enzymes are an epimerase, a cyclase and a ring expansion enzyme extracted preferably from S. clavuligerus, S. cattleya or S. lipmanii. The support is preferably a diethylaminoethyl ion exchange resin.Type: GrantFiled: January 23, 1986Date of Patent: September 15, 1987Assignee: Queen's University at KingstonInventors: Saul Wolfe, Donald Westlake, Susan Jensen
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Patent number: 4579818Abstract: A process is disclosed for converting a peptide precursor of the ACV type, in which the valine moiety may be replaced by any readily available amino acid, to an unnatural cephalosporin of the type ##STR1## where R.sub.1 is H, lower alkyl or functionalized carboxylic group and R.sub.2 is H or lower alkyl, in which the precursor is reacted with cyclase, epimerase and a ring expansion enzyme isolated from a cell-free extract of a prokaryotic organism such as Streptomyces clavuligerus. The three enzymes may be immobilized on a suitable support medium and the conversion may be carried out in a continuous mode.Type: GrantFiled: February 6, 1985Date of Patent: April 1, 1986Assignee: Queen's University at KingstonInventors: Saul Wolfe, Donald Westlake, Susan Jensen
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Patent number: 4536476Abstract: Cyclase, epimerase and a ring expansion enzyme are isolated separately from a cell free extract of a prokaryotic beta-lactam producing organism to provide three separate and stable enzyme reagents for commercial production of cephalosporins from peptide precursors. Isolation is carried out by using ammonium sulfate, gel filtration and ion exchange chromatography. The enzymes may be immobilized on a suitable support and the production of cephalosporins may be carried out continuously.Type: GrantFiled: June 27, 1983Date of Patent: August 20, 1985Assignee: Queen's University at KingstonInventors: Saul Wolfe, Donald Westlake, Susan Jensen
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Patent number: 4533632Abstract: A process for greatly improving the yields of cephalosporin nucleus produced by fermentation is described. Cephalosporin C-producing microorganisms are fermented in the presence of an acetylesterase enzyme so that cephalosporin C formed is immediately converted into desacetyl cephalosporin C before any non-enzymic degradation occurs. Fermentation to produce desacetyl cephalosporin C enables yield increases of cephalosporin nucleus of about 40% to be realized, and greater increases are possible if the fermentation is extended. The preferred cephalosporin C-producing organism is Acremonium chrysogenum, and mutants thereof that are capable of producing esterases in situ. Alternatively, esterases obtained from a variety of other sources may be added or formed in situ, for example from a strain of Rhodosporidium.Type: GrantFiled: August 6, 1982Date of Patent: August 6, 1985Assignee: Glaxo Group LimitedInventors: Alan Smith, Peter J. Bailey
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Patent number: 4510246Abstract: The enzymes, cyclase, epimerase and a ring expansion enzyme, are isolated separately from a cell-free extract of a prokaryotic beta-lactam producing organism. The isolated enzymes are used to produce unnatural cephalosporins from polypeptide precursors. Isolation is carried out by adding ammonium sulfate to 40% saturation to the cell-free extract to precipitate contaminating proteins, adding ammonium sulfate to 70% saturation to the resultant supernatant to precipitate the enzymes, suspending the precipitated enzymes in a buffer, separating epimerase from the suspension by gel filtration, and separating cyclase and the ring expansion enzyme from each other by ion exchange chromatography.Type: GrantFiled: August 23, 1982Date of Patent: April 9, 1985Assignee: Queen's University at KingstonInventors: Saul Wolfe, Donald Westlake, Susan Jensen
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Patent number: 4474879Abstract: This invention relates to a process for preparing 7-(S)-acylamino-3-hydroxymethyl-3-cephem-4-carboxylic acid sulfones and the salts and esters thereof. The instant process involves contacting a 7-(S)-acylamino-3-acetoxymethyl cephalosporin sulfone in an aqueous solution buffered from about pH 6 to about pH 8 with citrus acetylesterase, which is immobilized on silica gel. The 3-hydroxymethyl cephalosporin sulfones produced by the process of this invention are intermediates in the synthesis of 1-oxa .beta.-lactam antibiotics.Type: GrantFiled: November 16, 1982Date of Patent: October 2, 1984Assignee: Eli Lilly and CompanyInventor: Richard E. Heiney
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Patent number: 4414328Abstract: Deacetylcephalosporin C is converted to cephalosporin C by contact with an acetylesterase produced by Aureobasidium pullanans strain 1F0 4466.Type: GrantFiled: July 16, 1981Date of Patent: November 8, 1983Assignee: Fujisawa Pharmaceutical Co., Ltd.Inventors: Hiroshi Imanaka, Toshio Miyoshi, Toshio Konomi, Yoshiaki Kubochi, Seiziro Hattori, Takeshi Kawakita
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Patent number: 4371612Abstract: Biological material such as enzymes is immobilized by adsorption or covalent bonding to a micro-porous water-insoluble acrylonitrile polymer containing from 20 .mu.M to 1000 .mu.M of amino groups per gram of the polymer. Covalent bonding may be carried out with a crosslinking or condensing agent. The amino groups may be introduced into the polymer by reduction of nitrile groups with lithium aluminum hydride in an inert non-solvent.Type: GrantFiled: December 1, 1980Date of Patent: February 1, 1983Assignee: Toyo Jozo Company, Ltd.Inventors: Kunio Matsumoto, Rokuro Izumi, Hideji Seijo, Hiroyuki Mizuguchi
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Patent number: 4368263Abstract: An improved strain of Acremonium chrysogenum may be produced by submitting parent strains of Acremonium chrysogenum to protoplast fusion and nuclear fusion and selecting said improved strain from the progeny or from a mutant thereof.The improved strains have operational advantages for the production of cephalosporins compared with the parental strains.The invention also relates to a method of producing a cephalosporin by culturing the improved strain of Acremonium chrysogenum.Type: GrantFiled: April 25, 1979Date of Patent: January 11, 1983Assignee: Glaxo Group LimitedInventors: Christopher Ball, John S. Collins, Paul F. Hamlyn
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Patent number: 4316955Abstract: The microorganism Streptomyces capillispira, NRRL 12279, produces an enzyme which deesterifies cephalosporin methyl esters.Type: GrantFiled: November 10, 1980Date of Patent: February 23, 1982Assignee: Eli Lilly and CompanyInventors: Bernard J. Abbott, Dennis R. Berry
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Patent number: 4307192Abstract: A cell-free process for converting isopenicillin N, .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine (hereinafter "LLD") and 6-substituted derivatives thereof to deacetoxycephalosporin C (DACPC) by the following reaction sequence: ##STR1## is disclosed. In addition to the starting material, the reaction system includes ATP and a fresh extract of Cephalosporium acremonium prepared and used in a manner to preserve the racemase agent or agents necessary for conversion of the isopenicillin N to penicillin N, a necessary intermediate step in the process.Type: GrantFiled: January 7, 1981Date of Patent: December 22, 1981Assignee: Massachusetts Institute of TechnologyInventors: Arnold L. Demain, Toshio Konomi, Jack E. Baldwin
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Patent number: 4283492Abstract: Production of antibiotic WS-3442 A, B, C, D and E, and their acyl derivatives thereof, the production of antibiotic WS-3442 A, B, C, D and E being a new method by culturing a new species of Streptomyces and their acyl derivatives of said antibiotics being prepared by acylating said antibiotics with an acylating agent. The acyl derivative of antibiotic WS-3442 A, B, C, D and E has antimicrobiological activity and is also useful for intermediate for preparing Cephalosporin derivatives having antimicrobiological activity.Type: GrantFiled: June 13, 1978Date of Patent: August 11, 1981Assignee: Fujisawa Pharmaceutical Co., Ltd.Inventors: Hiroshi Imanaka, Junzi Hosoda, Kazuyoshi Jomon, Heiichi Sakai, Ikuo Ueda, Daizou Morino
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Patent number: 4198338Abstract: Fermentation broths or impure solutions containing thienamycin, a substance having antibiotic activity against gram-negative and gram-positive microorganisms, are purified using a sequence of ion exchange resins, starting with an anion exchange resin of the polystyrene-trimethylammonium type in the HCO.sub.3 -cycle, and ending with absorption on and elution from a polymeric cross-linked polystyrene-type resin adsorbent. Variations in the intermediate steps, e.g. different resins and eluates are possible and are illustrated within.Type: GrantFiled: March 13, 1978Date of Patent: April 15, 1980Assignee: Merck & Co., Inc.Inventors: Laszlo R. Treiber, Vincent P. Gullo
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Patent number: 4178210Abstract: A cephalosporin antibiotic is produced from a precursor comprising the five membered thiazolidine ring and the .beta. lactam moiety characteristics of penicillins by contacting the precursor with a cell-free extract of Cephalosporium acremonium in the presence of ATP under conditions favoring high oxygen transfer. Preferably, an ATP regeneration system comprising a phosphate donor and a phosphotransferase enzyme is included in the reaction.Type: GrantFiled: March 17, 1978Date of Patent: December 11, 1979Assignee: Massachusetts Institute of TechnologyInventors: Arnold L. Demain, Masanobu Kohsaka