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WO1999033994A1 - Desacetoxycephalosporine c synthase (daocs) modifies et structure aux rayons x - Google Patents

Desacetoxycephalosporine c synthase (daocs) modifies et structure aux rayons x Download PDF

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Publication number
WO1999033994A1
WO1999033994A1 PCT/GB1998/003860 GB9803860W WO9933994A1 WO 1999033994 A1 WO1999033994 A1 WO 1999033994A1 GB 9803860 W GB9803860 W GB 9803860W WO 9933994 A1 WO9933994 A1 WO 9933994A1
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WIPO (PCT)
Prior art keywords
atom
anisou
arg
phe
leu
Prior art date
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PCT/GB1998/003860
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English (en)
Inventor
Christopher Joseph Schofield
Jack Edward Baldwin
Peter L. Roach
Matthew D. Lloyd
Karl Harlos
Inger Andersson
Janos Hajdu
Anke S. Terwisscha Van Scheltinga
Karin Valegard
S. Ramaswamy
Original Assignee
Isis Innovation Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GBGB9727370.0A external-priority patent/GB9727370D0/en
Priority claimed from GBGB9813644.3A external-priority patent/GB9813644D0/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Priority to PL98341396A priority Critical patent/PL341396A1/xx
Priority to JP2000526650A priority patent/JP2002500015A/ja
Priority to SK976-2000A priority patent/SK9762000A3/sk
Priority to CA002316401A priority patent/CA2316401A1/fr
Priority to EP98962587A priority patent/EP1047785A1/fr
Priority to AU17720/99A priority patent/AU1772099A/en
Publication of WO1999033994A1 publication Critical patent/WO1999033994A1/fr

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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis

Definitions

  • Penicillin and cephalosporin antibiotics are produced either directly by fermentation or by modification of fermentation derived materials containing a beta-lactam ring.
  • the biosynthetic pathway to the penicillins and cephalosporins has been extensively studied and reviewed (J. E. Baldwin and C. J. Schofield, in 'The Chemistry of ⁇ -lactams (Ed. M. I. Page), Chapter 1 , Blackie, London 1992; ingolia and Queener, Med. Res. Rev., 1989, 9, 245-264; Aharonowitz, Cohen and Martin, Ann. Rev.
  • D-valine (ACV) to isopenicillin N in a step catalysed by isopenicillin N synthase (IPNS). This step is common to both penicillin and cephalosporin biosynthesis.
  • isopenicillin N is converted by exchange of its L- ⁇ - ⁇ - aminoadipoyl side chain to penicillins with other side chains, which are normally more hydrophobic than the side chain of isopenicillin N. This conversion is catalysed by an amidohydrolase/ acyltransferase enzyme.
  • penicillins produced by this biosynthetic process include penicillin G (which has a phenylacetyl side chain) and penicillin V (which has a phenoxyacetyl side chain). These hydrophobic penicillins may be commercially produced via fermentation under the appropriate conditions.
  • DOCS deacetoxycephalosporin C synthase
  • DAOC/DACS deacetoxy/deacetylcephalosporin C synthase
  • DAOC deacetylcephalosporin C
  • Penicillins with hydrophobic side chains may be used for the preparation of cephalosporins or intermediates used in the preparation of cephalosporins, e.g. penicillins (including penicillin G and penicillin V) may be used to prepare C-3 exomethylene cephams which may be used as intermediates in the preparation of the commercial antibiotics, e.g. Cefachlor.
  • IPNS The enzymes IPNS, DAOCS, DACS and DAOC/DACS are members of an extended family of Fe(ll) utilising oxidase and oxygenase enzymes. Most of this family (including DAOCS, DACS and DAOC/DACS) utilise a 2-oxo acid (normally 2-oxoglutarate) as a cosubstrate in addition to dioxygen and the 'prime' substrate (e.g. penicillin N in the case of DAOCS). Since IPNS, does not use 2-oxoglutarate, it has a substantially different mechanism to the 2-oxoglutarate dependent oxygenases, and this gives rise to a significantly different active site.
  • 2-oxo acid normally 2-oxoglutarate
  • the 'prime' substrate e.g. penicillin N in the case of DAOCS
  • This invention is based on the determination of the three dimensional crystal structure of DAOCS and the information and developments which come from it.
  • the X-ray co-ordinates provide very detailed 3-dimensional information on the relationships between amino acid residues in the structure of DAOCS and on the binding modes of the Fe-cofactor and the substrates to DAOCS.
  • the structure allows the modification of DAOCS and related enzymes of penicillin and cephalosporin biosynthesis (including DACS and DAOC/DACS) in order to alter their substrate and product selectivities. Since the DAOCS structures are the first from the family of 2-oxoglutarate dependent dioxygenases they also allow for the design of new inhibitors of this family of enzymes.
  • IPNS gene sequence (and therefore the amino acid sequence) is related but significantly different to those of DAOCS, DACS, DAOC/DACS. It is likely that gross elements of the fold (i.e. significant elements within the 3-dimensional structure) of these enzymes will be conserved but that the active site architecture will be very significantly different. Structural elements conserved are likely to include the beta- barrel 'jelly roll' core and certain alpha-helices (including alpha helix-10, as defined in Roach et al., Nature, 1995, 375, 700-704). The degree of similarity is insufficient to define the precise structure of DAOCS, DACS, or DAOC/DACS from the IPNS structures.
  • the three-dimensional structure of DAOCS is defined by the X-ray co-ordinates set out below (Structure A).
  • the present invention relates to the use of the structures of DAOCS in order to make modifications to it or DACS or DAOC/DACS in order that the modified enzymes catalyse the conversion of unnatural penicillins (e.g. penicillin G and penicillin V) to cephalosporins more efficiently than the wild-type enzyme.
  • Further aspects of the invention relate to the use of the DAOCS structure in order to produce unnatural products in micro-organisms. Such products include exomethylene cephalosporins, with or without alpha-aminoadipoyl or hydrophobic side chain (e.g. phenylacetyl or phenoxyacetyl).
  • one aspect of this invention refers to the use of the structure of DAOCS for modifying DAOCS (or the closely related enzymes DACS or DAOC/DACS) in order to: (i) permit the enzyme to accept (or accept more efficiently) unnatural penicillin substrates for the preparation of new or commercially valuable antibacterial materials. (ii) enable the modified enzyme to produce unnatural (e.g. exomethylene cephams) or optimise the production of minor products (e.g. 3- ⁇ -hydroxycephams) for use as antibacterials or as intermediates in the preparation of antibacterials or commercially valuable compounds.
  • unnatural e.g. exomethylene cephams
  • optimise the production of minor products e.g. 3- ⁇ -hydroxycephams
  • this invention provides modified enzymes that result from application of the aforementioned techniques.
  • These are enzymes having significant (as defined below) sequence and thus structural similarity with DAOCS.
  • structures of these enzymes may be predicted on the basis of the DAOCS structures.
  • sequence similarity/identity between most of the modified enzyme and a major part of DAOCS.
  • two enzymes may have structures in which secondary structural elements are largely or wholly conserved, differences in the structures of the two enzymes may result from the side chains of the amino acids forming the secondary structural elements. The effect of these differences, which alter the substrate/product selectivities of the compared enzymes, is predictable once the three-dimensional structure of one of the enzymes is known.
  • the invention provides an enzyme having significant (as herein defined) sequence similarity to DAOCS wherein the side chain binding site of penicillin N or DAOC is modified and at at least one of the following sites at least one amino acid residue is changed to another amino acid residue or is deleted: Thr72, Arg74, Arg75, Glu156, Leu158, Arg160, Arg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301 , Tyr302, Val303, Asn304; and/or at least one additional amino acid residue is inserted within the region 300-311 ; provided that other residues interacting with the above may be changed in order to accommodate the change in one of the above.
  • the side chain binding pocket of DAOCS is made of residues from different parts of the peptide chain, so it is likely that more than one residue will have to be altered to make a better penicillin G/V expander. Nevertheless some residues are more important than others.
  • the penam C-3 carboxylate group probably occupies an analogous position to that of Ala- 311 from a symmetry related molecule in the active site, forming electrostatic interactions with Arg-162 and Arg-160.
  • the side chain of Arg-160 may also form a hydrogen bonding interaction with the ⁇ -lactam carbonyl.
  • Arg-266 This residue binds with the -aminoadipate side chain of the natural substrate and should be changed to a residue of more hydrophobic character, e.g. Phe, Ala, Val, Leu, He. b) Thr-72. This should be changed to a hydrophobic residue e.g.
  • Arg-74 may be usefully changed to a neutral or hydrophobic residue (Phe, Tyr, Val, Leu, He, Ala). Modification of Arg-75 may be necessary in addition because it forms a hydrogen-bonding network with Arg-74.
  • Glu-156 This residue binds with the ⁇ -aminoadipate side chain. It should be changed to one of Ala, Val, Leu, He, Phe, Tyr, Trp, Asn, Gin, Ser.
  • the insertion or deletion of residues into the DAOCS sequence may also be of use in constructing a hydrophobic binding pocket for the penicillin side chain. Insertion of hydrophobic residues into the
  • C-terminal region may assist in the construction of a hydrophobic binding pocket for penicillin side chains.
  • the invention provides an enzyme having significant (as herein defined) sequence similarity to DAOCS wherein the penicillin/cephalosporin binding site of penicillin N or DAOC is modified and at at least one of the following amino acid residues is changed or deleted: Ile88, Arg160, Arg162, Phe164, Met180, Thr190, Ile192, Phe225, Pro241 , Val245, Val262, Phe264, Asn304, Ile305, Arg306, Arg307; and/or at least one additional amino acid residue is inserted within the region 300-31 1 ; provided that other residues interacting with the above may be changed in order to accommodate the change in one of the above.
  • Another aspect of the invention refers to the use of the structure of DAOCS in order to modify its active site (or that of a structurally related 2-oxoglutarate dependent dioxygenase) in order that the modified enzyme accepts non beta lactam substrates in order to produce oxidised compounds of value.
  • Oxidised amino acids e.g. 4-hydroxyprolines, hydroxylysines, hydroxyaspartic acids and others
  • specific residues can be targeted for modification in order that the modified enzyme can be used to produce oxidised amino acids or peptides.
  • the process may include modification of the following residues: Arg74, Glu156, Leu158, Arg160, Arg162, Leu186, Ser187, Phe225, Phe264, Arg266, Asp301 , Tyr302, Val303, Asn304, Ile88, Arg162, Phe164, Met180, Thr190, Ile192, Pro241 , Val245, Val262, Ile305, Arg306, Arg307.
  • Another aspect of the invention refers to the use of the use of the
  • DAOCS structure for the design of selective inhibitors of 2-oxoglutarate dependent dioxygenases.
  • the 2-oxoglutarate dependent dioxygenase prolyl 4-hydroxylase has been the target of inhibition in order to provide a therapeutic treatment for fibrotic diseases (e.g. liver cirrhosis, arthritis).
  • fibrotic diseases e.g. liver cirrhosis, arthritis.
  • no inhibitors are in clinical use, probably because it is difficult to achieve selective inhibition of the target enzyme for inhibition over other enzymes (including 2-oxoglutarate dependent enzymes).
  • the structure of DAOCS provides a template for the design of inhibitors of 2-oxoglutarate dependent dioxygenases. Set out below are two high resolution crystal structures for
  • DAOCS from S. clavuligerus the structure of the iron-free apoenzyme (Structure A) and the structure of the complex with Fe(ll) and 2-oxoglutarate (Structure B).
  • Structure A the structure of the iron-free apoenzyme
  • Structure B the structure of the complex with Fe(ll) and 2-oxoglutarate
  • the results imply a mechanism by which the enzyme-Fe(ll) complex reacts with 2-oxoglutarate and dioxygen to give the reactive ferryl species, a process common to many non-haem oxygenases.
  • 2-oxoacid-dependent ferrous enzymes are prolyl hydroxylase, involved in collagen biosynthesis, gibberellin 3 ⁇ -hydroxylase, a mutation of which influences stem length in plants, and clavaminic acid synthase, involved in the biosynthesis of the ⁇ -lactamase inhibitor, clavulanic acid.
  • DAOCS belongs to a sub-family, the members of which show sequence similarity with IPNS and 1-aminocyclopropane-1-carboxylate oxidase (the ethylene forming enzyme), enzymes that do not use a 2-oxoacid in catalysis.
  • the iron-free form of DAOCS crystallises in space group R3 as a crystallographic trimer.
  • the main chain of the protein folds into a conserved jelly roll core with flanking helices.
  • Figure 1 the biosynthetic pathway to the penicillins and cephalosporins.
  • Figure 2 is a view of the active site of DAOCS showing
  • Structure A is a three-dimensional structure of DAOCS.
  • Structure B is a high resolution crystal structure for prokaryotic DAOCS from S. clavuligerus as a complex with Fe(ll) and 2- oxoglutarate.
  • DAOCS The peptide sequence of DAOCS (with the numbering used herein) is set out below:
  • ATOM 92 CD2 LEU 10 16.852 29.603 44.869 1.000 21.93
  • ATOM 271 CE2 PHE 32 19.267 22.348 49.138 1.000 15.49
  • ATOM 364 CD2 LEU 44 6.793 29.259 57.653 1.000 51.56

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  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne la ou les structures tridimensionnelles de cristaux de la désacétoxycéphalosporine C synthase (DAOCS). Les coordonnées obtenues par rayons X fournissent des informations tridimensionnelles précises des acides aminés se trouvant dans la structure de DAOCS. Certains de ceux-ci se présentent en complexes avec du fer et/ou des substrats. Les informations tirées des structures sont utilisées pour modifier les enzymes de la voie de biosynthèse de la céphalosporine comprenant DAOCS, la désacétylcéphalosporine C synthase DAOCS/DACS, de manière qu'elles acceptent des substrats synthétiques (par exemple des pénicillines G, V) afin d'améliorer la production de β-lactamines. Les structures peuvent être utilisées pour prédire les structures d'autres enzymes dépendantes de 2-oxoglutarate, permettant ainsi la conception d'inhibiteurs, et de nouveaux catalyseurs pour la production, par exemple, d'acides aminés/peptides oxydés. Des modifications spécifiques de restes d'acides aminés sont proposées et exemplifiées.
PCT/GB1998/003860 1997-12-24 1998-12-24 Desacetoxycephalosporine c synthase (daocs) modifies et structure aux rayons x WO1999033994A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
PL98341396A PL341396A1 (en) 1997-12-24 1998-12-24 Modified syntheses of diacetoxycephalosporin c (daocs) and its roentgenographic structure
JP2000526650A JP2002500015A (ja) 1997-12-24 1998-12-24 改変型デアセトキシセファロスポリンｃシンターゼ（ｄａｏｃｓ）およびｘ線構造
SK976-2000A SK9762000A3 (en) 1997-12-24 1998-12-24 Modified deacetoxycephalosporin c synthase (daocs) and x-ray structure
CA002316401A CA2316401A1 (fr) 1997-12-24 1998-12-24 Desacetoxycephalosporine c synthase (daocs) modifies et structure aux rayons x
EP98962587A EP1047785A1 (fr) 1997-12-24 1998-12-24 Desacetoxycephalosporine c synthase (daocs) modifies et structure aux rayons x
AU17720/99A AU1772099A (en) 1997-12-24 1998-12-24 Modified deacetoxycephalosporin c synthase (daocs) and x-ray structure

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9727370.0 1997-12-24
GBGB9727370.0A GB9727370D0 (en) 1997-12-24 1997-12-24 Processed based on the structure of deacetoxycephalosporin c synthase
GB9813644.3 1998-06-24
GBGB9813644.3A GB9813644D0 (en) 1998-06-24 1998-06-24 Structure of a cephalosporin synthase

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CN (1) CN1284997A (fr)
AU (1) AU1772099A (fr)
CA (1) CA2316401A1 (fr)
PL (1) PL341396A1 (fr)
SK (1) SK9762000A3 (fr)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085951A1 (fr) * 2000-05-09 2001-11-15 Acs Dobfar Uk Limited Expandase modifiee et utilisations
EP1348759A1 (fr) * 2002-03-26 2003-10-01 Synmax Biochemical Co., Ltd Expandase de penicillin mutée et un procédé de production de 7-ADCA en utilisant ladite expandase

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9404139B2 (en) * 2012-01-10 2016-08-02 Orchid Chemicals & Pharmaceuticals Limited Mutated cephalosporin hydroxylase and its application in deacetylcephalosporanic acid synthesis
CN114958877B (zh) * 2022-06-14 2024-02-20 河南省健康元生物医药研究院有限公司 去乙酰氧基头孢菌素c合成酶突变体及其编码基因与应用

Citations (4)

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Publication number Priority date Publication date Assignee Title
EP0366354A2 (fr) * 1988-10-24 1990-05-02 Eli Lilly And Company Déacétoxycéphalosporine C synthase purifiée
EP0532341A1 (fr) * 1991-09-11 1993-03-17 Gist-Brocades B.V. Nouveau procédé biologique de préparation de 7-AADC
WO1997020053A2 (fr) * 1995-11-27 1997-06-05 Gist-Brocades B.V. Procede ameliore de production de cephalosporines semi-synthetiques par l'activite de l'expandase sur la penicilline g
WO1998016648A2 (fr) * 1996-10-15 1998-04-23 Isis Innovation Limited Enzymes isopenicilline n synthetase et desacetoxycephalosporine c synthetase et procedes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0366354A2 (fr) * 1988-10-24 1990-05-02 Eli Lilly And Company Déacétoxycéphalosporine C synthase purifiée
EP0532341A1 (fr) * 1991-09-11 1993-03-17 Gist-Brocades B.V. Nouveau procédé biologique de préparation de 7-AADC
WO1997020053A2 (fr) * 1995-11-27 1997-06-05 Gist-Brocades B.V. Procede ameliore de production de cephalosporines semi-synthetiques par l'activite de l'expandase sur la penicilline g
WO1998016648A2 (fr) * 1996-10-15 1998-04-23 Isis Innovation Limited Enzymes isopenicilline n synthetase et desacetoxycephalosporine c synthetase et procedes

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BALDWIN J E ET AL: "HIGH-LEVEL SOLUBLE EXPRESSION AND PURIFICATION OF DEACETOXYCEPHALOSPORIN C-DEACETYLCEPHALOSPORIN C SYNTHASE.", BIOORG MED CHEM LETT, (1992) 2 (7), 663-668. CODEN: BMCLE8. ISSN: 0960-894X., XP002095824 *
BALDWIN J E ET AL: "HIGH-LEVEL SOLUBLE EXPRESSION AND PURIFICATION OF DEACETOXYCEPHALOSPORIN C-DEACETYLCEPHALOSPORIN C SYNTHASE.", BIOORG MED CHEM LETT, (1992) 2 (7), 663-668. CODEN: BMCLE8. ISSN: 0960-894X., XP002095825 *
CORTES, JESUS ET AL: "Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans", J. GEN. MICROBIOL. (1987), 133(11), 3165-74 CODEN: JGMIAN;ISSN: 0022-1287, 1987, XP000035085 *
DOTZLAF, JOE E. ET AL: "Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison wit the native enzyme purified from Streptomyces clavuligerus", J. BIOL. CHEM. (1989), 264(17), 10219-27 CODEN: JBCHA3;ISSN: 0021-9258, 1989, XP002095823 *
N. SHIBATA ET AL.: "Adipoyl-6-aminopenicillanic acid is a substrate for deacetoxycephalosporin C synthase (DAOCS).", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 6, no. 113, 1996, pages 1579 - 1584, XP002095827 *
ROACH P L ET AL: "CRYSTAL STRUCTURE OF ISOPENICILLIN N SYNTHASE IS THE FIRST FROM A NEW STRUCTURAL FAMILY OF ENZYMES", NATURE, vol. 375, no. 6533, 22 June 1995 (1995-06-22), pages 700 - 704, XP002059796 *
ROACH P L ET AL: "STRUCTURE OF ISOPENICILLINN SYNTHASE COMPLEXED WITH SUBSTRATE AND THE MECHANISM OF PENICILLIN FORMATION", NATURE, vol. 387, no. 6635, 19 June 1997 (1997-06-19), pages 827 - 830, XP002067787 *
ROLLINS M J ET AL: "ISOPENICILLIN N SYNTHASE AND DEACETOXYCEPHALOSPORIN C SYNTHASE ACTIVITIES DURING DEFINED MEDIUM FERMENTATIONS OF STREPTOMYCES-CLAVULIGERUS EFFECT OF OXYGEN AND IRON SUPPLEMENTS", CAN J MICROBIOL, (1989) 35 (12), 1111-1117. CODEN: CJMIAZ. ISSN: 0008-4166., XP002095822 *
ROLLINS, M. J. ET AL: "Purification and initial characterization of deacetoxycephalosporin C synthase from Streptomyces clavuligerus", CAN. J. MICROBIOL. (1988), 34(11), 1196-202 CODEN: CJMIAZ;ISSN: 0008-4166, 1988, XP002095821 *
SCOTT R A ET AL: "X-RAY ABSORPTION SPECTROSCOPIC STUDIES OF THE HIGH-SPIN IRON(II) ACTIVE SITE OF ISOPENICILLIN N SYNTHASE: EVIDENCE FOR FE-S INTERACTION IN THE ENZYME-SUBSTRATE COMPLEX", BIOCHEMISTRY, vol. 31, no. 19, 1992, pages 4596 - 4601, XP002067783 *
VALEGARD, KARIN ET AL: "Structure of a cephalosporin synthase", NATURE (LONDON) (1998), 394(6695), 805-809 CODEN: NATUAS;ISSN: 0028-0836, 1998, XP002095826 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085951A1 (fr) * 2000-05-09 2001-11-15 Acs Dobfar Uk Limited Expandase modifiee et utilisations
EP1348759A1 (fr) * 2002-03-26 2003-10-01 Synmax Biochemical Co., Ltd Expandase de penicillin mutée et un procédé de production de 7-ADCA en utilisant ladite expandase
US6905854B2 (en) 2002-03-26 2005-06-14 Synmax Biochemical Co., Ltd. Mutated penicillin expandase and process for preparing 7-ADCA using the same

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PL341396A1 (en) 2001-04-09
SK9762000A3 (en) 2001-02-12
JP2002500015A (ja) 2002-01-08
CN1284997A (zh) 2001-02-21
CA2316401A1 (fr) 1999-07-08
AU1772099A (en) 1999-07-19

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