[go: up one dir, main page]

WO2007023369A1 - Expandase-hydroxylase modifiee et applications correspondantes - Google Patents

Expandase-hydroxylase modifiee et applications correspondantes Download PDF

Info

Publication number
WO2007023369A1
WO2007023369A1 PCT/IB2006/002303 IB2006002303W WO2007023369A1 WO 2007023369 A1 WO2007023369 A1 WO 2007023369A1 IB 2006002303 W IB2006002303 W IB 2006002303W WO 2007023369 A1 WO2007023369 A1 WO 2007023369A1
Authority
WO
WIPO (PCT)
Prior art keywords
hydroxylase
lysine
expandase
threonine
substituted
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.)
Ceased
Application number
PCT/IB2006/002303
Other languages
English (en)
Inventor
Durairaj Micheal
Twinkle Jasmine Masilamani
Meghana Ravindranathan
Ramakrishna Padharthi
Vasu Vinayagam
Ramanan Thirumoorthy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Orchid Pharma Ltd
Original Assignee
Orchid Chemicals and Pharmaceuticals Ltd
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
Application filed by Orchid Chemicals and Pharmaceuticals Ltd filed Critical Orchid Chemicals and Pharmaceuticals Ltd
Publication of WO2007023369A1 publication Critical patent/WO2007023369A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • 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.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • the present invention provides modified expandase-hydroxylase from Cephalosporin.TM acremonium ⁇ Acremonium chrysogenum) having better ring-expansion and hydroxylation activity and increased specificity towards substrates such as Penicillin G, Penicillin N, Phenyl acetyl 7-ADCA and deacetoxy cephalosporin C (DAOC).
  • the modified expandase-hydroxylase from Cephalosporium acremonium is found useful in the bioprocess for the preparation of 7-ACA.
  • ⁇ -lactam antibiotics such as Penicillins and Cephalosporins are widely used for the treatment of a variety of infectious diseases.
  • Cephalosporins offer better protection than penicillins particularly against resistant organisms, significant thrust has been given to derivatizations of Cephalosporins to broaden their spectrum and enhance their efficacy.
  • 7-amino deacetoxy cephalosporanic acid (7-ADCA) and 7-amino cephalosporanic acid (7-ACA) serve as core intermediates for the synthesis of numerous semisynthetic cephalosporins such as Cephradine, Cephalexin, Cephadroxyl, Cefazolin, Cefotaxime, Ceftriaxone, Cefepime, Cefoperazone, and the like.
  • 7- ACA is derived from Cephalosporin C by the cleavage of the 7-aminoadipyl side chain by either a chemical process or by a two stage enzymatic process (US 5,424,196) and 7-ADCA is derived from the hydrolysis of Phenyl acetyl 7-ADCA by Penicillin G amidase, wherein the Phenyl acetyl 7-ADCA is manufactured by conventional chemistry of ring expansion of Penicillin G.
  • the industrial process of manufacture of these intermediates has become non-competitive, hazardous and generates considerable effluent treatment issues. Alternate technologies and processes, which offer cost-effective and environment friendly manufacture of these compounds, are needed.
  • Penicillins and Cephalosporins are produced by a variety of bacteria and fungal organisms and significant progress has been made on understanding their regulatory architecture (Aharonowitz, Y., et al., Annu. Rev. Microbiol. 46: 461-495, 1992; Axel A. Brakhage, Microbiol. MoL Biol. Rev. 62: 547-585, 1998).
  • DAC Deacetyl cephalosporin C gets acetylated to Cephalosporin C in Cephalosporium acremonium, while further deviation occurs resulting in Cephamycin C in bacteria such as Streptomyces clavuligerus.
  • the deacetoxy cephalosporin C synthase also known as expandase is encoded by a gene ce/E; wherein ce/E gene from S. clavuligerus contains 936 nucleotides and 311 amino acids.
  • ce/E gene from S. clavuligerus contains 936 nucleotides and 311 amino acids.
  • the deacetyl cephalosporin C synthase from S. clavuligerus has 318 amino acids and is encoded by ce/F.
  • the ce/EF gene that codes for 332 amino acids encodes the bifunctional expandase-hydroxylase in C. acremonium.
  • Dotzlaf. J. E. et ah Journal of Bacteriology, 169, 1611-1618, 1987 purified, characterized and studied the kinetics and stochiometry of this enzyme.
  • DAOCS-DACS nucleotide sequence of C. acremonium is 67% identical to that of DAOCS of S. clavuligerus and the amino acid sequences are 56.7% identical.
  • the DAOCS-DACS enzyme has 54% amino acid sequence identity with that of the hydroxylase enzyme; of S. clavuligerus ( Figure 1).
  • Expandase, Expandase-hydroxylase and hydroxylase are iron (II) and ⁇ - ketoglutarate dependent oxygenases and they are part of a subfamily of the mononuclear ferrous enzymes.
  • Penicillin N is the natural substrate for expandase and expandase-hydroxylase (DAOCS-DACS) and DAOC is the substrate for hydroxylase.
  • DOCS-DACS expandase-hydroxylase
  • DAOC is the substrate for hydroxylase.
  • altered substrate specificity has been detected for different substrates such as Penicillin G, Penicillin V, 6- ⁇ - MethylPenicillin N and adipoyl-6-APA (Lloyd, M. D., et. ah, Journal of Biological Cnemistry, 279, 15420-15426, 2004).
  • As a result development of green process technologies for the manufacture of Cephalosporin intermediates, thus, narrowed down to these enzymes.
  • deacetyl phenyl acetyl 7-ACA can be modified by conventional chemistry to develop desirable intermediates readily. Taking into account the commercial importance of producing Cephalosporin intermediates, it is imperative to identify a modified mutant expandase-hydroxylase from C.acremonium having increased substrate specificity for substrates such as Penicillin G and Phenyl acetyl 7-ADCA, when compared with the wild-type expandase-hydroxylase. Hence we have focused on this and have succeeded in identifying a modified expandase- hydroxylase from C.acremonium.
  • SEQ ID No: 1 describes wild-type nucleotide and amino acid sequence for expandase-hydroxylase of C.acremonium.
  • An objective of this invention is to provide a modified mutant expandase- hydroxylase from C.acremonium with improved capability for ring expansion and hydroxylation than that occurs in the natural wild type expandase-hydroxylase.
  • Another objective of this invention is to provide a modified expandase- hydroxylase having increased ring-expansion on substrates like Penicillin G, increased hydroxylation activity on substrates like Phenyl acetyl 7-ADCA and improved expansion and hydroxylation activity on substrates such as Penicillin G and Penicillin N.
  • the present invention provides a mutated expandase-hydroxylase from C.acremonium, which consists of amino acid substitution at one or more amino acid residues corresponding to the wild type expandase-hydroxylase for the following group of residues consisting of Lysine at position 14, Serine at position 15, Threonine at position 20, Threonine at position 45, Glutamic acid at position 49, Lysine at position 56, Aspartic acid at position 70, Asparagine at position 72, Alanine at position 73, Valine at position 87, Lysine at position 93, Lysine at position 131, Tyrosine at position 185, Isoleucine at position 190, Tyrosine at position 203, Glutamic acid at position 212, Phenylalanine at position 226, Threonine at position 232, Lysine at position 247, Threonine at position 260, Lysine at position 269, Asparagine at position 275, Asparagine at position 285, Tryptophan at position 282, I
  • the invention provides mutants with amino acid substitutions at one or more amino acid residues of S15G, T20A, K56E, D70A, N72S, N72D, N72T, V87A, K93E, K131R, Y185C, I190V, Y203C, K247R, T260A, K269R, N275D, W282R, I288T, I288C, T293M, T293A, R296G, N305T, N305I, ⁇ 310, T20A: T293A, K14R: T293A, S15G: R296G, N72S: F226L, N72S: E212G: K269R, E49G: I288T: R296G, K56E: N72S: T232A, N72S: F226L: E49G, N72S: F226L: W282R, N285D: T293A, A73T: T332
  • Another embodiment of the invention is to provide an expandase-hydroxylase protein with modified hydroxylation activity.
  • the invention provides a recombinant vector specifically an expression Vector, which comprises the modified expandase-hydroxylase gene.
  • the present invention further relates to a host strain that contains the expression vector with the modified expandase-hydroxylase (ce/EF) gene.
  • Another embodiment of the invention is to provide a method of expression of expandase-hydroxylase in a host strain that contains the expression vector with the modified expandase-hydroxylase (ce/EF) gene.
  • the primary embodiment of the present invention is to provide a mutant expandase-hydroxylase having increased activity and greater specificity for substrates such as Penicillin G and Phenyl acetyl 7-ADCA than the wild type expandase- hydroxylase from the filamentous fungi C. acremonium.
  • This enzyme and the corresponding gene (ce/EF gene) has been isolated and characterized (Dotzlaf J. E. et ah, Journal of Bacteriology, 169, 1611-1618, 1987).
  • the wild-type nucleotide and amino acid sequence of expandase-hydroxylase from C. acremonium is given in SEQ ID No: 1 ( Figure 2).
  • a mutated Penicillin expandase-hydroxylase which comprises amino acid substitution at one or more amino acid residues corresponding to the wild type expandase-hydroxylase for the group of residues consisting of Lysine at position 14, Serine at position 15, Threonine at position 20, Threonine at position 45, Glutamic acid at position 49, Lysine at position 56, Aspartic acid at position 70, Asparagine at position 72, Alanine at position 73, Valine at position 87, Lysine at position 93, Lysine at position 131, Tyrosine at position 185, Isoleucine at position 190, Tyrosine at position 203, Glutamic acid at position 212, Phenylalanine at position 226, Threonine at position 232, Lysine at position 247, Threonine at position 260, Lysine at position 269, Asparagine at position 275, Tryptophan at position 282, Asparagine at position 285, Isoleucine at position 288, Thre
  • the invention provides mutants with amino acid substitutions at one or more amino acid residues of S15G, T20A, K56E, D70A, N72S, N72D, V87A, K93E, K131R, Y185C, I190V, Y203C, K247R, T260A, K269R, N275D, W282R, I288T, I288C, T293M, T293A, R296G, N305T, N305I, ⁇ 310, T20A: T293A, K14R: T293A, S15G: R296G, N72S: F226L, A73T: T332I, K131R: Y185C, N285D: T293A, N72S: E212G: K269R, E49G: I288T: R296G, K56E: N72S: T232A, N72S: F226L: E49G, N72S: F226L:
  • Threonine at position 20 is substituted by Alanine and Threonine at position 293 is substituted by Alanine
  • the present invention provides a mutated Penicillin expandase-hydroxylase having enhanced ring-expansion activity for substrates such as Penicillin G containing the following mutations at one or more amino acid positions when compared with the wild-type: S15G, K56E, V87A, Kl 3 IR, T260A, K247R, K269R, W282R, N305T, N72S:
  • F226L E49G
  • N72S F226L: W282R
  • N72S E212G: K269R
  • E49G: I288T R296G.
  • the present invention also provides a mutated Penicillin expandase-hydroxylase having enhanced hydroxylation activity for substrates such a Phenyl acetyl 7-ADCA containing the following amino acid substitutions at one or more amino acid residues when compared with the wild- type: K93E, Y203C, N275D, D310 and K14R: T293A.
  • the present invention provides a modified Penicillin expandase-hydroxylase having enhanced catalytic activity for both ring expansion and hydroxylation on substrates like Penicillin G and Phenyl acetyl 7-ADCA when compared with the wild- type expandase-hydroxylase.
  • the modified Penicillin expandase-hydroxylase carries the following amino acid substitutions at one or more amino acid residue positions: T20A, D70A, N72S, N72T, N72D, Y185C, Il 90V, I288T, 1288C 5 T293M, T293A, R296G, N305I, T20A: T293A, S15G: R296G, N72S: F226L, A73T: T332I, K131R: Y185C, N285D: T293A, K56E: N72S: T232A, N72S: F226L: E212G, N72S: F226L: Y185C, N72S: F226L: M306L wherein the substitution at 306 position is not I, N72S: F226L: R296G, N72S: F226L: K93E, N72S: F226L: K269R, N72S: F226L:
  • Another embodiment of the invention is to provide an isolated nucleic acid molecule that codes for the mutated Penicillin expandase-hydroxylase.
  • this isolated nucleic acid molecule is obtained by mutating the wild type expandase-hydroxylase.
  • the mutagenesis technique could be by chemical, error- prone PCR or site- directed approach.
  • the suitable mutagenesis technique can be selected and used for introducing mutations and the mutated nucleic acid molecule can be cloned and expressed and the property of the polypeptide can be studied.
  • the mutated nucleic acid molecule may be incorporated into a recombinant vector, which is capable of expression or replication when transferred into a host cell. Expression of the polypeptide can be controlled by a regulatory sequence probably a promoter.
  • the recombinant vector can be introduced into a host strain to produce the mutated Penicillin expandase-hydroxylase.
  • the mutated expandase-hydroxylase when expressed in the host strain is capable of converting the substrate Penicillin G into Phenyl acetyl 7-ADCA by ring expansion and Phenyl acetyl 7-ADCA to deacetyl phenyl acetyl 7-ACA by hydroxylation.
  • Phenyl acetyl 7-ADCA when treated with a suitable enzyme such as PenG Amidase (EP0453047), the Phenyl acetyl side chain can be removed efficiently and 7- ADCA can be produced.
  • Deacetyl phenyl acetyl 7-ACA can lead to 7-ACA after acetylation by acetyl transferase or suitable chemical conversion followed by enzymatic hydrolysis using Pen G amidase. It has been found that the mutated expandase-hydroxylase when expressed in the host strain according to this invention has greater activity against the conversion of penicillin N into deacetoxy cephalosporanic acid or deacetylcephalsoporanic acid, followed by converting the ensuing product into 7-ACA via cephalosporin C. According to the present invention, the modified peptide has amino acid sequence different from that of SEQ ID NO: 1. This polypeptide is one, which has ring expansion activity i.e.
  • the invention provides a modified expandase-hydroxylase, which has an enhanced catalytic activity or increased specificity for other substrates such as Penicillin G, Phenyl acetyl 7-ADCA when compared with the wild-type expandase- hydroxylase.
  • polypeptides thus produced from the mutated nucleotide sequence can be used to produce chimeras from portions of other expandase-hydroxylase, expandase and hydroxylase polypeptides.
  • Polypeptides from the present invention can be purified with varying level of homogeneity and can be used for other purposes.
  • the modified expandase-hydroxylase produced according to the invention is found useful in the bioprocess of preparing Cephalosporin C or Deacetyl Cephalosporanic acid by utilizing the process known in the prior art.
  • the invention can be used for the manufacture of modified cephalosporins either as enzymatic or in vivo
  • Cephalosporium acremonium growth conditions
  • the C. acremonium strain (ATCC No. 11550) was grown under the following medium composition: Conidia was grown in plates containing complete media (sucrose 20 gm, peptone 4 gm, yeast extract 4 gm, NaNO 3 3 gm, KH 2 PO 4 0.5 gm, K 2 HPO 4 0.5 gm, KCl 0.5 gm, MgSO 4 .7H 2 O 0.5 gm, FeSO 4 JH 2 O 0.01 gm and agar 20 gm made up to 1 litre of distilled water at a pH of 6.6) at 25°C.
  • complete media sucrose 20 gm, peptone 4 gm, yeast extract 4 gm, NaNO 3 3 gm, KH 2 PO 4 0.5 gm, K 2 HPO 4 0.5 gm, KCl 0.5 gm, MgSO 4 .7H 2 O 0.5 gm, FeSO 4 JH 2 O 0.01 gm and agar
  • Genomic DNA was isolated from the mycelia using the DNeasy Plant mini gDNA isolation kit (Qiagen) as recommended by the supplier.
  • the gene coding for the expandase-hydroxylase (ce/EF) was amplified using 20 pmole of primers 5' TCCATATGACTTCCAAGGTCCCCGT 3' and 5' GAAACTCTTGACACTCATGCTTTGTGT 3', 200 ⁇ M dNTPs, 5% DMSO, 10x deep vent DNA polymerase buffer, 2.5U deep vent DNA polymerase enzyme and water in a final reaction volume of 100 ⁇ l.
  • PCR condition consists of an initial denaturation for 5 min at 95 0 C followed by 24 cycles consisting of denaturation at 95 0 C for 40 sec, annealing at 6O 0 C for 1 min, extension at 72°C for 5 min with a final extension at 72°C for 15 min.
  • An amplified product of length of approximately 1100 bp of the cefEF gene fragment was seen by agarose gel electrophoresis.
  • the native EF gene fragment cloned into pUC19 served as template for error-prone PCR mutagenesis.
  • the amplification was carried out with 20 pmole of 5' ATCGGTGCGGGCCTCTTCGCTATT 3' and 5'
  • coli BL21 (DE3) recombinants were screened for inserts by colony PCR using 20 pmoles of 5' TCCATATGACTTCCAAGGTCCCCGT 3', 5' GCTAGTTATTGCTCAGCGG 3' primers in a reaction mix containing 10% DMSO, 1Ox Taq DNA polymerase buffer, IU Taq DNA polymerase enzyme and 80 ⁇ M dNTPs in a total reaction volume of 50 Dl.
  • Glycerol stocks containing the putative mutant EF genes in pET24a (+) vector in E.coli BL21 (DE3) strain was inoculated in 96-well plate containing LB medium with Kanamycin (75 ⁇ g/ml) for overnight growth at 37°C at 220 rpm. Overnight culture was subcultured again in 96-well deep well plates and grown till OD 60O reached to 0.6 to 0.8 5 and the induction was carried out with 0.1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the culture was allowed to grow for 3 hours at 25°C and pellet was harvested by centrifugation in a micro plate centrifuge at 4,000 rpm for 10 min at 4 0 C.
  • the pellets were resuspended in a buffer containing 50 mM Tris. HCl (pH 7.5), 0.1 mM DTT, 0.01 mM EDTA, 10% Glycerol, 50 mM Glucose and stored at -8O 0 C. 0
  • Enzyme was released using bugbuster reagent and the ring expansion reaction was assayed with 45.7 mM Ammonium Sulfate, 0.9 mM ⁇ -keto glutarate, 0.9 mM Ascorbate, 0.9 mM DTT, 0.9 mM Ferrous sulfate, 12.8 mM Penicillin G and 16 mM Tris.HCl in a
  • the short-listed positive isolates were re-expressed in 50 ml conical flasks containing 10 ml LB with 75 ⁇ g /ml Kanamycin in which over night grown culture was inoculated. After OD 60O of 0.6 to 0.8 was reached, induction was initiated by adding 0.1 mM IPTG. Pellets were harvested from 1.5 ml culture after allowing the culture to grow for 3 hours at 25°C. The pellets were resuspended in 50 mM Tris. HCl (pH 7.5), 0.1 mM DTT, 0.01 mM EDTA, 10% Glycerol, 50 mM Glucose and stored at -8O 0 C.
  • Lysis of the resuspended pellet was carried out at 25 0 C for 25 min at 220 rpm using Bugbuster reagent.
  • the ring expansion reaction was assayed using 35.6 mM Ammonium Sulfate, 0.7 mM Ascorbate, 0.7 mM DTT, 0.7 mM ⁇ -keto glutarate, 0.07 mM Ferrous sulfate, 10 mM Penicillin G and 12 mM Tris-HCl at pH 7.5 in a reaction volume of 450 ⁇ l at 25 0 C for 30 minutes.
  • the reaction was quenched with 75 ⁇ l methanol, centrifuged and the supernatant was analyzed. Reconfirmation reaction for hydroxylation activity was performed using the same reagent concentrations as that used for screening.
  • Cis symmetry column was equilibrated with buffer A containing 3.12g of Sodium phosphate in water with pH adjusted to 2.4 using ortho phosphoric acid.
  • the assay components were eluted using buffer B containing 100% Acetonitrile: buffer A at a ratio of 80:20 using a flow rate of 1.5 ml/min.
  • the components were detected at 215nm and 260 nm and 40 ⁇ l of sample was injected.
  • the retention times of the compounds under these conditions are 2.1 min, 4.3 min and 5.2 min for deacetyl phenyl acetyl 7-ADCA, Phenyl acetyl 7-ADCA and Penicillin G respectively.
  • Site-directed mutagenesis Single-stranded templates of either native or mutant gene in pET24a vector were generated from E. coli CJ236 using M13K07 helper phage by standard procedure as described in "Molecular Cloning, A laboratory Manual, 2 nd Edition by Sambrook et al, Cold Spring Harbor Laboratory Press, 1989 and the mutagenesis was based on Kunkel mutagenesis principle (Kunkel, T. A. Proc. Natl. Acad. Sci. USA, 82, 488-492 1985) as follows.
  • oligonucleotides to induce desired mutations were annealed either alone or in multiple combinations to the single-strand templates and the in vitro second-strand synthesis was carried out in presence of 200 ⁇ M of each dNTPs, 0.2 mg/ml of BSA, 0.5 mM of ATP, 2 units of T4 DNA ligase, 3 units of T4 DNA polymerase and 10 niM MgCl 2 at 42 0 C for 20 minutes in a reaction volume of 20 ⁇ L. After terminating the reaction with 2 ⁇ l of 0.5 M EDTA, 1 ⁇ l was used for transformation of E. coli DH5 ⁇ . Mutants were confirmed with restriction analysis followed by DNA sequencing.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne une expandase-hydroxylase mutée présentant une activité améliorée et une spécificité de substrat supérieur pour la Pénicilline G et l'acétyle de phényle 7-ADCA pour la production d'acétyle de phényle - 7-ADCA et d'acétyle de phényle désacétyle 7-ACA, respectivement; comportant une ou plusieurs modifications d'acides aminés à des positions de résidus par rapport au type sauvage de l'expandase-hydroxylase choisis dans le groupe de résidus comprenant, Lysine à la position 14, Serine à la position 15, Threonine à la position 20, Threonine à la position 45, acide glutamique à la position 49, Lysine à la position 56, acide aspartique à la position 70, Asparagine à la position 72, Alanine à la position 73, Valine à la position 87, Lysine à la position 93, Lysine à la position 131, Tyrosine à la position 185, Isoleucine à la position 190, Tyrosine à la position 203, acide glutamique à la position 212, Phenylalanine à la position 226, Threonine à la position 232, Lysine à la position 247, Threonine à la position 260, Lysine à la position 269, Asparagine à la position 275, Asparagine à la position 285, Tryptophane à la position 282, Isoleucine à la position 288, Threonine à la position 293, Arginine à la position 296, Lysine à la position 310 et Threonine à la position 332.
PCT/IB2006/002303 2005-08-25 2006-08-24 Expandase-hydroxylase modifiee et applications correspondantes Ceased WO2007023369A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1177/CHE/2005 2005-08-25
IN1177CH2005 2005-08-25

Publications (1)

Publication Number Publication Date
WO2007023369A1 true WO2007023369A1 (fr) 2007-03-01

Family

ID=37771266

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2006/002303 Ceased WO2007023369A1 (fr) 2005-08-25 2006-08-24 Expandase-hydroxylase modifiee et applications correspondantes

Country Status (1)

Country Link
WO (1) WO2007023369A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107782A3 (fr) * 2007-03-06 2008-11-13 Orchid Chemicals & Pharm Ltd Hydroxylase modifiée et ses applications
WO2017181809A1 (fr) * 2016-04-18 2017-10-26 百瑞全球有限公司 Expandase de pénicilline mutante, adn codant pour la mutante, et trousse de réactif contenant la mutante et son utilisation
CN113337494A (zh) * 2020-01-17 2021-09-03 浙江大学 一种l-苏氨酸醛缩酶突变体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107782A3 (fr) * 2007-03-06 2008-11-13 Orchid Chemicals & Pharm Ltd Hydroxylase modifiée et ses applications
WO2017181809A1 (fr) * 2016-04-18 2017-10-26 百瑞全球有限公司 Expandase de pénicilline mutante, adn codant pour la mutante, et trousse de réactif contenant la mutante et son utilisation
US10988742B2 (en) 2016-04-18 2021-04-27 Bioright Worldwide Co., Ltd. Penicillin expandase mutants, DNA coding the mutants, reagent kit containing the mutants and the application
CN113337494A (zh) * 2020-01-17 2021-09-03 浙江大学 一种l-苏氨酸醛缩酶突变体及其应用
CN113337494B (zh) * 2020-01-17 2023-12-26 浙江大学 一种l-苏氨酸醛缩酶突变体及其应用

Similar Documents

Publication Publication Date Title
CN105543201B (zh) 一种头孢菌素c酰化酶突变体
US20030100065A1 (en) NADH oxidase from Lactobacillus
JP5142268B2 (ja) 改良型没食子酸合成酵素および没食子酸の製造法
WO2018165881A1 (fr) Mutants de céphalosporine c acylase et leurs applications
WO2008107782A2 (fr) Hydroxylase modifiée et ses applications
WO2001085951A1 (fr) Expandase modifiee et utilisations
WO2007023369A1 (fr) Expandase-hydroxylase modifiee et applications correspondantes
US10988742B2 (en) Penicillin expandase mutants, DNA coding the mutants, reagent kit containing the mutants and the application
US9404139B2 (en) Mutated cephalosporin hydroxylase and its application in deacetylcephalosporanic acid synthesis
US20050158818A1 (en) Cephalosporin C acylases
EP1799817B1 (fr) Expandases mutantes
US6699699B2 (en) Mutated penicillin expandases
JP2007533316A (ja) 修飾エキスパンダーゼ酵素とその使用
RU2829362C1 (ru) РЕКОМБИНАНТНАЯ ПЛАЗМИДА pNhaDAAO, ОБЕСПЕЧИВАЮЩАЯ СИНТЕЗ ОКСИДАЗЫ ОБЫЧНЫХ И N-ЗАМЕЩЕННЫХ D-АМИНОКИСЛОТ (NhaDAAO) АРХЕЙ Natrarchaeobius halalkaliphilus AArcht4 (NhaDAAO) В КЛЕТКАХ Escherichia coli, И РЕКОМБИНАНТНЫЙ ШТАММ Escherichia coli BL21(DE3)/pNhaDAAO ВКПМ В-14556 - ПРОДУЦЕНТ NhaDAAO
KR102363768B1 (ko) 세파졸린 생산성이 증가된 페니실린 g 아실라제 변이체 및 이의 이용
WO2009013611A2 (fr) Estérase modifiée et ses applications
WO2011021717A2 (fr) Procédé de production d'acides aminés hydroxylés
JP4639235B2 (ja) Ｌ−カルニチン生合成関連遺伝子を含む腸内細菌属微生物、およびその微生物を用いたｌ−カルニチンの産生方法
JP5578598B2 (ja) 新規な芳香環水酸化酵素及びその遺伝子
CN117363591A (zh) 用于合成g-7-adca的扩环酶突变体
KR20080028924A (ko) 변이주 익스팬다제 및 베타-락탐 화합물의 생성에 있어서이들의 용도
CN119144591A (zh) 一种具有底物特异性的头孢菌素c酰化酶突变体及其应用
CN1965083A (zh) 经修饰的扩展酶及其用途
HK1125973A (en) Mutant expandases and their use in the production of beta-lactam compounds
EP1851309A2 (fr) Mutants de acyltransferase de isopenicillin n

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06795322

Country of ref document: EP

Kind code of ref document: A1