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
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)

Definitions

• IPC International Patent Classification
• SHERMAN DR ET AL "Mycobacte ⁇ ' um avi u al kyl hydroxperoxidase C (ahpC) gene
• HAHN JS AND ROE JH "Strepto yces coelicolor A3(2) ahpD, ahpC, and oxyR genes"
• PAGAN-RAMOS E ET AL Mycobacterium marinum ahpC and oxyR genes
• KRAMER R "Genetic and physiological approaches for the production of amino acids
• Present claim la relates to a polynucleotide having at least 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
• the invention provides nucleotide sequences from coryneform bacteria which code for the oxyR gene and a process for the fermentative preparation of amino acids, in particular L- lysine, using bacteria in which the oxyR gene is enhanced.
• the oxyR gene codes for the transcription regulator OxyR, which belongs to the LysR family.
• L-Amino acids in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
• amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
• fermentation measures such as, for example, stirring and supply of oxygen
• the composition of the nutrient media such as, for example, the sugar concentration during the fermentation
• the working up to the product form by, for example, ion exchange chromatography or the intrinsic output properties of the microorganism itself.
• Methods of utagenesis, selection and mutant selection are used to improve the output properties of these microorganisms.
• Strains which are resistant to anti etabolites, such as e.g. the lysine analogue S-(2- aminoethyl) -cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-lysine are obtained in this manner.
• Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
• the inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-lysine.
• amino acids including their salts, chosen from the group consisting of L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.
• L-asparagine L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.
• L-lysine or lysine are mentioned in the following, this also means the salts, such as e.g. lysine monohydrochloride or lysine sulfate.
• the invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the oxyR gene chosen from the group consisting of
• polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
• polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70 % to the amino acid sequence of SEQ ID No.2,
• polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c),
• polypeptide preferably having the activity of the transcription regulator OxyR.
• the invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:
• the invention also provides
• polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1 and 490;
• polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 491 and 1471; and c) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1472 and 1675.
• the invention also provides
• polynucleotide in particular DNA, which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No. 1;
• polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID 10 No. 2;
• coryneform bacteria serving as the host cell, which contain 15. the vector or in which the oxyR gene is enhanced.
• the invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which 20 comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotide according to the invention according to SEQ ID No.l or a fragment thereof, and isolation of the polynucleotide sequence mentioned.
• Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for the transcription regulator OxyR, or to isolate those nucleic acids or
• polynucleotides or genes which have a high similarity of sequence with that of the oxyR gene are also suitable for incorporation into so-called “arrays”, “micro arrays” or “DNA chips in order to detect and determine the corresponding polynucleotides.
• Polynucleotide sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for the transcription regulator OxyR can be prepared with the polymerase chain reaction (PCR) .
• PCR polymerase chain reaction
• Such oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides.
• Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are optionally also suitable.
• Polynucleotide in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
• the polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom and also those which are at least in particular 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom.
• Polypeptides are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
• polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those o IV) ) I- 1 H 1 o c ⁇ O ⁇ o n
• Suitable strains of the genus Corynebacterium in particular of the species Corynebacterium glutamicum (C. glutamicum) , are in particular the known wild-type strains
• Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and
• the inventors have succeeded in isolating the new oxyR gene of C. glutamicum which codes for the transcription regulator OxyR.
• E. coli Escherichia coli
• the setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Amsterdam Einf ⁇ hrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example.
• a well-known gene library is that of the E. coli K-12 strain W3110 set up in ⁇ vectors by Kohara et al. (Cell 50, 495 -508 (1987)).
• strain DH5 ⁇ mcr which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649).
• the long DNA fragments _ cloned with the aid of cosmids can in turn be subcloned in the usual vectors suitable for sequencing and then sequenced, as is described e.g. by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
• the resulting DNA sequences can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217-232 (1986)), that of Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).
• known algorithms or sequence analysis programs such as e.g. that of Staden (Nucleic Acids Research 14, 217-232 (1986)), that of Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).
• H H 1 Hi 3 • c ⁇ ⁇ ⁇ 3 C ⁇ tr ro 3 ⁇ 3 C ⁇ o H ⁇ - rt H ⁇ rt H 1 CO Hi Hi O ⁇ ⁇ O ⁇ o H 1 ⁇ X ⁇ . tr > C ⁇ ⁇ co ⁇ H O 3 C ⁇ O rt ⁇ Q ⁇ Hi 3 ⁇ ⁇ ⁇
• the hybridization takes place under stringent conditions, that is to say only hybrids in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 70 % identical are formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .
• a 5x SSC buffer at a temperature of approx. 50 - 68°C, for example, can be employed for the hybridization reaction.
• Probes can also hybridize' here with polynucleotides which are less than 70 % identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2x SSC and subsequently 0.5x SSC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approx. 50 - 68°C being established. It is optionally possible to lower the salt concentration to O.lx SSC.
• Polynucleotide fragments which are, for example, at least 70 % or at least 80 % or at least 90 % to 95 % identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise in steps of approx. 1 - 2°C. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558). CO CO ro ro
• Suitable plasmids are those which are replicated in coryneform bacteria.
• Numerous known plasmid vectors such as e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl . (Eikmanns et al., Gene 102:93-98 (1991)) or ' pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLl or pGAl .
• plasmid vectors such as e.g. those based on pCG4 (US-A 4,489,160) or pNG2. (Serwold-Davis et al., FEMS Microbiology Letters 66, 119- 124 (1990)) or pAGl (US-A 5,158,891), can be used in the same manner.
• a plasmid with the aid of which the oxyR gene can be over-expressed is the E. coli-C. glutamicum shuttle vector pT-oxyRexp. It contains the replication region rep of the plasmid pGAl including the replication effector per (US-A- 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance- imparting tetA(Z) gene of the plasmid pAGl (US-A- 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with accession number AF121000, the replication origin oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979)), the lacZ ⁇ gene fragment including the lac promoter and a "multiple cloning site" (mcs) (Norrander,
• the plasmid pT-oxyRexp is shown in figure 2.
• Plasmid vectors which are furthermore suitable are also those with the aid of which the process of gene amplification by integration into the chromosome can be used, as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994) ) for duplication or amplification of the hom-thrB operon.
• the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum.
• Possible vectors are, for example, pSUP301.
• amino acids in particular L-lysine
• enzymes of the particular biosynthesis pathway of glycolysis, of anaplerosis, of the pentose phosphate cycle, of the citric acid cycle or of amino acid export and optionally regulatory proteins, in addition to the oxyR gene.
• amino acids in particular L-lysine
• amino acids in particular L-lysine
• oxyR gene for one or more genes chosen from the group consisting of
• the term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme (protein) , and optionally combining these measures.
• microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids, in particular L-lysine.
• batch culture batch culture
• feed process fed batch
• repetitive feed process repetition feed process
• a summary of known culture methods is described in the textbook by Ch iel (Bioreatechnik 1. Einfiihrung in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere bamboo (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) .
• the culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
• Sugars and carbohydrates such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty ' acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substance can be used individually or as a mixture.
• oils and fats such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat
• fatty ' acids such as e.g. palmitic acid, stearic acid and linoleic acid
• alcohols such as e.g. glycerol and ethanol
• organic acids such as e.g. acetic acid
• Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep CO CO ro ro c ⁇ o c ⁇ o c ⁇ O c ⁇
• the process according to the invention is used for the fermentative preparation of amino acids, in particular L-lysine ._
• composition of the usual nutrient media such as LB or TY medium, can also be found in the handbook by Sambrook et al. CO ro ro H> H» o C ⁇ o C ⁇ O c ⁇
• the cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02) .
• the DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
• the cosmid fragments in the size range of 1500 to 2000 bp were isolated with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
• the DNA of the sequencing vector pZero-1 obtained from Invitrogen (Groningen, Holland, Product Description Zero Background Cloning Kit, Product No. K2500-01) , was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04) .
• the ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany) . This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strain DH5 ⁇ MCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 21
• the plasmid preparation of the recombinant clones was carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) .
• the sequencing was carried out by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067).
• the "RR dRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosyst ' ems (Product No. 403044, Rothstadt, Germany) was used.
• the raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0.
• the individual sequences of the pZerol derivatives were assembled to a continuous contig.
• the computer-assisted coding region analysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).
• the resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 981 base pairs, which was called the oxyR gene. The oxyR gene codes for a protein of 327 amino acids. 22
• chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817 -1828 (1994) ) .
• the sequence of the oxyR gene known for C. glutamicum from example 2 was chosen for the polymerase chain- reaction (see SEQ ID No. 3 and SEQ ID No. 4) .
• the primer OxyR (oxy-exp) contains the sequence for the cleavage site of the restriction endonuclease EcoRI, and the primer OxyR (oxy R2) the cleavage site of the restriction endonuclease Xbal, which are marked by underlining in the nucleotide sequence shown above .
• the amplified DNA fragment of approx. 1.43 kb which carries the oxyR gene was ligated with the Zero BluntTM Kit of
• E. coli strain- ToplO (Grant et al., Proceedings of the National ' Academy of Sciences USA, 87 (1990) 4645-4649) was then transformed with the ligation batch in accordance with the instructions of the manufacturer . of the kit (Invitrogen Corporation, Carlsbad, CA, USA) . Selection for plas id- carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 25 mg/1 kanamycin.
• Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and checked by treatment with the restriction enzyme Xbal and EcoRI with subsequent agarose gel electrophoresis (0.8 %) . The DNA sequence of the amplified DNA fragment was checked by sequencing.
• the plasmid was called pCR-oxyRexp.
• the strain was called E. coli ToplO / pCR-oxyRexp.
• the E. coli - C. glutamicum shuttle vector was constructed according to the prior art.
• the vector contains the replication region reg of the plasmid pGAl including the replication effector per (US-A- 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance-imparting tetA(Z) gene of the plasmid pAGl (US-A- 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with the accession number AF121000), the replication region oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979)), the lacZ ⁇ gene fragment including, the lac...
• Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and Hindlll and subsequent agarose gel electrophoresis (0.8 %) .
• the plasmid was called pEC-T18mob2 and is shown in figure 1.
• the E. coli - C. glutamicum shuttle vector pEC-Tl8mob2 described in example 3.2 was used as the vector.
• DNA of this plasmid was cleaved completely with the restriction enzymes EcoRI and Xbal and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH,
• the oxyR gene was isolated from the plasmid pCR-oxyRexp described in example 3.1. by complete cleavage with the enzymes EcoRI and Xbal.
• the oxyR fragment approx. 1400bp in size was isolated from the agarose gel with the QiaExII.Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
• the oxyR fragment obtained in this manner was mixed with the prepared vector pEC-T18mob2 and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04) .
• T4 DNA ligase Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04
• the ligation batch was transformed in the E. coli strain DH5 ⁇ (Hanahan, In: DNA cloning. A Practical 25
• Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and cleaved with the restriction enzymes EcoRI and Xbal to check the plasmid by subsequent agarose gel electrophoresis. The resulting plasmid was called pT- oxyRexp. It is shown in figure 2.
• the strain DSM5715 was transformed with the plasmid pT- oxyRexp using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989)). Selection of the transformants took place on LBHIS agar comprising 18.5 g/1 brain-heart infusion broth, 0.5 M sorbitol, 5 g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast extract, 5 g/1 NaCl and 18 g/1 Bacto-agar, which had been supplemented with 5 mg/1 tetracycline. Incubation was carried out for 2 days at 33°C.
• Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915 -927), cleaved with the restriction endonucleases EcoRI and Xbal, and the plasmid was checked by subsequent agarose gel electrophoresis. The resulting strain was called DSM5715/pT-oxyRexp.

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