EP1570059A1 - Procede de production d'acide r-alpha-lipoique par fermentation - Google Patents

Procede de production d'acide r-alpha-lipoique par fermentation

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Publication number
EP1570059A1
EP1570059A1 EP03799471A EP03799471A EP1570059A1 EP 1570059 A1 EP1570059 A1 EP 1570059A1 EP 03799471 A EP03799471 A EP 03799471A EP 03799471 A EP03799471 A EP 03799471A EP 1570059 A1 EP1570059 A1 EP 1570059A1
Authority
EP
European Patent Office
Prior art keywords
lipoic acid
activity
cell
culture medium
gene
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.)
Withdrawn
Application number
EP03799471A
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German (de)
English (en)
Inventor
Tobias Dassler
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.)
Consortium fuer Elektrochemische Industrie GmbH
Original Assignee
Consortium fuer Elektrochemische Industrie GmbH
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Publication date
Application filed by Consortium fuer Elektrochemische Industrie GmbH filed Critical Consortium fuer Elektrochemische Industrie GmbH
Publication of EP1570059A1 publication Critical patent/EP1570059A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers

Definitions

  • the invention relates to a method for the fermentative production of the R- ⁇ -lipoic acid and cells particularly suitable for the method.
  • R- ⁇ -lipoic acid is an essential cofactor of certain multienzyme complexes in a large number of pro and eukaryotes.
  • the R- ⁇ -lipoic acid with its carboxyl group is covalently bound to the ⁇ -amino group of a specific lysine residue of the corresponding enzyme to form a so-called lipoa id.
  • R- ⁇ -lipoic acid is part of the E2 subunit of pyruvate dehydrogenase (PDH) [EC 2.3.1.12] or ⁇ -ketoglutarate dehydrogenase (KGDH) [EC 2.3.1.61] and plays there as Redox partners and acyl group carriers play a crucial role in the oxidative decarboxylation of ⁇ -keto acids.
  • PDH pyruvate dehydrogenase
  • KGDH ⁇ -ketoglutarate dehydrogenase
  • R- ⁇ -lipoic acid also acts as an aminomethyl carrier in glycine-cleavage enzyme systems.
  • ⁇ -Lipoic acid is an optically active molecule with a chiral center at the carbon atom C ⁇ .
  • the R configuration of ⁇ -lipoic acid is the naturally occurring enantiomer. Only this form shows physiological activity as a cofactor of the corresponding enzymes.
  • ⁇ -Lipoic acid can occur both in an oxidized (5- [1, 2] -dithiolan-3-yl-pentanoic acid) and in a reduced form (6, 8-dimercapto-octanoic acid).
  • ⁇ -lipoic acid such as. B. to understand the calcium, potassium, magnesium, sodium or ammonium salt.
  • octanoic acid which is covalently bound to the acyl carrier protein (ACP) serves as a specific precursor in lipoic acid synthesis.
  • ACP acyl carrier protein
  • two sulfur atoms are activated on the octanoic acid (octanoyl- ACP) transfer, whereby R- ⁇ -Lipoyl-ACP arises.
  • This reaction is catalyzed by the lipoic acid synthase [EC 2.8.1.-], the lip ⁇ gene product.
  • the amino acid L-cysteine ultimately serves as the sulfur donor.
  • the subsequent transfer of the R- ⁇ -lipoic acid from R- ⁇ -lipoyl-ACP to the E2 subunit of the ⁇ -keto acid dehydrogenases is carried out by the lipoyl protein ligase B [EC 6.-.-.-], the lipS Gene product, catalyzed, but without R- ⁇ -lipoyl-ACP or R- ⁇ -lipoic acid occurring as free intermediates (Miller et al., 2000, Biochemistry 39: 15166-15178).
  • E. coli can also take up free R- ⁇ -lipoic acid from the surrounding medium and use it for the formation of functional ⁇ -keto acid dehydrogenases.
  • R- ⁇ -lipoic acid is first activated by ATP to R- ⁇ -lipoyl-AMP and then transferred to the corresponding enzyme subunits (see FIG. 2). Both activities are catalyzed by Lipoyl protein ligase A [EC 6.-.-.-], the IpI ⁇ gene product (Morris et al., 1994, J. Biol. Chem. 269: 16091-16100).
  • this LplA activity is not essential for wild-type strains of E.
  • ⁇ -lipoic acid Due to its two thiol groups, ⁇ -lipoic acid has a pronounced effectiveness as an antioxidant and can therefore protect the organism from harmful processes that are induced by oxidative stress.
  • ⁇ -dihydro-lipoic acid the reduced form of ⁇ -lipoic acid, due to its property as a strong reducing agent, is able to regenerate other oxidized natural antioxidants in the body, such as ascorbic acid or ⁇ -tocopherol, directly or indirectly, or if they are deficient, too replace.
  • ⁇ -lipoic acid is of central importance in interaction with ascorbic acid, ⁇ -tocopherol and glutathione, the so-called “network of antioxidants”.
  • ⁇ -Lipoic acid is also used for the prevention and control of type II diabetes mellitus and its consequential damage, such as. B. polyneuropathy, cataract or cardiovascular disease.
  • the mitochondrial NADH-dependent lipoamide reductase of mammals shows an almost 20-fold higher activity with the R enantiomer than with the S form. Furthermore, compared to the S enantiomer, R- ⁇ -lipoic acid has a significantly stronger effect on insulin-mediated glucose uptake and the glucose metabolism of skeletal muscle cells in insulin-resistant rats. The R-shape also showed in animal experiments an anti-inflammatory effect, while the S-shape had an analgesic effect. In order to avoid undesirable side effects, it is therefore extremely desirable to apply ⁇ -lipoic acid only in the enantiomerically pure form.
  • ⁇ -lipoic acid takes place exclusively by means of chemical processes, the racemate always being formed from the R and S forms as the end product (Yadav et al., 1990, J. Sei. Ind. Res. 49: 400-409 ).
  • Various processes have been developed to obtain enantiomerically pure R- ⁇ -lipoic acid.
  • the racemate of ⁇ -lipoic acid or one of the synthetic intermediates can either be chemically by means of chiral auxiliary substances (Walton et. Al. 1954, J. Amer. Chem. Soc. 76: 4748; DE 4137773) or enzymatically (Adger et al., 1995, J. Chem.
  • This object is achieved by a method which is characterized in that a cell which has an attenuated lipoyl protein ligase A activity is cultured in a culture medium, the cell being enantiomerically pure R- ⁇ -lipoic acid in the free form Culture medium excretes and the enantiomerically pure R- ⁇ -lipoic acid is separated from the culture medium.
  • an attenuated lipoyl protein ligase A activity is preferably to be understood to mean that the intracellular activity of the LplA protein in the cell is 25 to 100%, particularly preferably around, compared to a wild-type cell 75 to 100%, is reduced. Completely the intracellular activity of the LplA protein is particularly preferably completely switched off.
  • Lipoyl protein ligase A is not involved in the de novo synthesis of R- ⁇ -lipoic acid, rather the activity of this enzyme is the coupling of free R- ⁇ -lipoic acid to the E2 subunits of ⁇ -keto acid dehydrogenases .
  • reducing or completely switching off the lipoyl protein ligase A activity in a wild-type strain leads to the accumulation of free, enantiomerically pure R- ⁇ -lipoic acid in the culture medium of these cells, although both in an E.
  • the elimination of free R- ⁇ -lipoic acid from the cells allows simple isolation of the product from the culture medium after separation of the biomass, without the cells having to be broken up beforehand or without the R- ⁇ -lipoic acid being expensive and lossy Hydrolysis step from the bound carrier protein (ACP or the E2 subunit of the ⁇ -keto acid dehydrogenases) must be split off.
  • the lipoyl protein ligase A activity encoded by the IplA gene is to be understood as that lipoyl protein ligase activity of a cell which has a clear substrate preference for free R- ⁇ -lipoic acid in comparison to R- ⁇ -lipoyl-ACP having.
  • the LplA protein has about 100 times higher activity with free R- ⁇ -lipoic acid than with R- ⁇ -lipoyl-ACP.
  • the lipoyl protein ligase A activity of a cell thus clearly differs from the lipoyl protein ligase B activity, which R- ⁇ -lipoyl-ACP prefers as the substrate over free R- ⁇ -lipoic acid (see FIG. 1 and 2).
  • the lipoyl protein ligase A gene is preferably a gene with the sequence SEQ ID NO: 1 or a functional variant of this gene.
  • a functional variant is to be understood as a DNA sequence which is derived from the sequence shown in SEQ ID NO: 1 by deletion, insertion or substitution of nucleotides, the enzymatic activity and specificity of the sequence shown by the Gene encoded lipoyl protein ligase A is preserved.
  • the lipoyl protein ligase A gene encodes a protein comprising the sequence ID NO: 2 or functional variants with a sequence homology to SEQ ID NO: 2 greater than 35%.
  • sequence homology to SEQ ID NO: 2 is preferably greater than 60%, particularly preferably the sequence homology to SEQ ID NO: 2 is greater than 80%.
  • a downturn chung can be achieved, for example, by reducing the expression of the corresponding gene or by replacing the chromosomal wild-type gene with a mutated allele which codes for an enzyme with a reduced activity. In extreme cases, the enzyme activity can also be switched off completely.
  • the expression of a gene can be reduced or prevented, for example, by the following measures: - weakening of the promoter by means of suitable base substitutions
  • Mutated alleles of a gene that code for an enzyme with a reduced activity can be generated, for example, by the following measures:
  • Mutated alleles of the Ipl ⁇ gene can be generated using standard molecular biology methods. A preferred way of doing this is to introduce specific base substitutions into the gene. This can take place, for example, in that during the amplification of the 2pl gene by means of the polymerase chain reaction (PCR) by using special mutagenic primers, the base sequence of the gene or its promoter are specifically changed at one or more positions (site-specific mutagenesis).
  • PCR polymerase chain reaction
  • the introduction of a deletion into the Ipl ⁇ gene is particularly preferred. This can be achieved by first cloning the gene into a plasmid vector (e.g. pUC18, pBR322, pACYC184) after amplification by means of PCR using specific primers which detect the complete Ipl ⁇ gene. By restricting the plasmid obtained in this way with suitable restriction endonucleases which only cut in the region of the Ipl ⁇ gene, internal regions of the gene can be removed. In this way, after religation of the restricted plasmid, an internal deletion can be introduced into the Ipl ⁇ gene. As an alternative to religation of the plasmid restricted in the Ipl ⁇ gene, an antibiotic resistance cassette can also be cloned into the Ipl ⁇ gene.
  • a plasmid vector e.g. pUC18, pBR322, pACYC184
  • cells are used which secrete enantiomerically pure R- ⁇ -lipoic acid in a culture medium and have an attenuated lipoyl protein ligase A activity, where they have an IplA allele instead of a wild-type IplA gene , which has a base substitution in the region of base pairs 367-465, which leads to the LplA protein having at least 50% reduced activity, or a deletion in the Ipl ⁇ gene.
  • the present invention thus also relates to a cell with the aforementioned properties.
  • the activity of the LplA protein is preferably reduced by 50 to 100%, particularly preferably by 75% to 100%.
  • base substitution in the gene region mentioned means that no more activity of the LplA protein can be detected.
  • the cells according to the invention there is only one fragment of the IplA gene generated by an internal deletion on the chromosome of the host organism, which fragment can no longer code for a functional lipoyl protein ligase A activity.
  • Cells with weakened lipoyl protein ligase A activity can be produced by replacing the Ipl ⁇ wild-type gene with an Ipl ⁇ allele coding for an LplA protein with an at least 50% compared to the wild-type Protein reduced activity is introduced.
  • cells according to the invention can thus preferably be produced from cells of pro- or eukaryotic organisms which are able to synthesize R- ⁇ -lipoic acid themselves (starting cell), which are accessible to recombinant processes and which can be cultivated by fermentation. Plant or animal cells that can be grown in cell culture are thus also suitable for producing cells according to the invention. Starting cells which have not previously been manipulated can be used to produce cells according to the invention.
  • those cells according to the invention are particularly suitable which, by virtue of an increased expression of the lipA gene compared to the wild type, already have an increased lipoic acid synthase activity compared to the wild type and / or already an increased expression of the lipB gene compared to the wild type have increased lipoyl protein ligase B activity compared to the wild type.
  • the production of cells with an increased lipoic acid synthase compared to the wild type is particularly suitable which, by virtue of an increased expression of the lipA gene compared to the wild type, already have an increased lipoic acid synthase activity compared to the wild type and / or already an increased expression of the lipB gene compared to the wild type have increased lipoyl protein ligase B activity compared to the wild type.
  • the invention thus also relates in particular to cells which, in addition to the activity of the LplA protein which is reduced or absent by at least 50%, by an increased expression of the lipA gene via an increased lipoic acid synthase activity or by an increased expression of the lipB gene have increased lipoyl protein ligase B activity.
  • the cells are preferably microorganisms, such as, for example, yeast or bacterial strains.
  • the strains of the Enterobacteriaceae family are particularly preferred, and strains of the type Escherichia coli are very particularly preferred.
  • R- ⁇ -lipoic acid can be obtained from the culture medium by methods known to those skilled in the art, such as, for example, centrifuging the cell-containing culture medium in order to separate the cells and by subsequent extraction and / or precision of the product.
  • the cells according to the invention for the production of R- ⁇ -lipoic acid are preferably cultivated in a minimal salt medium known from the literature (Herbert and Guest, 1970, Meth. Enzymol. 18A, 269-272).
  • a minimal salt medium known from the literature (Herbert and Guest, 1970, Meth. Enzymol. 18A, 269-272).
  • all usable sugars, sugar alcohols or organic acids or their salts can be used as the carbon source.
  • Aspartic acid malic acid, succinic acid, pyruvic acid, fumaric acid, glutamic acid, glucose, glycerol or oxaloacetic acid are preferably used. Succinic acid and oxaloacetic acid are particularly preferred. Combined feeding of several different carbon sources is also possible. Furthermore, short-chain fatty acids with a chain length of C2-C8, preferably with a chain length of C6-C8 (hexanoic or octanoic acid), can be added to the medium as specific precursors for the ⁇ -lipoic acid synthesis. The concentration of the carbon source added is preferably 0.1-30 g / l.
  • the cells according to the invention are preferably incubated under aerobic cultivation conditions over a period of 16-150 h and in the range of the optimal growth temperature for the respective cells.
  • 15 - 55 ° C is preferred as the optimal temperature range.
  • a temperature between 30 and 37 ° C. is particularly preferred.
  • the detection and quantification of the R- ⁇ -lipoic acid produced in the method according to the invention is carried out, for example, by means of a bioassay using a lipoic auxotrophic indicator strain (lip ⁇ mutant).
  • lipoic auxotrophic indicator strain lip ⁇ mutant
  • This type of turbidimetric quantification of R- ⁇ -lipoic acid is known from the literature (Herbert and Guest, 1970, Meth. Enzymol. 18A, 269-272).
  • the indicator strain W14851ip2 (ATCC 25645) used in the context of the present invention would also grow without supplemented R- ⁇ -lipoic acid if the medium also contains acetate and succinate in addition to glucose.
  • the R- ⁇ -lipoic acid producer is preferably grown with sucrose as the only carbon source.
  • This strain is supplemented with the culture supernatant of a cell culture according to the invention; The lipoic acid content in the culture medium can then be determined on the basis of the growth of the indicator strain.
  • the bacterial strain Escherichia coli W3110 ⁇ lpl ⁇ which was used for the execution of the examples, was deposited with the DSMZ (German Collection for Microorganisms and Cell Cultures GmbH, D-38142 Braunschweig) under the number DSM 15299 in accordance with the Budapest Treaty.
  • the plasmids pKP477 and pBAD-lipB are described in patent application DE 10245993.
  • Example 1 Construction of a chromosomal mutation in the Ipl ⁇ gene of the host organism A) Amplification of the IplA gene
  • the Ipl ⁇ gene from E. coli was amplified by means of the polymerase chain reaction (PCR) using the Pwo DNA polymerase according to common practice known to the person skilled in the art.
  • the chromosomal DNA of the E. coli wild-type strain 3110 (ATCC 27325) served as the template.
  • the 3'-phosphorothioate-protected oligonucleotides lplA-fwd and lplA-rev were used as primers with the following sequences:
  • the DNA fragment with a length of approx. 1.6 kb obtained during the PCR was then analyzed by means of a DNA adsorption column of the QIAprep Spin Miniprep Kit (Qiagen, Hilden) cleaned according to the manufacturer's instructions.
  • the purified PCR fragment was cut with the restriction endonuclease BamHl under the conditions specified by the manufacturer, then separated on an agarose gel and then isolated from the agarose gel using the GENECLEAN kit (BIO 101 Inc., La Jolla, California, USA) according to the manufacturer's instructions .
  • the vector pUC18 was cut with the restriction enzyme BamHl under the conditions specified by the manufacturer, then dephosphorylated by treatment with alkaline phosphatase at the 5 'ends and then like the PCR Fragment cleaned using the GENECLEAN method.
  • the ligation of the PCR fragment with the cut and dephosphorylated vector was carried out according to the manufacturer's instructions using the T4 DNA ligase.
  • the transformation of E. coli cells of the DH5 ⁇ strain with the ligation mixture was carried out by means of electroporation in a manner known to the person skilled in the art. The transformation approach was on
  • LB ampicillin agar plates (10 g / 1 tryptone, 5 g / 1 yeast extract, 10 g / 1 NaCl, 15 g / 1 agar, 100 mg / 1 ampicillin) were applied and incubated overnight at 37 ° C.
  • the desired transformants were identified by means of a restriction analysis after plasmid isolation using a QIAprep Spin Miniprep Kit (Qiagen, Hilden). The plasmid obtained in this way is called pUCl8-Ipl ⁇ .
  • the vector pUC18-lpIA was digested with the restriction enzymes Nrul and Stul, which each cut once within the IplA gene, and the vector was described using T4-D ⁇ A- as described above. Ligase religated, then transformed and checked. This deleted a central region of the IplA gene by 197 base pairs and at the same time introduced a reading frame shift, whereby the gene was inactivated.
  • the resulting plasmid pUC18- ⁇ lpl ⁇ which now contains the shortened reading frame "AlplA”
  • the plasmid obtained in this way is called pK03- ⁇ lpl ⁇ .
  • the lipB overexpression plasmid pBAD-lipB was transformed into the E. coli strains W3110 ⁇ lplA and W3110 by electroporation and, after selection on LB agar plates with 100 mg / 1 ampicillin, the plasmid was reisolated from one of the transformants in each case and cleaved with restriction endonucleases and checked.
  • the control plasmid pKP477 which in addition to the ampicillin resistance gene contains only the regulatory sequences of the ara binose operon from E. coli (araC gene, araBAD promoter region), was carried out in an analogous manner.
  • Example 3 Fermentative production of R- ⁇ -lipoic acid
  • the strains mentioned in Example 2 were used both with and without a plasmid.
  • 5 ml of LB liquid medium containing 100 mg / 1 ampicillin were inoculated with the respective strain and incubated for 16 h at 37 ° C. and 160 rpm on a shaker.
  • the cells were then harvested by centrifugation and washed twice with the appropriate volume of sterile saline (0.9% NaCl).

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Abstract

L'invention concerne un procédé de production d'acide R-alpha-lipoïque par fermentation. Ledit procédé est caractérisé en ce qu'une cellule, possédant une activité lipoyl-protéine-ligase A réduite, est cultivée dans un milieu de culture ; cette cellule dégage de l'acide R-alpha-lipoïque énantiomériquement pur sous forme libre dans ledit milieu de culture puis cet acide R-alpha-lipoïque énantiomériquement pur est séparé du milieu de culture.
EP03799471A 2002-12-12 2003-12-04 Procede de production d'acide r-alpha-lipoique par fermentation Withdrawn EP1570059A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10258127A DE10258127A1 (de) 2002-12-12 2002-12-12 Verfahen zur fermentativen Herstellung von R-α-Liponsäure
DE10258127 2002-12-12
PCT/EP2003/013728 WO2004053131A1 (fr) 2002-12-12 2003-12-04 PROCEDE DE PRODUCTION D'ACIDE R-α-LIPOIQUE PAR FERMENTATION

Publications (1)

Publication Number Publication Date
EP1570059A1 true EP1570059A1 (fr) 2005-09-07

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Application Number Title Priority Date Filing Date
EP03799471A Withdrawn EP1570059A1 (fr) 2002-12-12 2003-12-04 Procede de production d'acide r-alpha-lipoique par fermentation

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US (1) US20060234359A1 (fr)
EP (1) EP1570059A1 (fr)
AU (1) AU2003299300A1 (fr)
DE (1) DE10258127A1 (fr)
TW (1) TW200426218A (fr)
WO (1) WO2004053131A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10332623A1 (de) * 2003-07-17 2005-02-03 Consortium für elektrochemische Industrie GmbH Zellen und Verfahren zur fermentativen Herstellung von R-alpha-Liponsäure
WO2006042666A1 (fr) * 2004-10-18 2006-04-27 Meda Pharma Gmbh & Co. Kg Utilisation d'acide alpha-lipoique r-(+) pour prevenir le diabete
US20090078581A1 (en) * 2007-09-24 2009-03-26 Applied Intellectual Capital Configurations and Methods of Reduction of Lipoic Acid
US20110262976A1 (en) * 2008-01-17 2011-10-27 Indigene Pharmaceuticals, Inc. PRODUCTION OF R-a-LIPOIC ACID BY FERMENTATION USING GENETICALLY ENGINEERED MICROORGANISMS
KR101153400B1 (ko) 2009-01-12 2012-06-05 건국대학교 산학협력단 신규 리포산 합성효소와 리포산 단백질 리가제를 이용한 알파-리포산의 생산방법
KR101250994B1 (ko) 2011-04-14 2013-04-11 건국대학교 산학협력단 리포산 합성 관련 효소를 이용한 알파-리포산의 생산방법
KR101124617B1 (ko) * 2011-04-14 2012-03-20 건국대학교 산학협력단 신규 리포산 합성효소와 리포산 단백질 리가제를 이용한 알파-리포산의 생산방법
US9944960B2 (en) 2015-11-17 2018-04-17 Premier Research Labs, Lp Process for microbial production of dihydrolipoic acid and extraction of dihydrolipoic acid with edible oils
TW202146641A (zh) * 2020-03-02 2021-12-16 新加坡國立大學 用於類脂酸之生產的代謝工程技術
CN115581808B (zh) * 2022-11-29 2023-08-18 郑州大学 一种在心脑血管支架材料表面制备聚硫辛酸铜涂层的方法及含有该涂层的心脑血管支架材料

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AU2002307432A1 (en) * 2001-04-20 2002-11-05 Cargill, Incorporated Production of alpha-lipoic acid
DE10235270A1 (de) * 2002-08-01 2004-02-12 Consortium für elektrochemische Industrie GmbH Zellen, die R-alpha-Liponsäure sekretieren und Verfahren zur fermentativen Herstellung der R-alpha-Liponsäure

Non-Patent Citations (1)

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Title
See references of WO2004053131A1 *

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US20060234359A1 (en) 2006-10-19
AU2003299300A1 (en) 2004-06-30
TW200426218A (en) 2004-12-01
WO2004053131A1 (fr) 2004-06-24
DE10258127A1 (de) 2004-07-08

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