EP1499728A2 - Sequence nucleotidique codant pour une mannitol-2-deshydrogenase et procede de production de d-mannitol - Google Patents

Sequence nucleotidique codant pour une mannitol-2-deshydrogenase et procede de production de d-mannitol

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Publication number
EP1499728A2
EP1499728A2 EP03737872A EP03737872A EP1499728A2 EP 1499728 A2 EP1499728 A2 EP 1499728A2 EP 03737872 A EP03737872 A EP 03737872A EP 03737872 A EP03737872 A EP 03737872A EP 1499728 A2 EP1499728 A2 EP 1499728A2
Authority
EP
European Patent Office
Prior art keywords
nucleotide sequence
mannitol
mdh
microorganisms
dehydrogenase
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Withdrawn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
EP03737872A
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German (de)
English (en)
Inventor
Hermann Sahm
Gerald Hahn
Stephanie Bringer-Meyer
Björn KAUP
Claudia Hemmerling
Martin Walter
Dieter Wullbrandt
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.)
Innosweet GmbH
Forschungszentrum Juelich GmbH
Original Assignee
Forschungszentrum Juelich GmbH
Nordzucker Innocenter GmbH
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Filing date
Publication date
Application filed by Forschungszentrum Juelich GmbH, Nordzucker Innocenter GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP1499728A2 publication Critical patent/EP1499728A2/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Withdrawn legal-status Critical Current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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • the invention relates to a nucleotide sequence coding for mannitol -2-dehydrogenase and a method for producing D-mannitol.
  • D-mannitol sugar alcohol D-mannitol
  • the worldwide annual requirement for sugar alcohol D-mannitol (D-mannitol) amounts to 30,000 tons per year.
  • D-mannitol is used in the food sector as a tooth-preserving sweetener, in medicine as a plasma expander and vasodilator (hexanitro derivative), and in the pharmaceutical industry for the production of tablets.
  • D-mannitol The large-scale production of D-mannitol has hitherto been carried out by catalytic hydrogenation on metal catalysts of glucose / fructose mixtures of sucrose as starting materials. Due to the lack of stereo-specificity of the catalytic hydrogenation, the yield of D-mannitol is only 25-30% with a triple excess of D-sorbitol (42, 21, 43). D-mannitol and D-sorbitol differ only in their configuration at the carbon atom C-2.
  • An alternative is the production of D-mannitol by the enzymatic hydrogenation of D-fructose in a microbial biotransformation process. Enzymes catalyze their reactions stereospecifically. In Slatner et al.
  • (47) describes, for example, an enzymatic process in which a recombinant mannitol Dehydrogenase is isolated from Pseudo onas fluorescens and is incubated together with a formate dehydrogenase from Candi-da boidinii and NAD in a membrane reactor.
  • formate dehydrogenase creates a reduction-oxidation cycle for NADH, which is retained by the membrane in the reaction vessel.
  • 70-90% of the fructose could be converted into D-mannitol.
  • the authors report the lack of stability of mannitol dehydrogenase (50 h half-life; after stabilization with
  • Dithiothreitol 100h
  • sensitivity to high temperatures> 30 ° C and to shear forces stated as a disadvantage of the method.
  • the reduction equivalents required for the reduction of fructose to D-mannitol came from the oxidation of glucose to organic acids (36). In addition to the problem of only 85% conversion of the substrate fructose to D-mannitol is the
  • mannitol -2-dehydrogenase The key enzyme for the enzyme-catalyzed reduction reaction from D-fructose to D-mannitol is mannitol -2-dehydrogenase.
  • Three mannitol-2-dehydrogenases are known from the literature and are also described with regard to their biochemical properties and nucleotide / amino acid sequences. These include mannitol - 2 dehydrogenase from Pseudomonas fluorescens DSM 50106 (4, 34), from Rhodobacter sphaeroides Si4 (32) and from Agaricus bisporus (11, 38,). The first two belong to the group of the long-chain dehydrogenase / reductase protein family (LDR), the latter to the
  • SDR short chain dehydrogenase / reductase protein family
  • D-mannitol is also to be understood below as D-mannitol.
  • all nucleotide sequences which code for a mannitol-2 dehydrogenase are summarized below under the name "md gene sequence”.
  • the enzyme mannitol -2-dehydrogenase is summarized below under the name "MDH”.
  • ii) comprises at least one nucleotide sequence which corresponds to the nucleotide sequence (i) within the range of the degeneration of the genetic code;
  • iii) comprises at least one nucleotide sequence which hybridizes with a nucleotide sequence which is complementary to the nucleotide sequence (i) or (ii), and if appropriate
  • function-neutral meaning mutations means the exchange of chemically similar amino acids, such as. B. glycine by alanine or serine by threonine.
  • nucleotide sequences according to the invention are distinguished by the fact that they are from the family Lactobacteri - aceae, preferably from the genus Leuconostoc, particularly preferably from Leuconostoc pseudomesenteroides, are particularly preferably isolated from Leuconostoc pseudomesenteroides ATCC 12291.
  • a nucleotide sequence, a nucleic acid or a nucleic acid fragment is to be understood as a polymer made from RNA or DNA, which can be single or double-stranded and optionally contain natural, chemically synthesized, modified or artificial nucleotides.
  • the term DNA polymer also includes genomic DNA, cDNA or mixtures thereof.
  • the (5 "or upstream) and / or subsequent (3" or downstream) sequence regions preceding the coding regions are also included.
  • this includes sequence areas with a regulatory function. You can influence the transcription, the RNA stability or the RNA processing as well as the translation. Examples of regulatory sequences include a. Promoters, enhancers, operators, terminators or translation enhancers.
  • the invention furthermore relates to a gene structure comprising at least one of the nucleotide sequences described above coding for MDH and also with regulatory sequences operatively linked thereto which express the expression of the coding sequences in the Control the host cell.
  • An operative link is understood to mean the sequential arrangement of, for example, the promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can perform its function as intended in the expression of the coding sequence.
  • a promoter that can be induced by IPTG (isopropyl- ⁇ -thiogalactoside) is mentioned here as an example.
  • a gene structure is produced by fusing a suitable promoter with at least one nucleotide sequence according to the invention using common recombination and cloning techniques as described, for example, in (24).
  • the present invention relates to a vector containing at least one nucleotide sequence of the type described above coding for an MDH, with the regulatory nucleotide sequences operatively linked to it and additional nucleotide sequences for selecting transformed host cells, for replication within the host cell or for integration into the corresponding host cell genome ,
  • the vector according to the invention can contain a gene structure of the aforementioned type.
  • Suitable vectors are those that are replicated in the host cells
  • probes or primers can be synthesized and used for this purpose, for example with the help of the PCR technique, analogous genes from others Microorganisms, preferably from the genus Leuconostoc, to be amplified and isolated.
  • the present invention thus also relates to a probe for identifying and / or isolating genes coding for proteins involved in the biosynthesis of D-mannitol, this probe being produced on the basis of the nucleotide sequences of the type described above and a label suitable for detection contains.
  • the probe can be a partial section of the sequence according to the invention, for example from a conserved area, which can hybridize specifically with homologous nucleotide sequences under stringent conditions. Suitable markings are well known from the literature. The person skilled in the art can find instructions for this in the following references (8, 18, 28).
  • the object of the invention is an MDH, encoded by a nucleotide sequence according to the invention, according to SEQ ID No
  • this activity is measured photometrically via the decrease in the NADH concentration for the reduction reaction: D-fructose + NADH + H + -
  • the present invention also relates to an MDH with an amino acid sequence selected from the sequence according to SEQ ID No 2 or a modified form of this polypeptide sequence or isoform thereof or mixtures thereof.
  • the polypeptides according to the invention are distinguished by the fact that they come from the Lactobacteriaceae family, preferably from the Leuconostoc genus, particularly preferably from the Leuconostoc pseudomesenteroides species, very particularly preferably from Leuconostoc pseudomesenteroides ATCC 12291.
  • Isoforms are to be understood as enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure.
  • modified forms are understood to mean enzymes in which there are changes in the sequence, for example at the N- and / or C-terminus of the polypeptide or in the region of conserved amino acids, but without impairing the function of the enzyme. These changes can be made in the form of amino acid exchanges using known methods.
  • microorganisms containing, in replicable form, at least one nucleic acid according to the invention of the type described above, which is more strongly expressed than the correspondingly non-genetically modified microorganism and / or whose number of copies is increased.
  • the present invention also includes a genetically modified microorganism containing, in replicable form, a gene structure or a vector of the type described above.
  • the present invention furthermore relates to a genetically modified microorganism containing at least one Polypeptide according to the invention with the function of an MDH of the type described above, which has an increased activity compared to the correspondingly non-genetically modified microorganism.
  • the microorganisms according to the invention can originate from the genus Bacillus, Lactobacillus, Leuconostoc, Enterobacteriaceae or methylotrophic yeasts, fungi and from all microorganisms also used in the food industry.
  • the following suitable microorganisms may be mentioned by way of example: Achromobacter parvolus, Methylobacterium organophilum, Mycobacterium formicum, Pseudomonas spec. 101, Pseudomonas oxalaticus, Moraxella sp., Agrobacterium sp. , Paracoccus sp.
  • Ancylobacter aquaticus Pseudomonas fluorescens, Rhodobacter sphaeroides, Rhodobacter capsulatus, Lactobacillus sp. , Lactobacillus brevis, Leuconostoc pseudomesenteroides, Gluconobacter oxydans, Candida boidinii, Candida methylica or also Hansenula polymorpha, Aspergillus nidulans or Neurospora crassa or Escherichia coli.
  • the nucleotide sequence is introduced into a host cell using genetic engineering methods.
  • the preferred method here is the transformation and particularly preferably the transfer of DNA by electroporation.
  • the copy number of the corresponding genes can be increased.
  • the promoter and / or regulatory region and / or the ribosome binding site, which is located upstream of the structural gene can be changed accordingly so that expression takes place at an increased rate.
  • Expression cassettes which are installed upstream of the structural gene act in the same way. Inducible promoters also make it possible to increase expression in the course of microbial D-mannitol production. Expression is also improved by measures to extend the life of the mRNA.
  • genes or gene constructs can either be present in plasmids with different copy numbers or can be integrated and amplified in the chromosome. Furthermore, the activity of the enzyme itself can also be increased or increased by preventing the breakdown of the enzyme protein. Alternatively, overexpression of the genes in question can also be achieved by changing the media composition and culture management.
  • a host system is to be understood as microorganisms, all of which can be transformed with foreign DNA. According to the invention, this includes microorganisms into which the nucleotide sequence according to the invention is introduced and / or amplified and is accordingly expressed. Microorganisms which already have a nucleotide sequence coding for an MDH or the nucleotide sequence according to the invention, such as, for. B. Leuconostoc pseudomesenteroides, are therefore also to be understood as a host system.
  • a suitable host system into which the nucleotide sequence according to the invention is introduced is the bacterium Escherichia coli and preferably the strain E. coli JM109 (DE3), which can be cultivated under standard conditions.
  • a complex medium such as e.g. B. LB medium (24) or a mineral - salt medium (15) suitable.
  • the bacterial suspension can be harvested and used for further investigation, for example for the transformation or for the isolation of nucleic acids by conventional methods.
  • Lactobacillus or Enterobacteriacea and methylotrophic yeast preferred.
  • Some preferred microorganisms are listed below by way of example: Achromobacter parvolus, Methylobacterium organophilum, Mycobacterium formicum, Pseudomonas spec. 101, Pseudomonas oxalaticus, Moraxella sp., Agrobacterium sp. , Paracoccus sp., Ancylobacter aquaticus or Pseudomonas fluorescens, Rhodobacter sphaeroides, Rhodobacter capsulatus, Lactobacillus sp.
  • Lactobacillus brevis Lactobacillus brevis, Leuconostoc pseudomesenteroides, Gluconobacter oxydans or methylotrophic yeasts such as Candida boidinii, Candida methylica or also Hansenula polymorpha, fungi such as Aspergillus nidulans and Neurospora crassa and all of them in the
  • the present invention also includes strains of bacteria which are distinguished as D-mannitol-producing mutants or production strains. These can e.g. B. based on wild-type strains by classic (chemical or physical) or genetic engineering methods.
  • the genetically modified microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch process (batch cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of producing D-mannitol.
  • batch cultivation or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of producing D-mannitol.
  • the culture medium to be used must meet the requirements of the respective strains in a suitable manner. Descriptions of culture media of various microorganisms are contained in the manual "Manual of Methods for General Bacteriology" (1).
  • Can as a carbon source Sugar and carbohydrates such as B. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose can be used. These substances can be used individually or as a mixture.
  • Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used as the nitrogen source.
  • the nitrogen sources can be used individually or as a mixture.
  • Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus.
  • the culture medium must also contain salts of metals such.
  • essential growth substances such as amino acids and vitamins can be used in addition to the substances mentioned above.
  • Suitable precursors can also be added to the culture medium.
  • the feedstocks mentioned can be added to the culture in the form of a single batch or can be added in a suitable manner during the cultivation.
  • the addition of Zn 2+ to the medium in particular has proven to be advantageous since the cells are better supplied with the metal ion essential for MDH.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are used in a suitable manner to control the pH of the culture.
  • anti foaming agents such as B. fatty acid polyglycol esters.
  • suitable selectively acting substances z.
  • B. Antibiotics can be added.
  • oxygen or oxygen-containing gas mixtures such as e.g. B. Air entered into the culture.
  • the temperature of the culture is usually 20 ° C to 40 ° C and preferably 30 ° C to 37 ° C.
  • the culture is continued until a maximum of D-mannitol has formed. This goal is usually achieved within 12 hours to 50 hours.
  • the analysis of the D-mannitol concentration can be carried out enzymatically / photometrically using the method of K. Horikoshi
  • a considerable increase in the yield or the conversion rate of the substrate in mannitol is made possible by creating a cofactor regeneration system. It is no longer the substrate for the preparation of the necessary for the reduction of fructose to mannitol
  • Reduction equivalents consumed, but provided by a second enzyme system As a result, the substrate is increasingly available for conversion to mannitol.
  • One of the most frequently used systems is regeneration with a formate dehydrogenase, e.g. B. from Candida boidinii (46).
  • a formate dehydrogenase e.g. B. from Candida boidinii (46).
  • MDH preferably from Leuconostoc pseudomesenteroi- of or also from Rhodobacter sphaeroides
  • formate acts as an electron donor and D-fructose as an electron acceptor.
  • the enzyme formate dehydrogenase catalyzes the oxidation of formate to C0 2 and the enzyme MDH the reduction of D-fructose to D-mannitol (see Fig. 1).
  • the intracellular nicotinamide adenine dinucleotide (NAD) pool serves as an electron shuttle between the two enzymes.
  • the oxidation of formate to C0 2 is thermodynamically favorable, since the free standard formation energy ⁇ G ° "for C0 2 is clearly negative and the C0 2 is removed from the reaction equilibrium by outgassing.
  • D-glucose is used as a substrate for the microbial production
  • Enzyme D-glucose / xylose isomerase (EC 5.3.1.5) can be converted to D-fructose (2) .Extra-cellular conversion is also possible, but intracellular is preferred.
  • D-glucose as a substrate in a microbial process for the production of D-mannitol improves the economy of the process, since di e Cost of D-glucose is only about a quarter of the price of D-fructose.
  • microorganisms into which a formate dehydrogenase and an MDH are introduced and / or strengthened suitable for the process described but also microorganisms which already have a formate dehydrogenase, such as, for. B. Achromobacter parvolus, Methylobacterium organophilum, Mycobacterium formicum, Pseudomonas spec.
  • Pseudomonas oxalaticus Moraxellas.
  • Agrobacterium sp. Paracoccus sp.
  • Ancylobacter aquaticus or also have an MDH.
  • microorganisms such as Pseudomonas fluorescens,
  • Rhodobacter sphaeroides Rhodobacter sphaeroides, Rhodobacter capsulatus, Lactobacillus sp., Lactobacillus brevis, Gluconobacter oxydans and preferably also Leuconostoc pseudomesenteroides or microorganisms, which already have both enzymes and are each enhanced in their activity.
  • methylotrophic yeasts such as Candida boidinii, Candida methylica or Hansenula polymorpha
  • fungi such as Aspergillus nidulans and Neurospora crassa and all microorganisms also used in the food industry.
  • the object is achieved according to the invention with the features specified in the characterizing part of claim 1. Furthermore, based on the preamble of claim 4, the object is achieved according to the invention with the features specified in the characterizing part of claim 4. The object is also achieved on the basis of the preambles of claims 5, 6, 7, 8, 9, 12, 17 and 20 according to the invention with those in the characterizing part of claims 5, 6, 7, 8, 9, 12, 17 and 20 specified characteristics.
  • the nucleic acid according to the invention and the method it is now possible to achieve an improved conversion of the substrate into the product D-mannitol. Compared to previously known methods, increased productivity is achieved, in particular by amplifying the nucleotide sequence according to the invention, and an increased yield of D-mannitol.
  • Fig. 1 Oxidoreduction cycle with formate dehydrogenase and MDH
  • Fig. 2 Derivation of a degenerate 24 base oligonucleotide probe from the N-terminal amino acid sequence of the MDH subunit of Leuconostoc pseudomesenteroides ATCC 12291.
  • Leuconostoc pseudomesenteroides ATCC 12291 was used as the source for the isolation of the MDH.
  • E. coli JM 109 (DE 3) (Promega) served as the host organism for the production of a plasmid bank for the isolation of the genomic DNA from Leuconostoc pseudomesenteroides ATCC 12291.
  • a part of the plasmid bank was obtained by ligation of a 4.0-4.5 kb Eco RI fragment of genomic DNA made from Leuconostoc pseudomesenteroides ATCC 12291 in pUC18. b) Cultivation conditions
  • E. coli JM109 (DE 3) was run at 170 rpm at 37 ° C in Luria-Bertani medium with the addition of ampicillin (100 ⁇ g / ml) or carbenicillin (50 ⁇ g / ml) cultured.
  • the enzyme activity is determined photometrically via the decrease in the NADH concentration for the reduction reaction D-fructose + NADH + H + -> D-mannitol + NAD + .
  • the approach for measuring the activity of the MDH contained 200 ⁇ M NADH and 200 mM D-fructose in 100 mM potassium phosphate buffer at pH 6.5.
  • the specific activities of the crude extracts and the partially purified enzyme isolates are expressed as units per milligram of protein
  • the native molecular weight of the MDH was determined to be 177 kDa by size exclusion chromatography.
  • the isoelectric point of the enzyme is at pH 4.3-4.4.
  • the mdh gene from this fragment was amplified with suitable primers, ligated into the vector pET24a (+) and transformed and expressed in E. coli BL21 (DE3).
  • Cell extracts from E. coli BL21 (DE3) pET24a (+) Lmdh showed a strong overexpression band at 55.2 kDa and a specific activity of the mannitol -2-dehydrogenase of 102.23 U / mg protein, during induction in SDS polyacrylic electrophoresis the controls (cells without plasmid, cells with empty plasmid) showed no activity.
  • nucleotide sequence and the deduced amino acid sequence of the mdh gene from L. pseudomesenteroides ATCC 12291 is shown in sequence ID no. 1 and No. 2 shown.
  • the strains E. coli BL21 (DE3) Gold (Stratagene) and E. coli JM109 (DE3) (Promega) were used.
  • the vectors used were pET-28a (+) Rsp / ndhNC (10) coding for the ORF of mannitol -2 dehydrogenase from Rhodobacter sphaeroides Si4 and pBTac2FDH coding for the formate dehydrogenase from Candida boidinii (35).
  • E. coli BL21 (DE 3) gold was co-transformed with pET28a (+) RspmdhNC and pBTac2FDH and selected on LB agar plates with 50 ⁇ g / ml carbenicillin and 30 ⁇ g / ml kanamycin.
  • E. coli BL21 (DE 3) gold was further transformed with either pET-- 28a (+) Rsp-md.NC or pBTac2FDH alone.
  • the transformants were selected on LB agar plates with either 50 ⁇ g / ml carbenicillin (pBTac2FDH) or with 30 ⁇ g / ml kanamycin (pET-28a (+) Rspmd NC.
  • pBTac2FDH carbenicillin
  • pET-28a (+) Rspmd NC kanamycin
  • LB agar plates for E. coli BL21 (DE 3) gold transformed with pET-28a (+) RspmdhNC additionally contained 1% (v / v)
  • Enzymatic activities of formate dehydrogenase and MDH in the cell-free extract were measured photometrically 340 nm measured.
  • the test batch for the formate dehydrogenase contained 2 mM NAD + and 200 mM sodium formate in 100 mM potassium phosphate buffer with pH 6.5. These high coenzy and substrate concentrations were necessary to achieve the maximum speed due to the high K m values of formate dehydrogenase (35).
  • the pH corresponded to the conditions of the biotransformation.
  • the approach for measuring the activity of the MDH was as described under Ic). Both determinations were carried out at 30 ° C. After measuring the basal activity without substrate for 2 min, the enzyme-specific activity was measured for a further 2 min after addition of the substrate.
  • the biotransformation of D-fructose to D-mannitol shown above with a mannitol-2 dehydrogenase from Rhodobacter spaeroides can also be carried out in a comparable manner for the mannitol -2 dehydrogenase from Leuconostoc pseudomesenteroides.
  • the nucleotide sequence according to the invention can be introduced into the corresponding host organism using methods known to those skilled in the art. BE coli are transformed and expressed and this is then used for the microbial production of D-mannitol. literature
  • Storhas bioreactors and peripheral devices, Vieweg Verlag, Braunschweig / Wiesbaden, (1994))

Abstract

L'invention concerne une séquence nucléotidique codant pour une mannitol-2-déshydrogénase, ainsi qu'un procédé de production de D-mannitol. Les procédés biocatalytiques classiques de production de D-mannitol permettent d'obtenir uniquement des taux de production limités en raison des activités spécifiques réduites des mannitol-2-déshydrogénases utilisées pour transformer du D-fructose en D-mannitol. Selon l'invention, la production de D-mannitol peut être améliorée grâce à une séquence nucléotidique codant pour une mannitol-2-déshydrogénase présentant une activité spécifique supérieure. Les taux de production de D-mannitol peuvent être augmentés de manière particulière grâce à la création d'un système de régénération pour des équivalents de réduction par introduction et/ou concentration d'une formiate déshydrogénase dans un micro-organisme.
EP03737872A 2002-05-08 2003-05-06 Sequence nucleotidique codant pour une mannitol-2-deshydrogenase et procede de production de d-mannitol Withdrawn EP1499728A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10220848 2002-05-08
DE10220848A DE10220848A1 (de) 2002-05-08 2002-05-08 Für eine Mannitol-2-Dehydrogenase codierende Nukleotidsequenz sowie Verfahren zur Herstellung von D-Mannitol
PCT/DE2003/001456 WO2003095635A2 (fr) 2002-05-08 2003-05-06 Sequence nucleotidique codant pour une mannitol-2-deshydrogenase et procede de production de d-mannitol

Publications (1)

Publication Number Publication Date
EP1499728A2 true EP1499728A2 (fr) 2005-01-26

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EP (1) EP1499728A2 (fr)
JP (1) JP2005528098A (fr)
AU (1) AU2003245816A1 (fr)
CA (1) CA2485489A1 (fr)
DE (1) DE10220848A1 (fr)
WO (1) WO2003095635A2 (fr)

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Publication number Priority date Publication date Assignee Title
DE10247147A1 (de) * 2002-10-09 2004-04-22 Forschungszentrum Jülich GmbH Verfahren sowie Mikroorganismus zur Herstellung von D-Mannitol
US8642297B2 (en) 2005-08-10 2014-02-04 Zuchem, Inc. Production of L-ribose and other rare sugars
US10266862B2 (en) 2014-11-06 2019-04-23 Industry-Academic Cooperation Foundation Gyeongsang National University Method for preparing psicose
CN112680482B (zh) * 2021-01-12 2022-12-20 中国科学院天津工业生物技术研究所 一种甘露醇的生物制备方法

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Title
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WO2003095635A3 (fr) 2004-04-22
DE10220848A1 (de) 2003-12-04
AU2003245816A1 (en) 2003-11-11
WO2003095635A2 (fr) 2003-11-20
US20050239180A1 (en) 2005-10-27
JP2005528098A (ja) 2005-09-22
CA2485489A1 (fr) 2003-11-20

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