WO2001007631A2 - Glycosyltransferase, sa fabrication et son utilisation - Google Patents

Glycosyltransferase, sa fabrication et son utilisation Download PDF

Info

Publication number
WO2001007631A2
WO2001007631A2 PCT/DE2000/002514 DE0002514W WO0107631A2 WO 2001007631 A2 WO2001007631 A2 WO 2001007631A2 DE 0002514 W DE0002514 W DE 0002514W WO 0107631 A2 WO0107631 A2 WO 0107631A2
Authority
WO
WIPO (PCT)
Prior art keywords
acid sequence
nucleic acid
cell
polypeptide
sequence according
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/DE2000/002514
Other languages
German (de)
English (en)
Other versions
WO2001007631A3 (fr
Inventor
Joachim STÖCKIGT
Heribert Warzecha
Joachim Arend
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.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and Co KGaA
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 Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of WO2001007631A2 publication Critical patent/WO2001007631A2/fr
Publication of WO2001007631A3 publication Critical patent/WO2001007631A3/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
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a nucleic acid sequence according to SEQ ID NO: 1 or its derivative or fragment or the variant of the nucleic acid sequence according to SEQ ID NO: 1 or a derivative or fragment of the variant, coding a polypeptide with the biological activity of an arbutin synthase and one of these nucleic acid sequences encoded polypeptide.
  • the invention further relates to a method for producing the polypeptides according to the invention and a method for producing glycosides.
  • Glycosyltransferases are important enzymes in plant cells where they are responsible for cell detoxification e.g. by converting toxic compounds into easily accumulable vacuolar compartmentalized glycosides.
  • the glycoside arbutin is particularly common in the plant kingdom in Ericaceae, e.g. in the bearberry Arctostaphylos uva-ursi and the cranberry Vaccinium vitis-idaea, in Saxifragaceae e.g. Bergenia crassifolia, but also in Chimaphila umbellata (forest umbel), Origanum majorana (marjoram) or leaves of the pear tree. Because of its antibacterial effect, it is e.g.
  • the glycoside is also able to inhibit the enzyme tyrosinase, which catalyzes an important step in melanin biosynthesis, in vivo.
  • the glycoside is of biotechnological interest because it is used in Japan in considerable quantities (1-2 t per year) in cosmetic preparations due to its skin-lightening effect due to the inhibition of melanin biosynthesis. While in the European market the preparations containing arbutin are mainly used for age spots, liver spots or freckles, in the Asian market the beauty ideals of light, flawless skin are to be achieved by full-body treatment with preparations containing arbutin; see. Blume G., Teichmüller E., Orndorff S., (1999), S ⁇ FW-Journal 2/3, 20-24.
  • Glycoside is the collective name for an extensive group of plant substances and synthetic compounds that consist of one or more carbohydrates (mono- or
  • Oligosaccharides and one or more aglycones, ie non-sugars.
  • the Glycoside is the aglycone, which is also called genin, bound to a hemiacetal carbon atom of the carbohydrate by an oxygen atom in an ether bond called glycosidic bond here.
  • Enzymatic in- / tro-processes for the production of glycosides are usually limited to alkyl glycosides and show very low yields for aromatic glycosides, which are less than 10%; see. Vic G., Thomas D., Crout DHG, (1997), Solvent effect on enzyme-catalyzed synthesis of ß-D-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl ß-D -glucopyranosides. Enzyme Microb. Technol.
  • glycosides Another method of producing glycosides consists in the chemical preparation of glycosides from unprotected sugars.
  • the chemical production process usually leads to unspecific mixtures of mono- and poly-alkylated sugars, so that the introduction and removal of protective groups is necessary if a specific product is to be synthesized in a targeted manner.
  • activated derivatives such as alcohol-HCl often results in undesired by-products which pollute the environment, make processing more difficult and reduce the yields of the desired product.
  • variant denotes a gene homologous to a first nucleic acid sequence. Homologous means that the gene can be allelic. Homologous also means that the gene can come from another species, but the polypeptide encoded by this gene has the same biological activity as the polypeptide encoded by the first nucleic acid sequence.
  • derivative denotes a nucleic acid or amino acid sequence which is derived from another sequence by modification.
  • the modification comprises the deletion, addition, substitution, insertion or inversion of nucleotides or amino acids at one or more sites in the other sequence.
  • fragment denotes a section of a nucleic acid or amino acid sequence generated by deletion at one or more sites.
  • hybridize includes stringent and less stringent hybridization conditions, e.g. in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969-309-6.
  • An example of stringent hybridization conditions is: Hybridization in 4 x SSC at 65 ° C with subsequent 2-3 washing steps in 0.1 x SSC at 65 ° C for a total of about one hour.
  • An example of less stringent hybridization conditions is: Hybridization in 4 x SSC at 37 ° C, followed by 2-3 washing steps in 1 x SSC at room temperature.
  • vector denotes naturally occurring or artificially produced constructs for the uptake, multiplication, expression or transfer of nucleic acids, e.g. Plasmids, phagemids, cosmids, artificial chromosomes, bacteriophages, viruses or retroviruses.
  • transformation means any transfer of a nucleic acid sequence into a cell.
  • the transfer can be, for example, a transfection or lipofection or can be carried out by the calcium method or electroshock method. The term therefore does not only refer to the process that is usually understood by the term transformation.
  • FIG. 1 is a schematic growth curve which shows the tolerance of recombinant E. coh (transformed with the glycosyltransferase cDNA according to the invention) to hydroquinone in the culture broth.
  • the dashed line (- ⁇ -) shows the growth of recombinant E. coli in the presence of hydroquinone
  • the solid line (- ⁇ -) shows the growth of recombinant E. coli without hydroquinone.
  • HQ means hydroquinone.
  • OD600 means optical density at 600 n.
  • FIG. 2 is a schematic representation of the yield of arbutin synthase which was achieved with recombinant E. coli (transformed with the glycosyltransferase cDNA according to the invention).
  • the present invention relates to glycosyltransferases, especially glucosyltransferases, especially arbutin synthases.
  • One aspect of the invention relates to a nucleic acid sequence that encodes a polypeptide that catalyzes the conversion of hydroquinone and glucose to arbutin in Rauwolfia serpentina.
  • a nucleic acid sequence according to the invention is listed in SEQ ID NO: 1.
  • the invention further relates to the derivatives or fragments of the nucleic acid sequence according to SEQ ID NO: 1.
  • the invention further relates to the variants of the nucleic acid sequence according to SEQ ID NO: 1, which encode a polypeptide with the biological activity of an arbutin synthase.
  • Another aspect relates to derivatives and fragments of the variants of the nucleic acid sequence according to SEQ ID NO: 1, as long as the derivatives and fragments encode a polypeptide with the biological activity of an arbutin synthase.
  • the invention particularly relates to the nucleic acid sequences of the variants and their derivatives and fragments which hybridize with the nucleic acid sequence according to the invention.
  • the hybridization can be carried out under stringent to less stringent conditions.
  • Variants of the nucleic acid sequence according to the invention which encode a polypeptide with the biological activity of an arbutin synthase can originate from all organisms.
  • the variants can in particular be made from Rauwolfia serpentina, from Ericaceae, for example from the bearberry Arctostaphylos uva-ursi, and the cranberry Vaccinium vitis-idaea, from Saxifragaceae, for example Bergenia crassifolia, from Chimaphila umbellata (forest umbel), from Origanum majorana (marjoram) or from the pear tree.
  • the invention further relates to derivatives and fragments of the variants.
  • the nucleic acid sequences according to the invention can be of natural origin or have been produced synthetically.
  • the nucleic acid sequences according to the invention can e.g. for the identification and isolation of genes homologous to the nucleic acid sequence according to SEQ ID NO: 1 in other organisms or of homologous genes in Rauwolfia serpentina by means of hybridization or screening methods, e.g. as a probe for screening in DNA libraries.
  • the nucleic acid sequences according to the invention can be in vectors, e.g. Expression vectors are inserted, which are then transferred to a suitable host cell.
  • the cells then express the polypeptide encoded by one or more of the nucleic acid sequences according to the invention.
  • Suitable host cells can contain microorganisms such as bacteria, e.g. E. coli, or yeast, or animal cells, e.g. Insect cells, or plant cells, e.g. Seaweed.
  • the vectors which are possible for the incorporation of the nucleic acid sequences according to the invention and the transfer into the host cells are known to the person skilled in the art.
  • nucleic acid sequences according to the invention can be integrated into the genome of the host cells by homologous or illegitimate recombination with the aid of recombination elements.
  • the nucleic acid sequences according to the invention can be modified e.g. Substitution of certain nucleotides can be adapted to the respective host cell in order to ensure expression suitable for the host cell.
  • the invention further relates to the polypeptides encoded and expressed by the nucleic acid sequences according to the invention.
  • the invention particularly relates to the polypeptide with the amino acid sequence according to SEQ ID NO: 2 or its derivative or fragment.
  • the invention relates to polypeptides whose amino acid sequences have an identity of 60% or more to the amino acid sequence according to SEQ ID NO: 2.
  • Preferred are polypeptides with an identity of 80% or more, particularly preferred are polypeptides with an identity of 90% or more.
  • Suitable host cells for example Microorganisms, animal cells or plant cells are transformed with one or more of the nucleic acid sequences according to the invention.
  • the nucleic acid sequence can be integrated into an expression vector which is present episomally in the cell after the transformation.
  • the nucleic acid sequence can be functionally linked to transcription elements and can be integrated into the genome after introduction into the host cell.
  • the cell is cultivated and propagated in an appropriate culture medium which the person skilled in the art can easily find in the prior art for the respective host cell.
  • the transformed cell may also include selection markers, e.g. can be present on the transformed vector. Selection markers can also be cotransformed with one or more of the nucleic acid sequences according to the invention or related to one of the nucleic acid sequences and integrated into the genome. Other commonly used selection methods can also be used.
  • the polypeptide encoded and expressed by the nucleic acid sequence according to the invention is isolated from the cells and purified by conventional methods.
  • the polypeptides according to the invention can be used for the production of glycosides.
  • the aglycone substrates used to prepare the glycosides can be alkyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl alcohols having 1 to 26 carbon atoms and 0 to 10 heteroatoms, which can be unbranched or branched, mono- or polyunsaturated and substituents , for example, hydroxyl, halogen, amino or nitro radicals, can carry on the carbon skeleton and / or the heteroatoms.
  • Preferred aglycons are aromatic alcohols, for example hydroquinone, vanillin, p-nitrophenol, ⁇ -naphthol, thymol, methoxyphenol, resorcinol, phenol, hydroxyacetophenone, niclosamide, dimethoxymethylhydroquinone, flavonoids, flavones, catechol and catecholonic acid, anthate. Hydroquinone is particularly preferred.
  • the carbohydrate substrates used to produce the glycosides are sugars with a hemiacetal-forming carbonyl group and several hydroxyl groups in the molecular residue.
  • Preferred sugars are glucose, Fmctose, Galactose, Maimose, Lactose and Maltose.
  • Glucose is particularly preferred.
  • the following can also be used for the production of glycosides a) substrates whose toxicity to the host organism is too high for in vivo conversion (ie direct addition of the aglycones to the culture broth), b) aglycones which would be metabolized by the host organism used, c ) Glycosides that would be changed or broken down in the host organism or have a toxic effect.
  • glycosides especially glucosides
  • the polypeptide according to the invention can e.g. act on one or more aglycons and one or more sugars in a reaction vessel.
  • the cell which has been transformed with one or more of the nucleic acid sequences according to the invention can be added to the culture medium and, if appropriate, the sugar, so that the glycoside is produced in the cell.
  • a method is preferred in which the glycoside produced is excreted so that it can be isolated from the culture medium.
  • glycosides can be produced in a "one-pot process” or “one step process” for the production of glycosides while maintaining the enzyme activity in bound form.
  • the polypeptide of the invention via chelate complexes on metals e.g. Nickel bound or bound to a matrix due to other binding principles.
  • polypeptides according to the invention can be substituted, deleted or added by modification of their amino acid sequence.
  • the modification can be introduced by conventional methods, e.g. through targeted mutagenesis (site directed mutagenesis) or evolutionary protein design.
  • the polypeptides can also have a changed pH or temperature optimum or a modified substrate specificity. The substrate acceptance may vary due to the changed substrate specificity, so that novel glycosides can be produced.
  • glycosides prepared with the polypeptides according to the invention and / or the method according to the invention can be used in cosmetic or pharmaceutical preparations. Examples:
  • a cell suspension of R. serpentina was in 1 1 Erlenmeyer flask on Linsmaier and Skoog medium (Linsmeier EM, Skoog F., (1965), Organic growth factor requirements of tabacco tissue cultures. Physiol. Plant 18: 100-127) for 10 days cultivated under continuous light influence (600 lux) and shaking (100 rpm) at 24 ° C. After harvesting the cells by filtration, the cells were frozen in liquid nitrogen and stored at -26 ° C.
  • the protein precipitated at 30-70% (NH 4 ) 2 SO 4 was collected after centrifugation at 10,000 ⁇ g for 30 min and dissolved in 500 ml of buffer A and centrifuged again. The supernatant was dialyzed twice for 8 hours against 10 l buffer B (20 mM Tris-HCl, 10 mM EtSH, pH 7.5). After centrifugation at 10,000 xg for 20 min and redissolving of the precipitate, a soluble protein fraction was obtained (750 ml, 7.4 mg / ml).
  • the protein solution obtained was further purified by anion exchange chromatography (flow rate 10 ml / min, fast-flow DEAE-Sepharose column 20.3 x 5 cm, volume of 400 ml, XK50 / 30 column, Pharmacia), which was equilibrated with buffer B. has been.
  • anion exchange chromatography flow rate 10 ml / min, fast-flow DEAE-Sepharose column 20.3 x 5 cm, volume of 400 ml, XK50 / 30 column, Pharmacia
  • the enzyme was prepared with a linear KCl gradient (4 column volume, 0.03-0.15 M KCl) Buffer B and C (buffer B with 1 M KCl) were determined. Enzyme activity was measured at about 0.1 M KCl. Fractions with transferase activity were pooled and concentrated to 250 mM for the next purification step by adding (NH 4 ) 2 SO.
  • the protein solution thus obtained was pumped at a flow rate of 5 ml / min through a fast-flow phenyl-Sepharose column (38 ⁇ 2.6 cm, volume of 200 ml, XK26 / 40 column, Pharmacia).
  • the column was equilibrated with buffer D (buffer B with 250 mM (NH 4 ) 2 SO 4 ).
  • buffer D buffer B with 250 mM (NH 4 ) 2 SO 4
  • the enzyme was washed with a Gradients from 250 to 0 mM (NH) 2 SO 4 (from buffer D and B) eluted.
  • the concentrated protem solution was diluted to a volume of 56 ml with buffer E (10 mM K 2 HPO 4 / KH 2 PO 4 , 10 mM EtSH, pH 7.5) and onto a Macro-Prep Ceramic Hydroxyapatite (BioRad) column (1st , 5 x 1.6 cm, volume of 3 ml, XK16 / 20 column, Pharmacia), which had previously been equilibrated with buffer E.
  • buffer E 10 mM K 2 HPO 4 / KH 2 PO 4 , 10 mM EtSH, pH 7.5
  • a Macro-Prep Ceramic Hydroxyapatite (BioRad) column (1st , 5 x 1.6 cm, volume of 3 ml, XK16 / 20 column, Pharmacia), which had previously been equilibrated with buffer E.
  • the proteins were buffered with a potassium phosphate buffer gradient (10 to 400 mM, 35 column volumes, 105 ml) from buffer E and F (0.4 MK 2 HPO 4 / KH 2 PO 4 , 10 mM EtSH, pH 7.5). Three fractions (15 ml) with more than 4 nkat / mg were combined and dialyzed against 2 l buffer B overnight.
  • the dialyzed enzyme solution (55 ml) was pumped over a Mono P HR 5/20 column (Pharmacia) with 0.5 ml min, which had been previously equilibrated with buffer B.
  • the proteins were fractionated with 30 ml of buffer G (polybuffer 74, 1:20, 10 mM EtSH, pH 4.0) by spanning a linear pH gradient from pH 7.5 to 4.0. Transferase activity was measured at a pH of about 5.3.
  • Total RNA from R. serpentina was prepared using the lithium chloride method.
  • 5 g of tissue which had been snap frozen in nitrogen was homogenized in 15 ml of extraction buffer TLE (0.2 M Tris-HCl, 0.1 M LiCl, 5 mM EDTA, 1% SDS) and mixed with 5 ml of TLE-equilibrated phenol. After 10 seconds of digestion in the Ultraturax, 5 ml of chloroform were added and the mixture was homogenized again. The solution was incubated for 20 minutes at 50 ° C. in a water bath for cell lysis and then centrifuged at 8,000 ⁇ g for 20 minutes.
  • the upper, aqueous phase was drawn off, transferred to a new centrifuge tube and extracted repeatedly with TLE-saturated phenol and chloroform (5 ml each) until no more protein precipitated at the interface. As a final step, the mixture was extracted again with 10 ml of chloroform. The separated aqueous phase was then mixed with a third of its volume (approx. 5 ml) 8 M LiCl. The RNA was precipitated overnight at 4 ° C.
  • RNA content was determined.
  • Single-stranded cDNA which was to be used as a template in the PCR, could be prepared from total RNA preparations.
  • M-MLV reverse transcriptase was used as the RNA-dependent DNA polymerase.
  • RNA in max. 10 ⁇ l water: 10 ⁇ g
  • the reaction mixture was heated to 65 ° C. for 5 min and then cooled to 37 ° C. Only now was 1 ⁇ l of enzyme solution (200 U) added. The reaction proceeded at 37 ° C for one hour and was stopped by heating at 95 ° C for five minutes. The cDNA was then used to amplify part of the arbutin synthase cDNA. Degenerate oligonucleotides were derived from peptide fragments 7b and 30 and synthesized as primers.
  • the conditions of the PCR were as follows: dNTP mix (2 mM) 1 ul
  • the amplified fragment was separated electrophoretically and eluted from the gel. After radioactive labeling with 32 P-ATP (Random Priming, Gibco), the fragment was used as a probe for screening a cDNA library from R. serpentina.
  • the cDNA library was produced from mRNA of a six-day-old R. serpentina cell culture using the Lambda ZAP system from Stratagene. Positive clones were isolated and sequenced. Two vectors were used to express the isolated clones in E. coli.
  • the plasmid pSE280 (Invitrogen) was used to express the enzyme without additional amino acids, the vector pQE60 (Qiagen) to produce a fusion protein (6xHis-Tag).
  • the complete coding sequence of the cDNA was amplified by PCR. Vent polymerase (NEB) was used as the polymerase.
  • Primer HGTpSErev 5 '- GACTCTCGAGTTATGTACTGGAAATTTTG- 3' (SEQ ID NO: 5)
  • Primer HGTpSEfor 5 '- CGAAACCATGGAGCATACACCTCACA- 3' (SEQ ID NO: 6)
  • PCR products were digested with the appropriate restriction enzymes and ligated into the corresponding vectors. Transformation took place in TOP 10 cells, with pQE60 additionally in Ml 5 cells.
  • ToplO E. coli were plasmid pSE280 in LB-Amp medium (1 1 Erlenmeyer flask, reaction volume of 200 ml, 40 h), in which 200 mg / 1 hydroquinone was present, at different temperatures (25 ° C, 30 ° C, 37 ° C) cultivated.
  • the growth of the microorganism was not restricted by the presence of hydroquinone at any of the chosen temperatures. The same was true for the upscale in 50 1 reactors with oxygen, stirring and pH control, where much higher hydroquinone concentrations of up to 10% (w / w) were tolerated.
  • the maximum enzyme activities for E. coli Ml 5 (pQE60) are over 750 nkat / 1.
  • Example 4 The recombinant E. coli listed in Example 4 were cultivated under the same conditions as in Example 4 with the substrates listed in Table 3. The relative activity of arbutin synthase for the individual substrates is shown in Table 3.

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)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une séquence d'acide nucléique correspondant à SEQ ID NO:1 ou son dérivé ou fragment ou les variantes de la séquence d'acide nucléique correspondant à SEQ ID NO:1 ou un dérivé ou fragment de la variante, codant pour un polypeptide dont l'activité biologique est celle d'une arbutine synthase, ainsi qu'un polypeptide codé par ces séquences d'acide nucléique. Cette invention concerne également un procédé de fabrication des polypeptides de l'invention, ainsi qu'un procédé de préparation de glycosides.
PCT/DE2000/002514 1999-07-27 2000-07-27 Glycosyltransferase, sa fabrication et son utilisation Ceased WO2001007631A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19935140.6 1999-07-27
DE19935140A DE19935140A1 (de) 1999-07-27 1999-07-27 Neue Glykosyltransferase, ihre Herstellung und Verwendung

Publications (2)

Publication Number Publication Date
WO2001007631A2 true WO2001007631A2 (fr) 2001-02-01
WO2001007631A3 WO2001007631A3 (fr) 2001-08-23

Family

ID=7916155

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2000/002514 Ceased WO2001007631A2 (fr) 1999-07-27 2000-07-27 Glycosyltransferase, sa fabrication et son utilisation

Country Status (2)

Country Link
DE (1) DE19935140A1 (fr)
WO (1) WO2001007631A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7056742B2 (en) 2003-06-16 2006-06-06 E. I. Du Pont De Nemours And Company High Level production of arbutin in green plants
US7846697B2 (en) 2003-06-19 2010-12-07 BLMaps Method of producing a low molecular weight organic compound in a cell
WO2017050853A1 (fr) 2015-09-24 2017-03-30 Evolva Sa Production d'anthocyanine à partir de sucres simples
US11884781B2 (en) 2017-11-22 2024-01-30 Suprapolix B.V. Supramolecular biomedical polymers

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5208151A (en) * 1988-08-18 1993-05-04 Nihon Shokuhin Kako Co., Ltd. Process for the preparation of derivatives of maltooligosaccharides
ES2121947T3 (es) * 1992-05-15 1998-12-16 Shiseido Co Ltd Preparacion externa para la piel.
GB9223235D0 (en) * 1992-11-05 1992-12-16 Unilever Plc Cosmetic composition
GB9322007D0 (en) * 1993-10-26 1993-12-15 Unilever Plc Cosmetic composition

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AREND JOACHIM ET AL: "Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: Enrichment and partial amino acid sequences." PHYTOCHEMISTRY (OXFORD), Bd. 53, Nr. 2, Januar 2000 (2000-01), Seiten 187-193, XP000982825 ISSN: 0031-9422 *
DATABASE EMBL [Online] Accession AF007269, 24. Juni 1997 (1997-06-24) SCHEET P ET AL: "Arabidopsis thaliana BAC IG002N01" XP002163428 *
KITAO SATOSHI ET AL: "Alpha-D-Glucosyl transfer to phenolic compounds by sucrose phosphorylase from Leuconostoc mesenteroides and production of alpha-arbutin." BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Bd. 58, Nr. 1, 1994, Seiten 38-42, XP002163427 ISSN: 0916-8451 *
LUTTERBACH RALF ET AL: "High-yield formation of arbutin from hydroquinone by cell-suspension cultures of Rauwolfia serpentia." HELVETICA CHIMICA ACTA, Bd. 75, Nr. 6, 1992, Seiten 2009-2011, XP002163426 ISSN: 0018-019X in der Anmeldung erw{hnt *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7056742B2 (en) 2003-06-16 2006-06-06 E. I. Du Pont De Nemours And Company High Level production of arbutin in green plants
US7217810B2 (en) 2003-06-16 2007-05-15 E. I. Du Pont De Nemours And Company High level production of arbutin in green plants and microbes
US7846697B2 (en) 2003-06-19 2010-12-07 BLMaps Method of producing a low molecular weight organic compound in a cell
EP2388333A2 (fr) 2003-06-19 2011-11-23 Evolva SA Procédé pour la production d'un composé organique de faible poids moléculaire dans une cellule
US8105786B2 (en) 2003-06-19 2012-01-31 Evolva Sa Method of producing a low molecular weight organic compound in a cell
US8986950B2 (en) 2003-06-19 2015-03-24 Evolva Sa Method of producing a low molecular weight organic compound in a cell
WO2017050853A1 (fr) 2015-09-24 2017-03-30 Evolva Sa Production d'anthocyanine à partir de sucres simples
US11884781B2 (en) 2017-11-22 2024-01-30 Suprapolix B.V. Supramolecular biomedical polymers

Also Published As

Publication number Publication date
DE19935140A1 (de) 2001-02-15
WO2001007631A3 (fr) 2001-08-23

Similar Documents

Publication Publication Date Title
DE69736261T2 (de) Alpha-1-6-fucosyltransferasen
KR102338008B1 (ko) 당사슬 연장에 촉매 작용하기 위한 한 그룹의 udp-글리코실트랜스퍼라제 및 이의 응용
DE69535543T2 (de) Für enzyme, die die fähigkeit besitzen lineare alpha 1,4-glucane in pflanzen, pilzen und mikroorganismen zu synthesieren, kodierende dna sequenzen
DE69732628T2 (de) Beta 1-4 n-acetylglucosaminyltransferase sowie das für diese kodierende gen
DE3629890A1 (de) Mikroorganismen und plasmide fuer die 2,4-dichlorphenoxyessigsaeure (2,4-d)-monooxigenase - bildung und verfahren zur herstellung dieser plasmide und staemme
AT504347B1 (de) Verfahren zur herstellung von glucosederivaten
DE19610984A1 (de) Alkohol-Dehydrogenase und deren Verwendung zur enzymatischen Herstellung chiraler Hydroxyverbindungen
US20200181631A1 (en) Plants and Methods for Increasing and Decreasing Synthesis of Cannabinoids
CN115109763A (zh) 一种黄酮醇3-o-葡萄糖苷生物合成相关的黄酮醇3-o-葡萄糖基转移酶及其应用
DE69322240T2 (de) N-acetylglucosaminyltransferase v kodierende sequenzen
DE69333812T2 (de) Endo-Xyloglucan-Transferase
CN104830817B (zh) 一种类黄酮异戊烯基转移酶AhFDT1及其编码基因和应用
DE69816586T2 (de) Beta-Carotenhydroxylase Gen
DE3639725A1 (de) Human-mangansuperoxiddismutase-cdna, ihre expression in bakterien und verfahren zur reindarstellung enzymatisch aktiver human-mangansuperoxiddismutase
KR101808749B1 (ko) 진세노사이드 글리코시다제를 이용한 진세노사이드 20(S)-Rg3 및 20(S)-Rh2의 제조방법
CN116855465A (zh) 一种异鼠李素生物合成相关的黄酮醇3’-o-氧甲基转移酶及其应用
CN112831481B (zh) 糖基转移酶以及催化糖链延伸的方法
DE69937444T2 (de) Isolierte und aufgereinigte nukleinsäuren, welche ein gen enthalten, welches in hopfendrüsen exprimiert wird
DE19935140A1 (de) Neue Glykosyltransferase, ihre Herstellung und Verwendung
Mohammadparast et al. Enhancement of the production of terpenoid and flavonoid secondary metabolites in the ground and aerial parts of licorice composite plant in a hydroponic system
CN104894080B (zh) 一种类黄酮异戊烯基转移酶AhFDT3及其编码基因和应用
CN104830816B (zh) 一种类黄酮异戊烯基转移酶AhFDT2及其编码基因和应用
DE60032328T2 (de) Verfahren zur herstellung von coenzym q 10
CN116042547B (zh) 一种黄酮3′-羟基化酶及其应用
DE60008962T2 (de) Klonierung einer N-Methyltransferase, die an der Koffeinbiosynthese beteiligt ist

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP