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WO2007099994A1 - carbonyle reductase, gene pour la reductase, vecteur, transformant et procede de production d'alcool optiquement actif au moyen de ces materiaux - Google Patents

carbonyle reductase, gene pour la reductase, vecteur, transformant et procede de production d'alcool optiquement actif au moyen de ces materiaux Download PDF

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Publication number
WO2007099994A1
WO2007099994A1 PCT/JP2007/053739 JP2007053739W WO2007099994A1 WO 2007099994 A1 WO2007099994 A1 WO 2007099994A1 JP 2007053739 W JP2007053739 W JP 2007053739W WO 2007099994 A1 WO2007099994 A1 WO 2007099994A1
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Prior art keywords
polypeptide
dna
activity
seq
transformant
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PCT/JP2007/053739
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English (en)
Japanese (ja)
Inventor
Tozo Nishiyama
Noriyuki Kizaki
Yoshihiko Yasohara
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Kaneka Corporation
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Publication of WO2007099994A1 publication Critical patent/WO2007099994A1/fr

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    • 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
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • 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)

Definitions

  • Novel carbonyl reductase its gene, vector, transformant, and method for producing optically active alcohol using them
  • Optically active alcohols such as (R) — 1 — (3,4-dimethoxyphenyl) — 2 — propanol are useful compounds as synthetic raw materials and intermediates for agricultural chemicals and pharmaceuticals.
  • As a method of asymmetrically reducing 3,4_dimethoxyphenylacetone to produce (R) — 1- (3,4-dimethoxyphenyl) 1 2-propanol There is known a reduction method using cells of microorganisms belonging to the genus of media and Pseudomonas (Patent Document 1).
  • Patent Document 1 Patent No. 3587569
  • the present invention is a method using a microorganism of Patent Document 1 described above and a novel carbonyl reductase, a gene thereof, a vector containing the gene, and transformation using the vector, which are different from the enzymes disclosed in Non-Patent Document 1. It is an object of the present invention to provide a method for producing an optically active alcohol using the transformed transformant. Means for solving the problem
  • the present invention has one or more of the following features.
  • [0005] (1) One feature of the present invention is the following DNA (a) or (b).
  • polypeptide comprising an amino acid sequence showing 80% homology (identity) with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and asymmetric 3,4-dimethoxyphenylacetone A polypeptide having an activity of reducing to form (R) -l- (3,4-dimethoxyphenyl) -2-propanol.
  • Another feature of the present invention is that a compound encoded by the DNA according to any one of (1), (2), and (4) and having a carbonyl group is reduced.
  • Another feature of the present invention is encoded by the DNA described in either (2) or (4), and asymmetrically reduces 3,4-dimethoxyphenylacetone.
  • Another feature of the present invention is a vector comprising the DNA according to any one of (1), (2), and (4).
  • Another feature of the present invention is the vector according to claim 7, further comprising DNA encoding a polypeptide having glucose dehydrogenase activity.
  • Another feature of the present invention is a transformant obtained by transforming a host cell with the vector according to any one of (7) and (8).
  • polypeptide according to (3), (5) or (6), or the transformant according to (9) or (10) A method for producing an optically active alcohol, characterized by reacting with a compound having a ru group.
  • the present invention provides a novel carbonyl reductase, its gene, a vector containing the gene, a transformant transformed with the vector, and a method for producing an optically active alcohol using them.
  • FIG. 1 shows a production method and structure of a recombinant vector pNPSG as an embodiment of the present invention.
  • polypeptide of the present invention a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing encoded by the base sequence shown in SEQ ID NO: 1 of the sequence listing can be mentioned.
  • a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing It has a certain degree of homology (identity) with the peptide, and asymmetrically reduces 3,4-dimethoxyphenyl ketone, and (R) — 1- (3, 4-dimethoxyphenyl) 1)
  • a polypeptide having an activity to produce 2-propanol is equivalent to the polypeptide and is included in the present invention.
  • sequence homology is obtained when, for example, two amino acid sequences are compared and analyzed using the homology search program FASTA (WR Pearson & DJ Lipman PNAS (1988) 85: 2444-2448). Represented by the Identity value for.
  • FASTA WR Pearson & DJ Lipman PNAS (1988) 85: 2444-2448.
  • the homology with the polypeptide is 80% or more, preferably 90% or more, more preferably May include polypeptides that are 95% or more.
  • the substrate specificity of dehydrogenases belonging to this category 3 is 4 to 16 carbon atoms. It has only been reported to have a reducing activity against ⁇ -ketoesters such as 3-ketosil CoA (Akio Kobayashi et al, J. Biochem. (1996) 119 775-782). Based on the technical common knowledge of those skilled in the art, the polypeptide of the embodiment of the present invention showing high homology with a 3-hydroxylacyl CoA dehydrogenase belonging to Category 3 is significantly different in structure from ⁇ -ketoesters. In general, asymmetric reduction of dimethoxyphenylacetone cannot be achieved.
  • the "polypeptide" of the present invention is, for example, a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence IJ shown in SEQ ID NO: 1 in the sequence listing. After ligation to an appropriate vector, it is obtained by introducing it into an appropriate host cell and expressing it.
  • amino acid substitution is performed on a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. It can also be obtained by causing insertions, deletions or additions.
  • the number of amino acids causing substitution, insertion, deletion or addition is not limited as long as the activity of the polypeptide of the embodiment is not lost, but it is preferably 50 amino acids or less, more preferably 30 No more than amino acids, more preferably no more than 10 amino acids, most preferably no more than 5.
  • the microorganism that is the origin of the polypeptide of the present invention is not particularly limited, and examples thereof include bacteria belonging to the genus Pseudomonas, which is particularly preferred, as Pseudomonas' Sulle (Pseudomonas ⁇ ⁇ ⁇ ii) List NBRC 13596 shares.
  • the microorganisms can be obtained from the Biological Genetic Resource Department (NBRC: Kiyotsutsu Kazusa 2_5 -8 Chiba Prefecture), National Institute of Biotechnology, Biotechnology Division, National Institute for Product Evaluation and Technology (NBRC).
  • Isolation of the polypeptide from the microorganism that is the source of the polypeptide of the present invention can be carried out by appropriately combining known protein purification methods. For example, it can be implemented as follows. First, the microorganism is cultured in an appropriate medium, and centrifuged from the culture solution. Or, the cells are collected by filtration. The obtained cells are crushed by a physical method using an ultrasonic breaker or glass beads, and then the cell residue is removed by centrifugation to obtain a cell-free extract.
  • the polypeptide of the present invention is isolated from the cell-free extract.
  • the “DNA” of the present invention is asymmetric with a DNA encoding a polypeptide having an activity of reducing a compound having a carbonyl group to produce an optically active alcohol, preferably 3,4-dimethoxyphenylacetone. It is a DNA that encodes a polypeptide having the activity of reducing and producing (R) -1- (3,4-dimethoxyphenyl) -2-propanol.
  • Any untranslated region may be included as long as it can express the polypeptide.
  • a person skilled in the art can obtain the DNA of the present invention from a microorganism that is the origin of the polypeptide by a known method. For example, the DNA of the present invention can be obtained by the method shown below.
  • the isolated polypeptide of the present invention is digested with an appropriate endopeptidase, and the resulting peptide fragment is fractionated by reverse phase HPLC. Then, for example, a part or all of the amino acid sequences of these peptide fragments are determined by ABI492 type sequencer (Applied Biosystems).
  • a PCR (Polymerase Chain Reaction) primer for amplifying a part of DNA encoding the polypeptide is synthesized.
  • chromosomal DNA of the microorganism that is the origin of the polypeptide is prepared by a conventional DNA isolation method, for example, the method of Visser et al. (Appl. Microbiol. Biotechnol., 53, 415 (2000)).
  • PCR is performed using the PCR primers described above, a part of the DNA encoding the polypeptide is amplified, and the nucleotide sequence is determined.
  • ABI373A DNA Sequencer Applied Biosystems
  • a DNA containing the base sequence shown in SEQ ID NO: 1 in the Sequence Listing can be mentioned. Further, it has a base sequence in which one or several bases are substituted, inserted, deleted and / or added in SEQ ID NO: 1, and 3,4-dimethoxyphenylacetone is asymmetrically reduced.
  • (R) -1- (3,4-dimethoxyphenyl) -2-DNA encoding a polypeptide having activity to produce propanol is included in the present invention.
  • the “several bases” is not limited as long as the polypeptide encoded by DNA does not lose the above activity, but is preferably 150 bases or less, more preferably 100 bases or less, and even more preferably 50 No more than bases, most preferably no more than 25 bases.
  • polypeptide comprising the base sequence represented by SEQ ID NO: 1 having a homology of 80% or more, preferably 90% or more, more preferably 95% or more and having the above activity.
  • DNA encoding a tide is included in the present invention.
  • DNA of the present invention is a DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing, and asymmetric with 3,4-dimethoxyphenylacetone.
  • the DNA of the present invention also includes a DNA that encodes a polypeptide having an activity of being reduced to form (R) -1- (3,4-dimethoxyphenyl) -2-propanol.
  • a DNA encoding a polypeptide having the above is also included in the DNA of the present invention.
  • a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing and a DNA that hybridizes under stringent conditions include colony'hybridization method, plaque'hybridizer. DN consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing when the hybridization method or Southern hybridization method is performed. A force A DNA that specifically forms a hybrid.
  • the stringent conditions are, for example, 75 mM trisodium citrate, 750 mM sodium chloride, 0.5% sodium dodecyl sulfate, 0.1% ushi serum albumin, 0.1% polyvinylinole.
  • the stringent conditions are, for example, 75 mM trisodium citrate, 750 mM sodium chloride, 0.5% sodium dodecyl sulfate, 0.1% ushi serum albumin, 0.1% polyvinylinole.
  • 15 mM trisodium citrate 150 mM sodium chloride
  • washing is performed at 65 ° C.
  • the washing is performed at 65 ° C. using an aqueous solution composed of 1.5 mM trisodium citrate, 15 mM sodium chloride, and 0.1% sodium dodecyl sulfate.
  • the “vector” of the present invention is not particularly limited as long as it can express the gene encoded by the DNA in a suitable host cell.
  • examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors capable of exchanging genes with other host strains can also be used.
  • Such vectors usually contain regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter, etc., and are operable with the DNA of the present invention. It can be suitably used as an expression vector comprising an expression unit linked to the. For example, PUCN18 described later can be used preferably.
  • the regulatory elements include functional promoters and any associated transcription elements (eg, Enhancer, CCAAT box, TATA box, SPI site, etc.).
  • operably linked means that a gene regulatory force such as a promoter that regulates the expression of a gene, an enhancer, etc. is linked in a state capable of operating in a host cell.
  • a gene regulatory force such as a promoter that regulates the expression of a gene, an enhancer, etc.
  • the type and kind of the control factor can vary depending on the host.
  • a plasmid pNPS described later in which the DNA shown in SEQ ID NO: 1 is introduced into the above pUCN18 can be mentioned (see Example 3).
  • Examples of the “host cell” described in the present specification include bacteria, yeast, filamentous fungi, plant cells, animal cells and the like, but Escherichia coli, which is preferred by bacteria from the introduction and expression efficiency, is particularly preferable.
  • the vector containing the DNA of the present invention can be introduced into a host cell by a known method. When Escherichia coli is used as a host cell, the vector can be introduced into the host cell by using, for example, a commercially available coli HB101 recombinant cell (manufactured by Takara Bio Inc.).
  • the “transformant” of the present invention can be obtained by incorporating DNA encoding the polypeptide of the present invention into the vector and introducing it into a host cell.
  • the “transformant” of the present invention includes not only cultured cells but also processed products thereof.
  • treated products include, for example, cells treated with a surfactant or an organic solvent, dried cells, disrupted cells, crude cell extracts, etc., and those obtained by immobilizing them by known means. This means that as long as the activity of asymmetrically reducing 3,4-dimethoxyphenylacetone to produce (R) — 1- (3,4-dimethoxyphenol) —2_propanol remains included.
  • Culture of the transformant of the present invention can be performed using a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like as long as it grows.
  • Examples of the transformant of the present invention include coli HBlOl (pNPS) described later (see Example 5).
  • Method for producing optically active alcohol is a transformant comprising a compound having a carboxy group serving as a substrate and a polypeptide of the present invention or DNA encoding the polypeptide in a suitable solvent. Can be added. If necessary, a coenzyme such as NADH may be added.
  • an aqueous solvent may be used, or an aqueous solvent and an organic solvent may be mixed and used.
  • the organic solvent include toluene, ethyl acetate, n-butyl acetate, hexane, isopropanol, diisopropyl ether, methanol, acetone, dimethyl sulfoxide and the like.
  • the reaction is carried out at a temperature of 10 ° C to 70 ° C, for example, and the pH of the reaction solution is maintained at 4 to 10 for example.
  • the reaction can be carried out batchwise or continuously. In the case of a batch system, the reaction substrate is added at a charge concentration of, for example, 0.1% to 70% (w / v).
  • Examples of the “compound having a carbonyl group” as a substrate include, for example, 3, 4-dimethoxyphenylacetone and the like, which are reduced under the above reaction conditions and converted into “optically active alcohol” If it is, it will not be specifically limited.
  • Optically active alcohols generated by the reaction can be purified by a conventional method. For example, a reaction solution containing optically active alcohols produced in the reaction is extracted with an organic solvent such as ethyl acetate or toluene, and the organic solvent is distilled off under reduced pressure, followed by distillation, recrystallization, chromatography, etc. It can refine
  • an organic solvent such as ethyl acetate or toluene
  • the compound having the carbonyl group By contacting and reacting the polypeptide of the present invention, a compound having a carbonyl group, and a coenzyme such as NADH as necessary, the compound having the carbonyl group is reduced asymmetrically. Optically active alcohols can be produced. At this time, as the reaction proceeds, coenzymes such as NADH are converted to oxidized forms. At this time, a polypeptide having the ability to convert this oxidized coenzyme into a reduced form (hereinafter referred to as coenzyme regeneration ability) and a compound serving as a substrate for the polypeptide are converted to the polypeptide of the present invention. The amount of coenzyme used can be reduced by carrying out the reaction in the presence of.
  • polypeptide having coenzyme regeneration ability examples include hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose 6-phosphate dehydrogenase, and gnolecose dehydrogenase.
  • glucose dehydrin enzyme is used.
  • An example of a transformant containing both the DNA encoding the polypeptide of the present invention and the DNA encoding a polypeptide capable of coenzyme regeneration is obtained by transforming coli HB101 with the above-described pNPSG.
  • Examples thereof include coli HBlOl (pNPSG) described later (see Example 5).
  • the culture of a transformant containing both the DNA encoding the polypeptide of the present invention and the DNA encoding the polypeptide having the coenzyme regeneration ability is a normal carbon source as long as it grows. , Using a liquid nutrient medium containing nitrogen sources, inorganic salts, organic nutrients, etc.
  • the above reaction composition contains a polypeptide having a coenzyme regenerating ability (for example, glucose dehydrogenation).
  • Enzyme and its substrate compound (eg, dalcose) are further added.
  • Optically active alcohols can be produced in the same manner using a transformant containing both the DNA encoding the polypeptide of the present invention and the DNA encoding a polypeptide having coenzyme regeneration ability.
  • the polypeptides of the present invention are produced by combining the polypeptide of the present invention and a polypeptide having a coenzyme regenerating ability.
  • a transformant containing both a DNA encoding a peptide and a DNA encoding a polypeptide having a coenzyme regeneration ability or a processed product thereof a polypeptide having a coenzyme regeneration ability (for example, glucose
  • a polypeptide having a coenzyme regeneration ability for example, glucose
  • the polypeptide of the present invention can be efficiently produced, and by using it, for example, (R) — 1 — (3,4-dimethoxyphenyl) )
  • An excellent method for producing useful optically active alcohols including 2_propanol is provided (see Example 7).
  • the reduction activity for 3,4-dimethoxyphenylacetone was obtained by adding 2 mM substrate 3,4-dimethoxy to lOOmM phosphate buffer (pH 6.5) containing 0.4% (v / v) dimethylol sulfoxide. Add siphenylacetone, 0.167 mM coenzyme NADH, and crude enzyme 30. It was calculated from the rate of decrease in absorbance at a wavelength of 340 nm when reacted with C for 1 minute. Under this reaction condition, the activity to oxidize 1 / i mol NADH to NAD per minute is defined as limit.
  • Bacteria were collected from the culture broth by centrifugation and washed with 0.85% aqueous sodium chloride solution.
  • the cells are suspended in a 40 mM phosphate buffer (pH 7.5) containing a protease inhibitor cocktail (Roche), crushed using a SONIFIER250 ultrasonic crusher (BRANSON), and then centrifuged. The cell residue was removed to obtain a cell-free extract.
  • the cell-free extract obtained above was treated at 45 ° C. for 20 minutes, and then the insoluble fraction was removed by centrifugation to obtain a heat-treated cell-free extract.
  • Ammonium sulfate was added to the heat-treated cell-free extract obtained above to a final concentration of 1 M and stirred for 1 hour, and then the precipitate was removed by centrifugation. Ammonium sulfate was added to the supernatant to a final concentration of 3M, and after stirring for 1 hour, a precipitate was obtained by centrifugation. This precipitate was dissolved in 40 mM phosphate buffer (pH 7.5) and dialyzed overnight against the same buffer.
  • the active fraction of the ammonium sulfate fraction was applied to a DEAE-TOYOPEARL 650M (Tosohichi Co., Ltd.) column (30 ml) pre-equilibrated with 40 mM phosphate buffer (pH 7.5) to elute the active fraction. .
  • the active fractions, 10 mM phosphate buffer solution (P H7. 5) was dialyzed overnight at, 10 mM phosphate buffer (pH7. 5) (manufactured by Tosoh Corporation) DEAE-TOYOPEARL 650M, previously equilibrated with The column was applied to a column (30 ml) to adsorb the active fraction. After washing the strength ram with the same buffer, the active fraction was eluted with a NaCl linear gradient (from 0 M to 0.2 M).
  • the active fraction obtained by Phenyl-TOYOPEARL column chromatography was applied to a Blue Sepharose 6 Fast Flow (Amersham Biosciences) column (2 ml) pre-equilibrated with 10 mM phosphate buffer (pH 7.5). The active fraction was adsorbed. After washing the column with the same buffer, the active fraction was eluted with NaCl stepwise (from 0M to 2M every 0.2M) to obtain a purified preparation of a single polypeptide by electrophoresis.
  • the purified polypeptide obtained in Example 1 was denatured in the presence of 8M urea, and then digested with achromobacterium-derived ricinoleendopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The amino acid sequence of the obtained peptide fragment was converted to ABI492. Type protein sequencer (manufactured by PerkinElmer).
  • Primer 1 5 ′ —ATGCARATHMGNGAYAARGT— 3 ′ (SEQ ID NO: 3 in the sequence listing) for amplifying a part of the gene encoding the polypeptide by PCR based on the DNA sequence predicted from this amino acid sequence
  • Primer 2 5′—GTCATNACNCGDATNCCRAA—3 ′ (SEQ ID NO: 4 in the sequence listing) was synthesized.
  • the chromosomal DNA of Pseudomonas stutzeri NBRC135 96 strain prepared above was completely digested with restriction enzyme Pstl, and the resulting mixture of DNA fragments was intramolecularly cyclized with T4 ligase.
  • Pstl restriction enzyme
  • T4 ligase T4 ligase
  • Example 2 Obtained in Example 2 using primer 3: 5'—GGGAGAGCCATATGCAGATTCGCGACAAGGTA— 3 ′ (sequence table 1J number 6) and primer 4: 5′-TCTCTGGAATTCTCACTTGGCGGCCATGC GCAA-3 ′ (sequence number 7 in the sequence table) Pseudomonas ⁇ ⁇ ⁇ ⁇ Perform PCR using the chromosomal DNA of NBRC13596 strain as a saddle.
  • SEQ ID NO: 2 in the sequence listing shows the amino acid sequence encoded by the gene consisting of the base sequence shown in SEQ ID NO: 1.
  • PCR was performed using Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual.
  • the DNA fragment obtained by the above PCR was digested with Ndel and EcoRI, and the 185th T of plasmid pUCNl8 (pUC18 (manufactured by Takara Bio Inc.) was changed to A by PCR to change the Ndel substrate.
  • the plasmid was newly introduced with the Ndel site by modifying the GC of positions 471 to 472 to TG, and inserted between the Ndel recognition site downstream of the lac promoter and the EcoRI recognition site.
  • a replacement vector pNPS was constructed.
  • the description of the 185th chome and the 471_472th 0th used here was in accordance with the description of 0611: 6 & 1 ⁇ Accession No. L09136.
  • Primer 5 5 '-CAGGAGCTCTAAGGAGGTTAACAATGTATAAAG-3' (SEQ ID NO: 8 in the sequence listing) and primer 6: 3 '_CACGGATCCTTATCCGCGTCCTGCTTGG 1 5' (SEQ ID NO: 9 in the sequence listing) were used to create plasmid pGDKl (Eur. J. Biochem., 186, 389 (1989), which can be obtained and prepared by those skilled in the art), and the glucose is dehydrogenase derived from Bacillus megaterium IAM 1030 (hereinafter referred to as GDH). ) Obtain double-stranded DNA with a ribosome binding sequence of E. coli 5 bases upstream from the start codon of the gene, a Sacl recognition site added just before it, and a BamHI recognition site added just after the stop codon. did.
  • coli HB101 complex cell manufactured by Takara Bio Inc. was transformed to obtain £ coli HBlOl (pNPS).
  • YT medium tryptone 1.6%, yeast extract 1.0% NaCl 0.5% pH 7.0
  • the cells were collected by centrifugation and suspended in 5 ml of lOOmM phosphate buffer (pH 6.5).
  • GDH activity was measured by adding glucose 0 ⁇ 1340, coenzyme NAD 2 mM, and crude enzyme solution to 1M Tris-HCl buffer ( ⁇ 8 ⁇ 0) and reacting at 25 ° C for 1 minute, and increasing the absorbance at a wavelength of 340 nm. Calculated from speed. Under these reaction conditions, the enzyme activity that reduces 1 / i mol of NAD to NADH per minute was defined as limit.
  • Example 7 Production of (R) -1 mono (3,4-dimethoxyphenyl) 2-propanol using a transformant To 1 ml of E coli HBlOl (pNPSG) culture solution cultured in the same manner as in Example 6, 10 mg of dalcose, 5 mg of NADlmg, 3,4 dimethoxyphenenoreacese was added and stirred at 30 ° C for 20 hours. .
  • reaction solution was extracted with ethyl acetate, and the obtained organic layer was dried over anhydrous sodium sulfate. After removing sodium sulfate by centrifugation and distilling off the organic solvent under reduced pressure, (R) -1- (3,4-dimethoxyphenyl) -1-propanol was obtained by TLC. The optical purity of this product is 98.3. /. It was ee.

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Abstract

L'invention a pour objet un procédé de production de (R)-1-(3,4-diméthoxyphényl)-2-propanol à bonne efficacité. Pour cela, l'invention divulgue : un polypeptide isolé à partir de Pseudomonas stutzeri pouvant réduire asymétriquement le 3,4-diméthoxyphénylacétone pour produire du (R)-1-(3,4-diméthoxyphényl)-2-propanol ; un ADN encodant le polypeptide ; un transformant pouvant produire le polypeptide et un procédé de production d'un alcool optiquement actif par réduction d'un composé carbonyle au moyen du polypeptide ou du transformant.
PCT/JP2007/053739 2006-03-02 2007-02-28 carbonyle reductase, gene pour la reductase, vecteur, transformant et procede de production d'alcool optiquement actif au moyen de ces materiaux WO2007099994A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012063843A1 (fr) 2010-11-09 2012-05-18 株式会社カネカ Indénones halogénées et procédé pour la production d'indanones optiquement actifs ou d'indanoles optiquement actifs utilisant lesdites indénones
US8404461B2 (en) 2009-10-15 2013-03-26 SK Biopharmaceutical Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US8501436B2 (en) 2009-06-22 2013-08-06 Sk Biopharmaceuticals Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] COPELAND A. ET AL., XP003017352, Database accession no. (AALM01000019) *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501436B2 (en) 2009-06-22 2013-08-06 Sk Biopharmaceuticals Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US8404461B2 (en) 2009-10-15 2013-03-26 SK Biopharmaceutical Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US9068207B2 (en) 2009-10-15 2015-06-30 Sk Biopharmaceuticals Co. Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
US9434970B2 (en) 2009-10-15 2016-09-06 Sk Biopharmaceuticals Co., Ltd. Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester
WO2012063843A1 (fr) 2010-11-09 2012-05-18 株式会社カネカ Indénones halogénées et procédé pour la production d'indanones optiquement actifs ou d'indanoles optiquement actifs utilisant lesdites indénones

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