WO2003093477A1 - Carbonyl reductase, gene de celle-ci et son utilisation - Google Patents
Carbonyl reductase, gene de celle-ci et son utilisation Download PDFInfo
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- WO2003093477A1 WO2003093477A1 PCT/JP2003/005500 JP0305500W WO03093477A1 WO 2003093477 A1 WO2003093477 A1 WO 2003093477A1 JP 0305500 W JP0305500 W JP 0305500W WO 03093477 A1 WO03093477 A1 WO 03093477A1
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- 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)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
Definitions
- the present invention provides the following formula (1)
- the asymmetric reduction is performed by the asymmetric reduction of the 1- (3,1-chlorophenyl) represented by the formula (2)
- the present invention also relates to a method for producing an optically active alcohol, particularly an optically active 1-phenylethanol derivative or an optically active 3-hydroxyestenole derivative, using the transformant.
- Optically active 1-phenylethanol derivatives and optically active 3-hydroxyester derivatives are useful compounds as raw materials for synthesis of pharmaceuticals, agricultural chemicals and the like. Background art
- the method for producing the optically active 1-fuunylethanol derivative includes:
- 2-Halo1-1 (substituted phenyl) Ethanone is treated with a microorganism belonging to the genus Asibia or Pogatataea or a processed product thereof to obtain an optically active 2-halo1-1 (substituted phenyl).
- a method for obtaining ethanol JP-A-4-218384, JP-A-111-215995
- the present invention provides a polypeptide useful for producing an optically active 1-phenylethanol derivative or an optically active 3-hydroxyester derivative, a polynucleotide encoding the polypeptide, the polynucleotide It is an object of the present invention to provide an expression vector comprising: and a transformant transformed by the expression vector. Another object of the present invention is to provide a method for efficiently producing an optically active 1-funilethanol derivative or an optically active 3-hydroxyxester derivative using the transformant.
- the present inventors asymmetrically reduced 2-chloro-1- (3'-chlorophenyl) ethanone to give (R) _2-chloro-1- (3'-chlorophenyl) ethanol.
- (R) _2-chloro-1- (3'-chlorophenyl) ethanol By isolating a polypeptide having the activity from a microorganism having the activity to be produced, and utilizing the polypeptide, not only (R) _2_chloro_1- (3,1-chlorophene) ethanol but also ethanol can be obtained.
- optically active alcohols such as optically active 1-phenylethanol derivatives such as toluene, and optically active 3-hydroxyester derivatives represented by (R) —4-chloro-3-hydroxybutyrate, etc. It has been found that it can be manufactured.
- optically active 1-phenylethanol derivatives such as toluene
- optically active 3-hydroxyester derivatives represented by (R) —4-chloro-3-hydroxybutyrate, etc. It has been found that it can be manufactured.
- the polynucleotide encoding the polypeptide was isolated, and an expression vector and a transformant were successfully created. Thus, the present invention was completed.
- the present invention provides an asymmetric reduction of 2-cyclo-1- (3'-chlorophenyl) ethanone to give (R) _2-chloro-1- (3'-chlorophenyl) It is a polypeptide that can produce ethanol.
- the present invention is also a polynucleotide encoding the above polypeptide. Further, the present invention is an expression vector containing the polynucleotide. Further, the present invention is a transformant which highly produces the polypeptide.
- the present invention provides a method for producing a (R) -2-chloro-11- (3′-chlorophenyl) ethanol and (S) -1- (2,1-fluorophenyl) ethanol using the transformant. It is also a practical method for producing an optically active 1-phenylethanol derivative or an optically active 3-hydroxyester derivative represented by (R) -4 monochloro-3-ethylhydroxybutyrate.
- polypeptide of the present invention has the following formula (1)
- Such a polypeptide can be isolated from a microorganism having the activity.
- the microorganism used as the origin of the polypeptide is not particularly limited, and examples thereof include yeast of the genus Rhodotorula, and particularly preferred are Rhodotorula goninitinis. Bar direnensis IFO 0 415 strains can be mentioned.
- the microorganism producing the polypeptide of the present invention may be either a wild type or a mutant type. Alternatively, a microorganism derived by a genetic technique such as cell fusion or genetic manipulation can also be used.
- a microorganism producing the genetically engineered polypeptide of the present invention may be, for example, a step of isolating and / or purifying these polypeptides to determine a part or all of the amino acid sequence thereof, To determine the base sequence of the polynucleotide encoding the polypeptide, and to introduce the polynucleotide into another microorganism to obtain a recombinant microorganism.
- Purification of the polypeptide from the microorganism having the polypeptide of the present invention can be performed by a conventional method.
- the cells of the microorganism are cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation.
- the obtained cells are disrupted by, for example, an ultrasonic disrupter, and the cell residue is removed by centrifugation to obtain a cell-free extract.
- salting out ammonium sulfate precipitation, sodium phosphate precipitation, etc.
- solvent precipitation protein fraction precipitation with acetone or ethanol, etc.
- dialysis gel filtration, ion exchange, reverse phase, etc.
- Polypeptides can be purified using techniques such as column chromatography and ultrafiltration alone or in combination.
- the enzyme activity was measured by adding the substrate 2-chloro-1-1 (3, -chlorofuran) to 100 mM phosphate buffer (pH 6.5) containing 0.3% (v / v) dimethyl sulfoxide.
- Enyl) Ethanone lmM, 0.25 mM coenzyme NADPH and enzyme are added, and the decrease in absorbance at 340 nm at 30 ° C can be measured.
- polypeptide of the present invention examples include polypeptides having the following physicochemical properties (1) to (4).
- polypeptide of the present invention for example, (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or (b) the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing contains an amino acid sequence having at least one amino acid substitution, insertion, deletion, or addition, and the amino acid sequence has at least one amino acid sequence.
- (Phenyl phenyl) Polypeptides having the activity of asymmetrically reducing ethanone to produce (R) -2-chloro-1- (3,1-chlorophenyl) ethanol.
- a polypeptide comprising an amino acid sequence having at least one amino acid substitution, insertion, deletion or addition in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing is obtained by Curent Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989), etc. t can be prepared according to a known method described in this publication, and the asymmetric reduction of 2- (1,1-chlorophenyl) ethanone to (R ) — 2_cloguchi 1— (3, chlorophenyl) As long as it has an activity of producing ethanol, it is included in the polypeptide of the present invention.
- any polynucleotide can be used as long as it encodes the above polypeptide.
- a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing and a polynucleotide hybridizing under stringent conditions are complementary to the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.
- the polynucleotide shown in SEQ ID NO: 2 has a sequence identity of 60% or more, preferably 80 ° / 0 or more, more preferably 90% or more, More preferably, 95% or more, and most preferably, 99% or more of the polynucleotide can be mentioned. As long as it has the activity of reducing to (R) -2-chloro-1- (3,1-chlorophenyl) ethanol.
- sequence identity refers to the optimal alignment of two contrasted polynucleotides and the presence of a nucleobase (eg, A, T, C, G, U, or I) in both sequences. The number of matching positions in the column is divided by the total number of comparison bases, and the result is multiplied by 100. Sequence identity can be calculated, for example, using the following sequence analysis tools: Unix-based GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group 575 Science Drive Madison Rice, P.
- the polynucleotide of the present invention is obtained by asymmetrically reducing 2- (1-, 3-chloro-2-phenyl) ethanone to give (R) —2-cyclo-1,1- (3,1-chloro).
- Mouth phenyl) can be obtained from a microorganism having an activity of producing ethanol. Examples of such microorganisms include yeast belonging to the genus Rhodotorula, and particularly preferred is Rhodotorula glutinis var. Dairenensis IFO0415 strain.
- a partial amino acid sequence of the purified polypeptide and a peptide fragment obtained by digesting the polypeptide with an appropriate endopeptidase is determined by the Edman method. Then, nucleotide primers are synthesized based on the amino acid sequence information.
- the polynucleotide of the present invention is isolated from the microorganism as the origin of the polynucleotide by a conventional DNA isolation method, for example, the method described in Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989). Prepare the chromosomal DNA of the microorganism.
- PCR polymerase chain reaction
- the nucleotide sequence of the amplified polynucleotide is determined by the dideoxy sequence method, the dideoxy chain method, etc. Can be determined by For example, it can be carried out using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and ABI 373A DNA Sequencer (Perkin Elmer).
- the entire nucleotide sequence can be determined by, for example, the i-PCR method (Nucl. Acids Res. 16, 8186 (1988)). Can be determined.
- the polynucleotide on the chromosome DNA contains an intron, for example, the nucleotide sequence of a mature polynucleotide containing no intron can be determined by the following method.
- a conventional nucleotide isolation method for example, a method described in Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1989), is used from a microorganism which is a source of the polynucleotide.
- the RT-PCR method Proc. Nati. Acad. Acad., Ltd.
- the vector used to introduce the polynucleotide of the present invention into a host microorganism and express it in the host microorganism into which the polynucleotide has been introduced is a vector capable of expressing the gene in the polynucleotide in an appropriate host microorganism. If so, any can be used. Examples of such a vector include those selected from a plasmid vector, a phage vector, and a cosmid vector. Further, the shuttle vector may be a shuttle vector capable of gene exchange with another host strain.
- Such vectors usually contain regulatory elements such as the 1 ac UV5 promoter, the trp promoter, the trc promoter, the tac promoter, the lpp promoter, the tuf B promoter, the rec A promoter, the p L promoter, and the like. It can be suitably used as an expression vector containing an expression unit operably linked to a nucleotide.
- the term “regulator” refers to a base sequence having a functional promoter and any associated transcription elements (eg, enhancers, CCAAT boxes, TATA bottus, SPI sites, etc.).
- the term “operably linked” refers to a polynucleotide that, when expressed in a host cell, contains various regulatory elements such as promoters, enhancers, etc., that regulate its expression such that the gene in the polynucleotide is expressed. It is connected in a state where it can be operated by. It is well known to those skilled in the art that the type and type of the regulatory element may vary depending on the host cell.
- Examples of host cells into which the expression vector containing the polynucleotide of the present invention is introduced include bacteria, yeast, filamentous fungi, plant cells, animal cells, etc., and Escherichia coli is particularly preferred.
- An expression vector containing the polynucleotide of the present invention can be introduced into a host cell by a conventional method.
- Escherichia coli is used as a host cell, an expression vector containing the polynucleotide of the present invention can be introduced by, for example, a calcium chloride method.
- the 2- (1-, 3-chlorophenyl) ethanone can be asymmetrically reduced using the polypeptide of the present invention to give the (R) -2-cyclo-1,1- (3,1-chlorophenyl) ethanone.
- coenzymes such as NADPH and NADH are required.
- a coenzyme regenerating ability an enzyme having an ability to convert the oxidized coenzyme to a reduced form
- enzymes having a coenzyme regeneration ability include, for example, hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, and glucose dehydrogenase. I can do it. Preferably, glucose dehydrogenase is used.
- the reaction can also be performed by separately adding the enzyme having the coenzyme regenerating ability to the asymmetric reduction reaction system, but the polynucleotide of the present invention and the polypeptide having the coenzyme regenerating ability can be used.
- the reaction can be carried out efficiently without separately preparing an enzyme having a coenzyme regeneration ability and adding it to the reaction system. I can do it.
- Such a transformant is a polynucleotide encoding the polynucleotide of the present invention and a polypeptide (eg, glucose dehydrogenase) capable of regenerating a capture enzyme.
- a polypeptide eg, glucose dehydrogenase
- these two polynucleotides are incorporated into two vectors having different incompatibility groups, respectively, and the two vectors are used. Can also be introduced into the same host cell.
- the expression vector of the present invention contains the above-mentioned polynucleotide.
- An expression vector that is preferably plasmid p NTRG is mentioned.
- the expression vector further include a polynucleotide encoding a polypeptide having the glucose dehydrogenase activity.
- the polypeptide having glucose dehydrogenase activity is glucose dehydrogenase derived from Bacillus megaterium.
- an expression vector which is plasmid p NTRGG1 is mentioned.
- the transformant of the present invention is obtained by transforming a host cell using the above expression vector. Escherichia coli is preferred as the host cell.
- E.coli HB101 (p NTRG) is the accession number of FERM BP-7857, effective January 22, 2002,
- E.coli HB101 (pNTRGG 1) is the accession number of FERM BP-7858, as of January 22, 2002,
- the activity of an enzyme capable of regenerating a coenzyme in a transformant can be measured by a conventional method. For example, to measure glucose dehydrogenase activity, add 0.1 M substrate glucose, 2 mM coenzyme NADP and enzyme to 1 M Tris-HCl buffer (pH 8.0), and absorb at 340 nm at 25 ° C. This can be done by measuring the increase in
- the culture of the above-mentioned transformant or its processed product was An optically active alcohol is produced by reacting with a compound having a group.
- a compound having a carbonyl group serving as a substrate first, in a suitable solvent, a compound having a carbonyl group serving as a substrate,
- a coenzyme such as NADP, a culture of the transformant or a processed product thereof is added, and the mixture is stirred and reacted under pH adjustment.
- Culture of the transformant can be carried out using a liquid nutrient medium containing a usual carbon source, nitrogen source, inorganic salts, organic nutrients and the like as long as the microorganism grows.
- the culture temperature is preferably 4 to 50 ° C.
- the processed product of the transformant means, for example, a crude extract, a cultured cell, a freeze-dried organism, an acetone-dried organism, or a ground product thereof. Further, they can be used by immobilizing the polypeptide itself or the cells as they are by known means.
- the transformant When performing this reaction, when a transformant that produces both the polypeptide of the present invention and an enzyme having coenzyme-reproducing ability (for example, glucose dehydrogenase) is used, the transformant is added to the reaction system.
- an enzyme having coenzyme-reproducing ability for example, glucose dehydrogenase
- the transformant is added to the reaction system.
- a substrate for enzyme regeneration eg, glucose
- RR 2 represents a hydrogen atom, a halogen atom, an alkoxy group, or a nitro group, and may be the same or different.
- R 3 has a hydrogen atom, a halogen atom, a hydroxyl group, or a substituent.
- R 4 represents a hydrogen atom, a halogen atom, an azide, a benzyloxy group, or an alkyl group which may have a substituent
- R 5 represents an alkyl group or a phenyl group.
- 2-chloro-1- monochloropheninole
- 1- (2, one-funolelophenyl) ethanone or 4-chloroacetate.
- Ethyl acetate and the like can be mentioned.
- Examples of the optically active alcohol obtained by the above method include, for example, the formula (4)
- R 4 and R 5 are the same as described above). Examples thereof include: (R) —2-chloro-1- (3, Ethanol, (S) —1— (2′—funolelophenyl) ethanol, or (R) —4—cloth — 3-ethylethyl butyrate.
- Examples of the halogen atom in R 1 , R 2 , R 3 , and R 4 include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
- the alkoxy group in R ⁇ R 2 Ri alkoxy groups der of 1 to 3 carbon atoms, for example, main butoxy group, an ethoxy group, a propoxy group. Preferably, it is a methoxy group.
- the alkyl group for R 3 and RR 5 is an alkyl group having 1 to 8 carbon atoms, for example, a methyl group, an ethyl group, a propyl group, a hexyl group, an octyl group and the like. Preferably, it is an alkyl group having 1 to 2 carbon atoms.
- the alkyl group in R 3 and R 4 may have a substituent, and examples of the substituent include a fluorine atom, a chlorine atom, a bromine atom, a hydroxyl group, an amino group and the like.
- an aqueous solvent may be used as the solvent, or an aqueous solvent and an organic solvent may be mixed and used.
- the organic solvent include toluene, hexane, diisopropyl ether, n-butyl acetate, ethyl acetate and the like.
- the reaction temperature is 10 ° C to 70 ° C, preferably 20 to 40 ° C, and the reaction time is 1 to 100 hours, preferably 10 to 50 hours.
- the pH of the reaction solution is maintained at 4 to 10, preferably 5 to 8, using, for example, an aqueous solution of sodium hydroxide or an aqueous solution of sodium carbonate.
- the reaction can be performed in a batch or continuous mode.
- the reaction substrate is added at a feed concentration of 0.1% to 70% (w / v).
- the optically active alcohol generated by the reaction can be purified by a conventional method.
- the optically active alcohol produced by the reaction is (R) —2_chloro-1- (3,1-chlorophenyl) ethanol, (S) -1- (2'-fluorophenyl) ethanol, or (R) If it is 3-ethyl ethyl 3-butyrate, centrifugation, filtration, etc. are performed as necessary to remove suspended cells such as bacterial cells from the reaction product, and then ethyl acetate, toluene, etc. Extract with organic solvent and remove the organic solvent under reduced pressure.
- purification can be performed by a treatment such as distillation or chromatography.
- the polypeptide of the present invention can be efficiently produced, and by utilizing the same, an excellent method for producing various useful optically active alcohols is provided. .
- FIG. 1 is a diagram showing a polynucleotide sequence of the present invention and a deduced amino acid sequence.
- FIG. 2 is a diagram showing a method and a structure of a recombinant plasmid pNTRGG1. BEST MODE FOR CARRYING OUT THE INVENTION
- glutamine variniensis Rhodotorula glutinis var. Dairenensis
- An enzyme having the activity of reducing and producing (R) -2-chloro-11- (3'-chlorophenyl) ethanol was purified solely. Unless otherwise noted, purification procedures were performed at 4 ° C.
- the culture was performed while adjusting the lower limit pH to 5.5. After 18 hours, 22 hours, and 26 hours from the start of the culture, 655 g of a 55% (w / w) aqueous glucose solution was added, and the cells were cultured for 30 hours.
- Ammonium sulfate was added to the cell-free extract obtained above to make 45% saturation, dissolved, and the resulting precipitate was removed by centrifugation. (At this time, the pH of the cell-free extract was adjusted to pH 7. 5 while maintaining). While maintaining the pH at 7.5 as before, ammonium sulfate was further added to the centrifuged supernatant to 60% saturation, dissolved, and the resulting precipitate was collected by centrifugation. This precipitate was dissolved in 1 OmM phosphate buffer (pH 7.5) and dialyzed against the same buffer overnight.
- the crude enzyme solution obtained above is applied to a DEAE-TOYOPEARL 65 OM (manufactured by Tosoichi Co., Ltd.) column (250 ml) pre-equilibrated with 1 OmM phosphate buffer (pH 7.5). Adsorbed. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of NaCl (from OM to 0.3M). Active fractions were collected and dialyzed against 1 OmM phosphate buffer (pH 7.5) overnight.
- DEAE-TOYOPEARL 65 OM manufactured by Tosoichi Co., Ltd.
- Ammonium sulfate was dissolved in the crude enzyme solution obtained above to a final concentration of 1 M (this was performed while maintaining the pH of the crude enzyme solution at pH 7.5 with aqueous ammonia), and 1 M ammonium sulfate was added.
- Phenyl-TOYOPEARL 65 OM (Tosoichi Co., Ltd.) column pre-equilibrated with 1 OmM phosphate buffer (pH 7.5) (100 ml) to adsorb the enzyme. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (from 1M to 0M). The active fractions were collected and analyzed overnight with 1 OmM phosphate buffer (pH 7.5).
- the crude enzyme solution obtained above was previously equilibrated with 1 OmM phosphate buffer (pH 7.5), and then applied to a column (20 ml) of Blusepharose CL TM (manufactured by Bharmacia Biotech). The enzyme was adsorbed. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of NaCl (from 0M to 1M). The active fractions were collected and dialyzed against 1 OmM phosphate buffer (pH 7.5) overnight to obtain a single purified enzyme sample by electrophoresis. From now on, this enzyme will be called RRG.
- Example 2 Measurement of enzyme properties
- the enzyme activity was measured in a 100 mM phosphate buffer (pH 6.5) containing 0.3% (v_ / v) dimethyl sulfoxide, and the substrate 2_chloro-1-1 (3, (Phenyl) Ethanone lmM, 0.25 mM coenzyme NADPH and enzyme were added, reacted at 30 ° C for 1 minute, and the decrease in absorbance at a wavelength of 340 nm was measured.
- NAD PH is used as a coenzyme to act on 2- (1-, 3-chlorophenyl) ethanone and has an optical purity of 99.9% ee or more.
- '—Chlorophenyl) Ethanol was produced.
- the enzyme activity was measured according to the above method using NADH as a coenzyme, it showed about 7% of the activity when NADPH was used as a coenzyme.
- the buffer was 10 OmM phosphate buffer containing dimethyl sulfoxide and 10 OmM acetate buffer, and the pH was in the range of 4.0 to 8.0. Enzyme activity was measured in the same manner as in the enzyme activity measurement method described above. As a result, the optimal pH acting on 2-chloro-1,1 (3,1-chlorophenol) ethanone was 5.0 to 6.0.
- the enzyme activity was measured in the same manner as the above-mentioned method for measuring the enzyme activity, except that the temperature was 20 ° C to 60 ° C.
- the optimal temperature acting on 2-chloro-1- (3'-chlorophenyl) ethanone was 40 ° C to 50 ° C.
- the purified RRG obtained in Example 1 was denatured in the presence of 8M urea, and then digested with achromobacter-derived lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The sequence was determined using an ABI type 492 protein sequencer (Perkin Elmer). Based on this amino acid sequence, two DNA primers (primer 1, SEQ ID NO: 3; primer 2, SEQ ID NO: 4) were synthesized according to a conventional method.
- an N-terminal DNA primer (primer 3, SEQ ID NO: 5) having an NdeI site added to the start codon of the RRG gene
- a C-terminal DNA primer (primer 4, SEQ ID NO: 6) was added with a stop codon (TAA) and an EcoRI site immediately after the 3 'end of the gene.
- TAA stop codon
- EcoRI EcoRI site
- Example 6 Preparation of Recombinant Plasmid Containing Both RRG Gene and Glucose Dehydrogenase Gene
- the Bacillus megaterium I AMI030 strain-derived glucose dehydrogenase (hereinafter referred to as GDH) contains the E. coli Shaine-Dalgarno sequence (9 bases) 5 bases upstream from the start codon of the gene, and immediately before that. Then, a SacI cleavage point was obtained, and a double-stranded DNA having a BamHI cleavage point added immediately after the stop codon was obtained by the following method. Based on the base sequence information of the GDH gene, a Shaine-Dalgarno sequence (9 bases) of E. coli was added 5 bases upstream from the start codon of the GDH structural gene, and an N-terminal DN with an Eco RI cleavage point added immediately before it.
- GDH Bacillus megaterium I AMI030 strain-derived glucose dehydrogenase
- a primer (Primer 5, SEQ ID NO: 7) and a C-terminal DNA primer (Primer 6, SEQ ID NO: 8) having a Sal1 site added immediately after the termination codon of the structural gene of GDH were synthesized by a conventional method.
- plasmid pG Double-stranded DNA was synthesized by PCR using DK1 (Eur. J. Biom em. 186, 389 (1899)) as a template.
- the resulting DNA fragment was digested with EcoRI and Sa1I, and inserted into the EcoRI and Sa1I sites (present downstream of the RRG gene) of the pNTRG constructed in Example 5 for recombination.
- the plasmid pN TRGG1 was obtained.
- FIG. 2 shows a method for preparing p NT RGG1 and its structure.
- Example 7 Preparation of recombinant E. coli
- E. coli HB101 (manufactured by Takara Shuzo Co., Ltd.) was transformed using the recombinant plasmids pNTRG and pNTRGGl obtained in Examples 5 and 6, and recombinant E. coli HB101 (pNTRG) and HB101 (pNTRGGl) were transformed. Obtained.
- the thus obtained transformants E. coli HB101 (pNTRG) and E. coli HB101 (NTRGGl) were respectively obtained as Accession Nos.
- the recombinant Escherichia coli HB101 (pNTRG) obtained in Example 7 was cultured in 2 XYT medium containing lZO / ig / ml ampicillin, and after collecting the cells, lO OmM phosphate buffer (pH 6.5) , And crushed using a UH-50 ultrasonic homogenizer (manufactured by SMT) to obtain a cell-free extract.
- the RRG activity of this cell-free extract was measured as follows.
- the measurement of RRG activity was performed by adding 10% OmM phosphate buffer (pH 6.5) containing 0.3% (vZv) dimethyl sulfoxide to the substrate 2—cloth—11 (3′—clohenyl) ethanone lmM, The coenzyme NADPH 0.25 mM and the enzyme were added, and the measurement was performed by measuring the decrease in absorbance at 340 nm at 30 ° C. Under these reaction conditions, the enzyme activity that oxidizes ⁇ 1 of NAD ⁇ to NAD ⁇ per minute was defined as 1 unit. The RRG activity in the cell-free extract measured in this way was expressed as specific activity, and compared with the transformant carrying vector plasmid.
- a mouth dotorula prepared in the same manner as in Example 1 RRG activity in the cell-free extract of Rhodotorula glutinis var. Dairenensis I FO041 5 strain was similarly compared.
- Table 1 shows the results.
- Escherichia coli HB101 (pNTRG) showed a marked increase in RRG activity as compared to Escherichia coli HB101 (pUCNT), which is a transformant containing only vector plasmid, indicating that Rhodotorella gonoretinis nodilelenensis (Rhodotorula g) lutinis var. dairenensis)
- Rhodotorella gonoretinis nodilelenensis Rhodotorula g
- the specific activity was about 150 times higher than that of the IFO0415 strain.
- the GDH activity of the cell-free extract obtained by treating the recombinant Escherichia coli HB101 (pNTRGG1) obtained in Example 7 in the same manner as in Example 8 was measured as follows. GDH activity was measured by adding substrate glucose 0-1 M, coenzyme NADP 2 mM and enzyme to 1 M Tris-HCl buffer (pH 8.0), and measuring the absorbance at 340 nm at 25 ° C. This was done by measuring the increase. Under these reaction conditions, the enzyme activity of reducing 1 ⁇ mo 1 of NADP to NADPH per minute was defined as 1 unit. Also, the RRG activity was measured in the same manner as in Example 8.
- E. coli HB101 pNTRG
- pUCNT the transformant containing only vector plasmid
- the culture solution of the recombinant Escherichia coli HB101 (pNTRG) obtained in Example 8 was sonicated using SONIFIRE 250 (manufactured by BRANSON).
- Glucose dehydrogenase manufactured by Amano Pharmaceutical Co., Ltd. 2000U, 2 g of glucose dehydrogenase, 2 g of NADP 2 mg, 2 g of 1- (3, 1 g of feninole) and 2 g of ethanone was added.
- the reaction solution was stirred at 30 ° C. for 18 hours while adjusting the pH to 6.5 by adding 5 M sodium hydroxide.
- the culture solution of the recombinant E. coli HB101 (pNTRGG1) obtained in Example 9 was sonicated using SON IFI RE 250 (manufactured by BRAN SON). 3 g of glucose, 2 mg of NADP, and 2 g of 2- (3'-chlorophenyl) ethanone were added to 2 Om 1 of the cell lysate. The reaction solution was stirred at 30 ° C. for 24 hours while adjusting the pH to 6.5 by dropwise addition of 5 M sodium hydroxide. After the completion of the reaction, the reaction solution was extracted with toluene, and the solvent was removed. The extract was analyzed in the same manner as in Example 10. Black mouth Feuer) Ethanol was obtained.
- the culture solution of the recombinant E. coli HB101 (pNTRGG1) obtained in Example 9 was sonicated using SON I FIRE 250 (manufactured by BRAN SON). 4 g of glucose and 3 mg of NADP were added to 20 ml of the cell lysate. The reaction solution was stirred at 30 ° C and adjusted to pH 6.5 by dropwise addition of 5 M sodium hydroxide, and a total of 2 g of 4-ethyl chloroacetoacetate was continuously added at a rate of 0.2 g per hour. Was added. After the addition was completed, the reaction was continued for another 12 hours. After the completion of the reaction, the reaction solution was extracted with ethyl acetate, and the solvent was removed.
- the culture solution of the recombinant Escherichia coli HB101 (pNTRGG1) obtained in Example 9 was sonicated using SON IFI RE250 (manufactured by BRAN SON). 15 g of glucose, 5 g of 1- (2'-fluorophenyl) ethanone and 5 mg of NADP were added to 10 Om1 of the cell lysate. The reaction solution was stirred at 30 ° C. for 24 hours while adjusting the pH to 6.5 with 5 M sodium hydroxide. After completion of the reaction, the reaction mixture was extracted with ethyl acetate, and the solvent was removed.
- the reducing activity of RRG on various carbonyl compounds was investigated. Under the basic reaction conditions for the measurement of RRG activity in Example 2, the activity was measured using various carbonyl compounds shown in Table 3 as substrates instead of 2-chloro-1- (3, -chlorophenyl) ethanone. The measurement results are shown in Table 3 as relative values when the reduction activity was 100% when 2-chloro-1- (3,1-chlorophenyl) ethanone was used as the substrate.
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Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003235980A AU2003235980A1 (en) | 2002-04-30 | 2003-04-30 | Novel carbonyl reductase, gene thereof and use of the same |
US10/512,908 US7332312B2 (en) | 2002-04-30 | 2003-04-30 | Carbonyl reductase, gene thereof and use of the same |
JP2004501613A JP4426437B2 (ja) | 2002-04-30 | 2003-04-30 | 新規カルボニル還元酵素、その遺伝子、およびその利用法 |
EP03720993A EP1505155B1 (en) | 2002-04-30 | 2003-04-30 | Carbonyl reductase, gene thereof and use of the same |
DE60325046T DE60325046D1 (de) | 2002-04-30 | 2003-04-30 | Carbonylreduktase, deren gen und verwendung |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2002128648 | 2002-04-30 | ||
JP2002-128648 | 2002-04-30 |
Publications (1)
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WO2003093477A1 true WO2003093477A1 (fr) | 2003-11-13 |
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ID=29397271
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Application Number | Title | Priority Date | Filing Date |
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PCT/JP2003/005500 WO2003093477A1 (fr) | 2002-04-30 | 2003-04-30 | Carbonyl reductase, gene de celle-ci et son utilisation |
Country Status (7)
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US (1) | US7332312B2 (ja) |
EP (1) | EP1505155B1 (ja) |
JP (1) | JP4426437B2 (ja) |
AT (1) | ATE416255T1 (ja) |
AU (1) | AU2003235980A1 (ja) |
DE (1) | DE60325046D1 (ja) |
WO (1) | WO2003093477A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1811021A1 (en) * | 2004-10-27 | 2007-07-25 | Kaneka Corporation | Novel carbonyl reductase, gene therefor and use thereof |
US7465842B2 (en) | 2004-08-26 | 2008-12-16 | Agouron Pharmaceuticals, Inc. | Enantioselective biotransformation for preparation of protein tyrosine kinase inhibitor intermediates |
JP5005672B2 (ja) * | 2006-02-28 | 2012-08-22 | 株式会社カネカ | 新規カルボニル還元酵素、その遺伝子、およびそれらを利用した光学活性アルコールの製造方法 |
CN107118986A (zh) * | 2017-05-16 | 2017-09-01 | 浙江医药高等专科学校 | 一种恶臭假单胞菌及在制备(r)‑1‑(2‑三氟甲基苯基)乙醇中的应用 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10327454A1 (de) * | 2003-06-18 | 2005-01-20 | Juelich Enzyme Products Gmbh | Oxidoreduktase aus Pichia capsulata |
AT501928B1 (de) * | 2004-10-27 | 2010-09-15 | Iep Gmbh | Verfahren zur herstellung von chiralen alkoholen |
BRPI0924997B1 (pt) * | 2009-06-22 | 2024-01-16 | Sk Biopharmaceuticals Co., Ltd | Método para preparar um composto de éster do ácido 1-aril-2- tetrazoil etil carbâmico |
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 |
CN109943597B (zh) * | 2019-03-06 | 2022-08-09 | 江苏惠利生物科技有限公司 | 一种利用酶膜反应器耦合萃取制备s-4-氯-3-羟基丁酸乙酯的方法 |
Citations (4)
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EP0493617A1 (en) * | 1990-07-24 | 1992-07-08 | Kanegafuchi Kagaku Kogyo Kabushiki Kaisha | Process for producing optically active (-)-2-halo-1-(substituted phenyl)ethanol and (-)-substituted styrene oxide |
JPH08103269A (ja) * | 1994-10-07 | 1996-04-23 | Denki Kagaku Kogyo Kk | カルボニルレダクターゼ遺伝子の塩基配列及びその利用法 |
JPH11215995A (ja) * | 1998-02-02 | 1999-08-10 | Kanegafuchi Chem Ind Co Ltd | 光学活性2−ハロ−1−(置換フェニル)エタノールの製造法 |
EP1153919A1 (en) * | 1999-02-16 | 2001-11-14 | Kaneka Corporation | Substituted acetylpyridine derivatives and process for the preparation of intermediates for optically active beta3 agonist by the use of the same |
Family Cites Families (1)
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JP3067817B2 (ja) | 1990-07-24 | 2000-07-24 | 鐘淵化学工業株式会社 | 光学活性(−)−2−ハロ−1−(置換フェニル)エタノールの製造法 |
-
2003
- 2003-04-30 AU AU2003235980A patent/AU2003235980A1/en not_active Abandoned
- 2003-04-30 JP JP2004501613A patent/JP4426437B2/ja not_active Expired - Fee Related
- 2003-04-30 DE DE60325046T patent/DE60325046D1/de not_active Expired - Lifetime
- 2003-04-30 WO PCT/JP2003/005500 patent/WO2003093477A1/ja active Application Filing
- 2003-04-30 EP EP03720993A patent/EP1505155B1/en not_active Expired - Lifetime
- 2003-04-30 US US10/512,908 patent/US7332312B2/en not_active Expired - Lifetime
- 2003-04-30 AT AT03720993T patent/ATE416255T1/de not_active IP Right Cessation
Patent Citations (4)
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EP0493617A1 (en) * | 1990-07-24 | 1992-07-08 | Kanegafuchi Kagaku Kogyo Kabushiki Kaisha | Process for producing optically active (-)-2-halo-1-(substituted phenyl)ethanol and (-)-substituted styrene oxide |
JPH08103269A (ja) * | 1994-10-07 | 1996-04-23 | Denki Kagaku Kogyo Kk | カルボニルレダクターゼ遺伝子の塩基配列及びその利用法 |
JPH11215995A (ja) * | 1998-02-02 | 1999-08-10 | Kanegafuchi Chem Ind Co Ltd | 光学活性2−ハロ−1−(置換フェニル)エタノールの製造法 |
EP1153919A1 (en) * | 1999-02-16 | 2001-11-14 | Kaneka Corporation | Substituted acetylpyridine derivatives and process for the preparation of intermediates for optically active beta3 agonist by the use of the same |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US7465842B2 (en) | 2004-08-26 | 2008-12-16 | Agouron Pharmaceuticals, Inc. | Enantioselective biotransformation for preparation of protein tyrosine kinase inhibitor intermediates |
EP1811021A1 (en) * | 2004-10-27 | 2007-07-25 | Kaneka Corporation | Novel carbonyl reductase, gene therefor and use thereof |
EP1811021A4 (en) * | 2004-10-27 | 2008-05-28 | Kaneka Corp | NEW CARBONYL REDUCTASE, GENE FOR IT AND USE THEREOF |
US8008461B2 (en) | 2004-10-27 | 2011-08-30 | Kaneka Corporation | Carbonyl reductase, gene therefor and use thereof |
JP5005672B2 (ja) * | 2006-02-28 | 2012-08-22 | 株式会社カネカ | 新規カルボニル還元酵素、その遺伝子、およびそれらを利用した光学活性アルコールの製造方法 |
CN107118986A (zh) * | 2017-05-16 | 2017-09-01 | 浙江医药高等专科学校 | 一种恶臭假单胞菌及在制备(r)‑1‑(2‑三氟甲基苯基)乙醇中的应用 |
CN107118986B (zh) * | 2017-05-16 | 2020-08-11 | 浙江医药高等专科学校 | 一种恶臭假单胞菌及在制备(r)-1-(2-三氟甲基苯基)乙醇中的应用 |
Also Published As
Publication number | Publication date |
---|---|
AU2003235980A1 (en) | 2003-11-17 |
EP1505155B1 (en) | 2008-12-03 |
JPWO2003093477A1 (ja) | 2005-09-08 |
EP1505155A4 (en) | 2006-05-03 |
DE60325046D1 (de) | 2009-01-15 |
US7332312B2 (en) | 2008-02-19 |
US20060046289A1 (en) | 2006-03-02 |
JP4426437B2 (ja) | 2010-03-03 |
EP1505155A1 (en) | 2005-02-09 |
ATE416255T1 (de) | 2008-12-15 |
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