JP2008212144A - Alcohol dehydrogenase, gene encoding the same and method for producing optically active (r)-3-quinuclidinol using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new enzyme for reducing 3-quinuclidinone stereo-selectively to (R)-3-quinuclidinol. <P>SOLUTION: The enzyme having a biochemical properties shown by the following (1) to (3) is provided. (1) To produce (R)-3-quinuclidinol by asymmetrically reducing the 3-quinuclidinone or its salt by using a reduced type nicotinamide adenine dinucleotide (NADH) as a coenzyme; (2) Substrate specificity; It acts on quinuclidinone, p-toluquinone and α-naphthoquinone, and its relative activity is higher in the order of 3-quinuclidinone, p-toluquinone and α-naphtoquinone; and (3) Optimum temperature; In the case of reacting at pH7, the action is optimum at the temperatures of 40 to 45°C. The enzyme may include a specific amino acid sequence or its modified sequences. By utilizing the enzyme, it is possible to produce the optically active (R)-3-quinuclidinol having high optical purity in a high yield. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the production of (R) -3-quinuclidinol which is useful as an optically active physiologically active compound used in medicines and agricultural chemicals. More specifically, alcohol dehydrogenase, microorganism producing the enzyme, gene encoding the enzyme, method for producing the enzyme, and production of optically active (R) -3-quinuclidinol or a salt thereof using the enzyme Regarding the method.

  Currently, a mixture of optical isomers is used for many compounds having physiological activity. However, the desired activity is often present only in one optical isomer. Furthermore, it is also known that the other optical isomer that does not have the desired activity can be toxic to living organisms. Therefore, in order to provide an effective and safe pharmaceutical or physiologically active compound, it is strongly desired to develop a method for producing an optically active substance having a high optical purity used as a raw material or a synthetic intermediate.

  3-quinuclidinol, a reduction product of 3-quinuclidinone, is known as a synthetic intermediate such as a therapeutic agent for arteriosclerosis having a squalene synthase inhibitory action, a bronchodilator having a muscarinic receptor antagonistic action, and a gastrointestinal motility inhibitor. It is a compound (see Patent Documents 1 to 5).

  Optical isomers exist in 3-quinuclidinol having an asymmetric carbon atom. Therefore, it is necessary to isolate optically active 3-quinuclidinol useful as a synthetic intermediate, and several attempts have been made so far.

  As a method for producing optically active 3-quinuclidinol, for example, 1-benzyl-3-hydroxyquinuclidium chloride is optically resolved with a D-tartaric acid derivative to produce (S)-(+)-3-quinuclidinol. A method (Non-Patent Document 1) and a method for synthesizing (S)-(+)-3-quinuclidinol from D-glucose have been reported (Non-Patent Document 2). A method of using a rhodium complex compound (Patent Document 6) or a ternium complex compound (Patent Documents 7 and 8) as a catalyst to produce an optically active 3-quinuclidinol by asymmetric hydrogenation of a Lewis acid adduct of 3-quinuclidinone However, none of them are practical production methods because of low optical purity.

  As a method using a microorganism or an enzyme, for example, (R, S) -quinuclidinyl butyrate is allowed to act on horse serum butylcholinesterase to produce optically active (S)-(+)-3-quinuclidinyl butyrate. A method of producing a high optical purity optically active (R)-(−)-3-quinuclidinol by reacting a rate (Non-patent Document 3) and a protease derived from a microorganism belonging to the genus Bacillus ( There exists patent document 9). As an example starting from racemic 3-quinuclidinol ester, a method for producing optically active (R) -3-quinuclidinol using subtilisin protease (Patent Document 10), Aspergillus genus or A method for producing optically active (R) -3-quinuclidinol and optically active (S) -3-quinuclidinol by hydrolyzing an ester using an esterolytic enzyme derived from a microorganism belonging to the genus Pseudomonas (Patent Document 11) ), And asymmetric hydrolysis by microorganisms belonging to the genus Aspergillus, Rhizopus, Candida, or Pseudomonas and enzymes derived from these microorganisms. , Optically active 3-quinuclidinol or optically active 3-quinuclidinol Method for producing ether (Patent Document 12) are also known.

  However, these methods include problems such as low optical purity of the product or difficulty in mass production due to the complexity of the synthesis process. In addition, any of these methods is a method of optically resolving racemic 3-quinuclidinol to obtain a target optical isomer, and therefore the other optical isomer that is not the target may remain. Therefore, in these methods, for the non-target optical isomer, the configuration of the asymmetric carbon is reversed and converted to the target optical isomer, or the non-target optical isomer is racemized and reused again. Since a process for obtaining the desired optical isomer by optical resolution is required, there is a problem that the production cost increases.

  Furthermore, a method (Patent Documents 13 to 16) for producing optically active 3-quinuclidinol from 3-quinuclidinone using an asymmetric reduction reaction by microorganisms or enzymes is known. In Patent Document 13, as microorganisms that produce (R) -3-quinuclidinol from 3-quinuclidinone, the genus Nakazawaea, the genus Candida, the genus Proteus, the genus Arthrobacter, It is described that the genus Pseudomonas and Rhodosporidium are included. Patent Document 14 includes the genus Rhodotorula, the genus Candida, the genus Sporidiobolus, the genus Rhodosporidium, the genus Schizosaccharomyces, and the genus Cryptococcus. The use of microorganisms of the genera Trichosporon, Gordona, Pichia and Nocardia is described. Further, Patent Document 15 includes Alcaligenes, Corynebacterium, Arthrobacter, Filobasidium, Rhodotorula, Aureobasidium ( The use of microorganisms belonging to the genus Aureobasidium or Yarrowia is described. Patent Document 16 includes the genus Geotrichum, the genus Tsukamurella, the genus Micrococcus, the genus Kurthia, the genus Microbacterium, the genus Kluyveromyces, and the acremonium. The use of microorganisms belonging to the genus (Acremonium) or the genus Mucor and having the ability to produce (R) -3-quinuclidinol from 3-quinuclidinone is described. These reactions are reactions in which a wild-type microorganism is allowed to act on a substrate compound to directly generate an optically active compound. The reaction process is a one-step reaction, and it can be said that the simplification of the reaction process is greatly improved. However, there are still problems such as low optical purity of the product and low accumulated concentration of the product.

  A method for producing (R) -3-quinuclidinol from 3-quinuclidinol by an asymmetric reduction reaction utilizing a plant-derived enzyme has been reported (Patent Document 17). In this method, a thrombinone reductase-I (TR-I) derived from Datura stramonium or Hyosyamus niger and glucose dehydrogenase are co-expressed in Escherichia coli to produce a coenzyme regeneration system. (R) -3-quinuclidinol is produced from 3-quinuclidinone by an asymmetric reduction reaction combined with the above.

  Non-Patent Document 4 describes the purification of thrombinone reductase-I and II (TR-I and TR-II) from cultured roots of Hyoscyamus niger and their characteristics. TR-I and TR-II are both reduced nicotinamide adenine dinucleotide phosphate (NADPH) -dependent oxidoreductases, but exhibit different substrate specificities. 3-quinuclidinone is accepted as a substrate for TR-I, but is hardly accepted as a substrate for TR-II. TR-I is NADPH-dependent as described above, but reduced nicotinamide adenine dinucleotide (NADH) can also be used as a coenzyme.

  It has been reported that a novel enzyme that catalyzes the asymmetric reduction reaction of 3-quinuclidinone has been found for the production of optically active 3-quinuclidinol (Patent Document 18). This enzyme is produced by a microorganism belonging to the genus Microbacterium and reduces 3-quinuclidinone or a salt thereof using NADH as a coenzyme to produce (R) -3-quinuclidinol.

Thus, in order to efficiently produce optically active 3-quinuclidinol, attempts have been made to discover an enzyme having an action of asymmetric reduction from 3-quinuclidinone to (R) -3-quinuclidinol.
Japanese Patent Laid-Open No. 8-134067 EP-A-404737 European Patent Application No. 424021 International Patent Application Publication No. 92/04346 International Patent Application Publication No. 93/06098 JP-A-9-194480 JP-A-2005-306804 JP 2006-63028 A US Pat. No. 5,215,918 German Patent Application No. 19715465 Japanese Patent Laid-Open No. 10-210997 Japanese Patent No. 3129663 Japanese Patent Laid-Open No. 10-243795 JP-A-11-196890 JP 2000-245495 A JP 2002-153293 A JP 2003-230398 A JP 2003-334069 A A. A. Kalir et al., Israel Journal of Chemistry, 1971, 9, pp.267-268 Gee. W. Jay. GWJ Fleet et al., Tetrahedron Letters, 1986, 27, pp. 3057-3058 M. M. Rehavi et al., Life Sciences, 1977, 21, pp.1293-130 Takashi Hashimoto et al., Plant Physiol., 1992, 100, pp.836-845

  The present invention provides a novel enzyme that stereoselectively reduces 3-quinuclidinone to (R) -3-quinuclidinol, and optically active (R) -3-quinuclidinol having high optical purity by using the enzyme. It aims at providing the method of manufacturing well.

  As a result of searching for microorganisms that produce the enzyme from the natural world for the purpose of obtaining an enzyme that reduces 3-quinuclidinone to produce optically active (R) -3-quinuclidinol, It was found that one strain, more purely isolated and named Agrobacterium tumefaciens NK1, stereoselectively reduced 3-quinuclidinone to produce optically active (R) -3-quinuclidinol. . Next, the enzyme having the desired activity was purified from this strain and its properties were clarified. This enzyme has the ability to stereoselectively reduce the carbonyl group of 3-quinuclidinone or a salt thereof using NADH as a coenzyme to produce (R) -3-quinuclidinol (hereinafter, this enzyme is simply referred to as “3 -Sometimes referred to as “quinuclidinone asymmetric reductase”). Furthermore, the present inventors cloned a gene encoding the enzyme, revealed its structure, and confirmed that this gene is a novel gene. This gene was highly expressed in a heterogeneous organism to obtain a transformant that stereoselectively produces (R) -3-quinuclidinol from 3-quinuclidinone using NADH as a coenzyme. The inventors have found that (R) -3-quinuclidinol can be obtained from 3-quinuclidinone with high yield and high optical purity by using this enzyme or cells producing the enzyme, and the present invention has been completed.

  The present invention provides 3-quinuclidinone asymmetric reductase.

The 3-quinuclidinone asymmetric reductase of the present invention has the biochemical properties shown in the following (1) to (3):
(1) Action: Using reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme, asymmetric reduction of 3-quinuclidinone or a salt thereof produces (R) -3-quinuclidinol;
(2) Substrate specificity: acting on 3-quinuclidinone, p-toluquinone and α-naphthoquinone, the relative activities of which are higher in the order of 3-quinuclidinone, p-toluquinone and α-naphthoquinone; and (3) optimum temperature: When the reaction is carried out at pH 7, the action is optimal at a temperature of 40 to 45 ° C.

  In one embodiment, the 3-quinuclidinone asymmetric reductase has a subunit molecular weight of 28,500 when measured by SDS-PAGE and a molecular weight of 102,000 when measured by gel filtration chromatography. is there.

The 3-quinuclidinone asymmetric reductase of the present invention also has an amino acid sequence of any of the following (a) to (c):
(A) the amino acid sequence set forth in SEQ ID NO: 2;
(B) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 2; or (c) 85% of the amino acid sequence described in SEQ ID NO: 2 Amino acid sequence having the above homology.

  The enzyme having the amino acid sequence (a) has the biochemical properties shown in the above (1) to (3). In the case of an enzyme having the amino acid sequence (b) or (c), the enzyme has at least the biochemical property (1) described above.

  In one embodiment, the 3-quinuclidinone asymmetric reductase is derived from Agrobacterium tumefaciens strain NK1 (FERM P-21144).

  The present invention also provides Agrobacterium tumefaciens strain NK1 (FERM P-21144).

  The present invention also provides a polynucleotide encoding the 3-quinuclidinone asymmetric reductase.

In one embodiment, the polynucleotide is any of the following (i) to (iii):
(I) a polynucleotide having the base sequence set forth in SEQ ID NO: 1;
(Ii) hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions and asymmetrically reduces 3-quinuclidinone to produce (R) -3-quinuclidinol A polynucleotide encoding an enzyme having activity; or (iii) a base sequence having at least 60% sequence identity with the base sequence set forth in SEQ ID NO: 1, and asymmetrically reducing 3-quinuclidinone A polynucleotide encoding an enzyme having an activity to produce (R) -3-quinuclidinol.

  The present invention also provides a vector comprising the polynucleotide.

In one embodiment, the vector further comprises a polynucleotide encoding an enzyme that regenerates oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) to NADH.

  The present invention also provides a transformant carrying the above vector so that it can be expressed.

The present invention also provides a transformant that retains the above vector and a vector containing a polynucleotide encoding an enzyme that regenerates NAD ⁺ into NADH so that each vector can be expressed.

  In one embodiment, the transformant has an E. coli host.

In another embodiment, the enzyme that regenerates NAD ⁺ to NADH is glucose dehydrogenase.

  The present invention also provides a process for producing (R) -3-quinuclidinol or a salt thereof, which comprises the 3-quinuclidinone asymmetric reductase, a microorganism producing the enzyme, the transformant, or the microorganism. Alternatively, it includes a step of allowing the culture solution or treated product of the transformant to act on 3-quinuclidinone or a salt thereof.

In one embodiment, NADH or NAD ⁺ is further added in the step of action.

  In another embodiment, the microorganism is Agrobacterium tumefaciens strain NK1 (FERM P-21144).

  According to the present invention, a novel enzyme that stereoselectively reduces 3-quinuclidinone to (R) -3-quinuclidinol and a microorganism that produces the enzyme are provided. Furthermore, according to the present invention, optically active (R) -3-quinuclidinol or a salt thereof can be produced with high yield and high optical purity using the enzyme.

  In the present invention, the “enzyme” is not limited to a purified enzyme (enzyme purified to almost a single unit), but includes a crude product, an immobilized product, and the like. For example, the enzyme of the present invention can be obtained by using methods well known to those skilled in the art, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, etc. using cells treated with acetone and disrupted cells. Depending on the treatment, enzymes with various degrees of purification can be obtained.

  In the present invention, “microorganism” refers to wild strains, mutant strains (for example, induced by ultraviolet irradiation, etc.), or recombinants induced by genetic engineering techniques such as cell fusion or genetic recombination. Any microorganism may be sufficient. Engineered microorganisms such as recombinants are known to those skilled in the art, for example, as described in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor laboratory Press, 1989). It can be easily made using technology. The culture solution of microorganisms means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like. Examples of processed microorganisms include, for example, acetone-treated products, cell disruptions and cell-free extracts by mechanical methods such as sonication or enzymatic methods, those treated with surfactants and organic solvents, and immobilization thereof. This includes chemicals. These can be prepared according to methods well known to those skilled in the art.

<3-Quinuclidinone asymmetric reductase>
The present invention provides 3-quinuclidinone asymmetric reductase. This enzyme is an alcohol dehydrogenase and has the ability to asymmetrically reduce 3-quinuclidinone or a salt thereof using reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme to produce (R) -3-quinuclidinol. Have. The enzyme of the present invention does not use NADPH as a coenzyme. As a coenzyme, as long as the ability to produce (R) -3-quinuclidinol can be exhibited, generally known enzymes other than NADH may be used in combination except NADPH.

  Examples of the 3-quinuclidinone asymmetric reductase of the present invention include natural enzymes of Agrobacterium tumefaciens NK1 described below. This enzyme may change slightly depending on electrophoresis conditions, etc., but the molecular weight of the subunit measured by SDS-PAGE (12.5% (w / v) polyacrylamide gel) is about 28,500. The measured molecular weight is about 102,000. This enzyme is a homotetramer.

  In addition, the optimum pH of the enzyme using 3-quinuclidinone as a substrate is in the range of 5.5 to 8.0, preferably 5.5 to 7.5.

  The optimum temperature of the enzyme is 25 to 50 ° C, preferably around 40 to 45 ° C. This enzyme is stable up to 35 ° C., for example, when treated for 30 minutes in 1M potassium phosphate buffer (pH 7.0).

The enzyme activity of the 3-quinuclidinone asymmetric reductase of the present invention can be confirmed, for example, as follows. The enzyme solution was incubated at 37 ° C. in 0.2 M potassium phosphate buffer (pH 7.0) containing 0.618 mM 3-quinuclidinone hydrochloride and 0.32 mM NADH, and the absorbance decreased at a wavelength of 340 nm. Measure. The enzyme amount of the 3-quinuclidinone asymmetric reductase is defined as 1 unit (U), which is the amount of enzyme that converts 1 micromole of NADH to NAD ⁺ per minute. Furthermore, the 3-quinuclidinone asymmetric reductase of the present invention can confirm that NADPH is not used as a coenzyme by measuring NADH instead of NADPH.

  The 3-quinuclidinone asymmetric reductase of the present invention exhibits substrate specificity for 3-quinuclidinone. The 3-quinuclidinone asymmetric reductase of the present invention can also reduce p-toluquinone and α-naphthoquinone. However, the 3-quinuclidinone asymmetric reductase of the present invention is an enzyme that clearly shows higher reactivity with respect to 3-quinuclidinone than p-toluquinone and α-naphthoquinone. The 3-quinuclidinone asymmetric reductase of the present invention has a higher relative activity in the order of 3-quinuclidinone, p-toluquinone and α-naphthoquinone. This relative activity is expressed as a relative value in the enzyme activity measured as described above, assuming that 3-quinuclidinone is used as a substrate, which is 100.

  The 3-quinuclidinone asymmetric reductase of the present invention is preferably a polypeptide having a full-length amino acid sequence from position 1 to position 260 of SEQ ID NO: 2. In the present specification, the full-length amino acid sequence from position 1 to position 260 of SEQ ID NO: 2 is simply referred to as “amino acid sequence described in SEQ ID NO: 2”. As long as this enzyme has 3-quinuclidinone asymmetric reductase activity (particularly, an activity for asymmetrically reducing 3-quinuclidinone to produce (R) -3-quinuclidinol), for example, a natural amino acid sequence (for example, The enzyme may have an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added to the amino acid sequence described in SEQ ID NO: 2. A person skilled in the art, for example, site-directed mutagenesis (Nucleic Acid Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1989; PCR: A Practical Approach, IRL Press, 1991, pp. 200), etc. The structure of the protein can be altered by introducing additional mutations. In the present invention, the number of amino acid residues that can be substituted, deleted, inserted, and / or added is usually 50 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, even more preferably. Is 0-3. In addition, since amino acid mutations may occur in nature, not only the artificially mutated enzyme but also the naturally mutated enzyme has 3 quinuclidinone asymmetric reductase activity. -Included in quinuclidinone asymmetric reductase.

  As long as the protein having an amino acid sequence having homology to the amino acid sequence of the natural enzyme (for example, the amino acid sequence described in SEQ ID NO: 2) also has 3-quinuclidinone asymmetric reductase activity, the 3- Included in quinuclidinone asymmetric reductase. The 3-quinuclidinone asymmetric reductase of the present invention is preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99% with the amino acid sequence set forth in SEQ ID NO: 2. It may be a protein having an amino acid sequence having the homology of Protein homology (homology) searches, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, DAD, or DNA databases such as DDBJ, EMBL, or Gene-Bank, etc. BLAST, FASTA, etc. This program can be used, for example, via the Internet. Confirmation of the activity of the protein can be performed using the procedure described above.

  Although it does not restrict | limit especially as a supply source of 3-quinuclidinone asymmetric reductase of this invention, It can obtain from biological cells, such as microorganisms. This microorganism is preferably Agrobacterium tumefaciens strain NK1 described below.

Agrobacterium tumefaciens strain NK1 has the following mycological properties:
A. Form (1) Cell shape Neisseria gonorrhoeae (2) Cell size 0.6-0.7μm × 1.5-2.5μm
(3) Presence or absence of mobility −
(4) Gram staining-
(5) Spore formation-
B. Growth state in standard agar plate culture Cream color, glossy, perfect circle, smooth, low convex Physiological properties (1) Reduction of nitrate nitrate
(2) Denitrification reaction −
(3) Generation of indole −
(4) Starch hydrolysis-
(5) Use of citric acid −
(6) Urease activity +
(7) Oxidase activity +
(8) Catalase activity +
(9) Growth range Temperature: 37 ° C +; 41 ° C-; 45 ° C-
(10) Attitude toward oxygen Aerobic (11) Degradation of amino acids Lysine −
Arginine −
Ornithine −
(12) Casein degradability −
(13) Degradability of gelatin −
(14) Growth in anaerobic medium −
(15) Macconkey medium growth +
(16) Utilization and productivity of sugar L-arabinose +
D-xylose +
D-glucose +
D-Mannose +
D-fructose +
D-galactose +
Maltose +
Sucrose +
Lactose +
Trehalose +
D-man knit +
Inosit +
Ethanol +
(17) Esculin hydrolysis +
(18) Oxidation of gluconic acid-

  As a result of searching the above properties from “Burger's Manual of Systematic Bacteriology” (1984), this strain was identified as a strain belonging to Agrobacterium tumefaciens and named Agrobacterium tumefaciens NK1. This strain was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Tsukuba City East 1-1-1, Tsukuba Center, Chuo No. 6) in Ibaraki Prefecture. On December 26, 2006, the accession number FERM P- 21144 is given.

  The enzyme of the present invention is not limited to the enzyme derived from the Agrobacterium tumefaciens NK1 strain, and can be obtained from any microorganism as long as it can produce the 3-quinuclidinone asymmetric reductase of the present invention.

  In order to obtain the 3-quinuclidinone asymmetric reductase of the present invention, microorganisms that naturally produce the 3-quinuclidinone asymmetric reductase of the present invention (for example, Agrobacterium tumefaciens NK1 strain) and natural or artificial mutants thereof , As well as other microorganisms (ie, genetically modified microorganisms) in which a gene fragment necessary for the expression of the 3-quinuclidinone asymmetric reductase of the present invention is artificially extracted and incorporated. These microorganisms are collectively referred to as “microorganisms producing the enzyme of the present invention”.

  A method for producing 3-quinuclidinone asymmetric reductase using a microorganism that produces the enzyme of the present invention will be described. The microorganism is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to accumulate the enzyme. Here, examples of the carbon source include starch and starch hydrolysates, sugars such as glucose and sucrose, alcohols such as glycerol, and organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt). Can be mentioned. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor and soybean flour, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. Culture is performed using a medium containing these nutrient components in appropriate amounts. The medium may be a liquid medium or a solid medium, but is preferably a liquid medium in terms of productivity. The culture temperature is a temperature at which the cultured microorganism can sufficiently grow, and is preferably 20 to 37 ° C. The culture time is a time during which the enzyme is sufficiently produced, and is preferably about 1 to 7 days. Culturing is preferably performed under aerobic conditions, for example with aeration or shaking.

  Microorganisms producing the enzyme of the present invention can usually accumulate the enzyme in the cells by liquid culture in a nutrient medium. Not only cultured microorganisms themselves, but also microorganisms obtained by acetone-drying or freeze-drying treatment, cell-free extracts with disrupted cell walls by mechanical or enzymatic methods, microorganisms treated with surfactants, organic solvents, etc., or appropriate These microorganisms immobilized on a simple carrier or cell-free extract can also be used. These are collectively referred to as a “processed product” of a microorganism that produces the enzyme of the present invention, but the “processed product” includes any form in which an enzyme produced by the enzyme-producing microorganism can be used for the reaction. Similarly, microbial broth can also be used for enzymatic reactions.

  The 3-quinuclidinone asymmetric reductase of the present invention is fractionated by protein solubility (precipitation with organic solvents, salting out by ammonium sulfate, etc.); chromatography such as cation exchange, anion exchange, gel filtration, hydrophobicity; Purification can be achieved by combining known methods such as affinity chromatography using chelates, dyes, antibodies, and the like. For example, by crushing the cells of the above microorganisms to prepare a cell-free extract, by repeating ammonium sulfate precipitation, anion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, and further anion exchange chromatography It can be purified until it becomes almost a single band in polyacrylamide gel electrophoresis (SDS-PAGE). The crudely purified enzyme generated in the middle of the above fractionation or purification may be used, or a purified enzyme may be used.

  The enzyme of the present invention, the microorganism producing the enzyme, and the culture solution and processed product thereof can be used for the production of (R) -3-quinuclidinol or a salt thereof as described in detail below.

<3-nucleotide encoding quinuclidinone asymmetric reductase>
Polynucleotides encoding the 3-quinuclidinone asymmetric reductase of the present invention described above are also included in the present invention. The polynucleotide of the present invention may be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA or RNA. The polynucleotide of the present invention may be a DNA-RNA chimeric molecule.

  The polynucleotide of the present invention can be obtained by determining the partial amino acid sequence of the purified enzyme, and then by methods commonly used by those skilled in the art, for example, PCR using multiple primers and hybridization using multiple probes.

  The polynucleotide of the present invention includes, for example, a polynucleotide represented by a full-length base sequence from position 1 to position 783 of SEQ ID NO: 1. In the present specification, the full-length base sequence from position 1 to position 783 of SEQ ID NO: 1 is simply referred to as “the base sequence described in SEQ ID NO: 1.” The base sequence shown in SEQ ID NO: 1 encodes a protein containing the amino acid sequence shown in SEQ ID NO: 2, and the protein containing this amino acid sequence represents a preferred form of the 3-quinuclidinone asymmetric reductase according to the present invention. Constitute. The polynucleotide of the present invention includes an amino acid in which one or more amino acids are substituted, deleted, inserted, and / or added to the amino acid sequence shown in SEQ ID NO: 2, and has 3-quinuclidinone asymmetric reductase activity Also included are polynucleotides that encode proteins having:

  The polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention preferably has at least 85%, more preferably at least 90%, still more preferably at least 95%, even more preferably the amino acid sequence set forth in SEQ ID NO: 2. A polynucleotide that preferably encodes a protein having an amino acid sequence having 99% homology and having 3-quinuclidinone asymmetric reductase activity is also mentioned. The protein homology search is as described above.

  As long as the encoded protein has substantially the same activity as the 3-quinuclidinone asymmetric reductase of the present invention (3-quinuclidinone asymmetric reductase activity), the polynucleotide of the present invention has the nucleotide sequence set forth in SEQ ID NO: 1. One or more bases may be substituted for the polynucleotide having

  Preferably, the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention has at least 60%, more preferably at least 70%, even more preferably at least 80%, and more preferably the nucleotide sequence set forth in SEQ ID NO: 1. More preferably at least 90%, even more preferably at least 95%, even more preferably 99% of the base sequence having sequence identity and encoding a protein having 3-quinuclidinone asymmetric reductase activity Also included are nucleotides. The determination and search of the sequence identity of the base sequence is also as described above.

  The polynucleotide of the present invention is a polynucleotide that can hybridize with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions and that has a 3-quinuclidinone asymmetric reductase activity. Also included are polynucleotides that encode.

  The polynucleotide of this invention can acquire the target gene from said microorganisms based on the base sequence information described in this specification. PCR and hybridization screening are used for gene acquisition. It is also possible to chemically synthesize the full length of a gene by DNA synthesis. Based on the base sequence information, a polynucleotide encoding the 3-quinuclidinone asymmetric reductase derived from an organism other than the above can also be obtained. For example, by designing a probe using the above base sequence or a part of the base sequence and performing hybridization under conditions stringent to DNA prepared from another organism, 3- A polynucleotide encoding a quinuclidinone asymmetric reductase can be isolated. Based on the above base sequence information, PCR primers can be designed from regions with high homology using sequence information registered in DNA databases such as DNA Databank of Japan (DDBJ), EMBL, and Gene-Bank. it can. By using such primers and performing PCR using chromosomal DNA or cDNA as a template, the polynucleotide encoding the 3-quinuclidinone asymmetric reductase can also be isolated from various organisms.

  The polynucleotide that can hybridize under stringent conditions is a sequence comprising at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive sequences in the nucleotide sequence set forth in SEQ ID NO: 1. One or a plurality of probes are selected and designed, for example, using ECL direct nucleic acid labeling and detection system (GE Healthcare), the conditions described in the manual (for example, washing conditions: 42 ° C., 0.5 × SSC) In the primary wash buffer).

  More specifically, the “stringent conditions” are usually conditions of 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, the conditions are 65 ° C., 0.1 × SSC and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can achieve optimal stringency by appropriately selecting these factors. .

  The polynucleotides of the invention can be expressed in homologous or heterologous hosts using genetic engineering techniques.

<Vector and transformant>
The vector of the present invention contains the above-described polynucleotide. The transformant of the present invention holds the vector of the present invention so that it can be expressed.

  A procedure for producing a transformant and construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). In order to express the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention in a microorganism, first, this DNA is introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and its genetic information is obtained. Must be transcribed and translated. For this purpose, a promoter corresponding to a unit that controls transcription / translation may be incorporated upstream of the 5 ′ side of the DNA strand of the present invention, more preferably a terminator downstream of the 3 ′ side. As the promoter and terminator, a promoter and terminator known to function in microorganisms used as hosts are used. Regarding vectors, promoters, terminators and the like that can be used in these various microorganisms, “Basic Course of Microbiology 8 Genetic Engineering”, Kyoritsu Publishing, especially yeast, Adv. Biochem. Eng., 1990, 43, pp. 75; Yeast, 1992, Vol. 8, pp. 423.

In the transformant of the present invention, it is preferable that a regeneration system of coenzyme NAD ⁺ into NADH is introduced. In the process of stereoselectively reducing 3-quinuclidinone to (R) -3-quinuclidinol, the 3-quinuclidinone asymmetric reductase of the present invention requires NADH. NAD ⁺ is generated from NADH accompanying the reduction reaction by the 3-quinuclidinone asymmetric reductase. NAD ⁺ can be regenerated back to the reduced form NADH by utilizing the oxidation reaction of an appropriate substrate. Therefore, in order to make the reduction reaction of the substrate more efficient, it is preferable that the transformant of the present invention expresses an enzyme that regenerates NAD ⁺ to NADH (“NADH regenerating enzyme”). The NADH regenerating enzyme in the present invention includes glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydrogenase. Enzymes, glycerol dehydrogenase and the like can be mentioned. Glucose dehydrogenase derived from Bacillus megaterium is preferable, and glucose dehydrogenase derived from Bacillus megaterium IWG3 and IAM1030 strains is more preferable. Genes encoding these have already been isolated, and their sequence information has been registered in a database (UniProtKB / Swiss-Prot Accession No. P39482 and P39482). It can also be obtained from the microorganism by performing PCR or hybridization screening based on the base sequence registered in the database.

  When introducing multiple genes into a single vector, a method of linking a region involved in expression control such as a promoter or terminator to each gene, or a method of expressing as an operon containing multiple cistrons such as lactose operon and so on.

  In order to introduce these two genes into a host, in order to avoid incompatibility, a method of transforming the host with a recombinant vector in which genes are separately inserted into a plurality of vectors having different origins of replication, a single vector A method of transforming a host with a recombinant vector into which both genes are inserted, a method of inserting one or both genes into the host chromosome, and the like can be used.

  The host to be transformed in order to express the polynucleotide of the present invention is not particularly limited as long as it is an organism that is transformed with a vector containing the polynucleotide and can express 3-quinuclidinone asymmetric reductase activity. There is no. For example, Escherichia genus, Bacillus genus, Pseudomonas genus, Serratia genus, Brevibacterium genus, Corynebacterium genus, Streptococcus genus, lacto Bacteria for which host vector systems such as Bacillus (Lactobacillus) have been developed; Actinomycetes for which host vector systems such as Rhodococcus and Streptomyces have been developed; Saccharomyces The genus Kluyveromyces, the genus Schizosaccharomyces, the genus Zygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genus Rhodosporidium, the genus Rhodosporidium Da (Candida) etc. Yeast for which host vector systems have been developed; molds for which host vector systems such as Neurospora, Aspergillus, Cephalosporium, and Trichoderma have been developed. It is done. E. coli is preferred because of the ease of genetic recombination operations.

  In addition to microorganisms, various host / vector systems have been developed in plants and animals. For example, insects using moths (Nature, 1985, 315, pp.592-594), rapeseed, corn, potato A system for expressing a large amount of a heterologous protein in plants such as these has been developed, and these may be used.

<Production method of (R) -3-quinuclidinol>
3-quinuclidinone asymmetric reductase of the present invention (including both the purified enzyme and the crudely purified enzyme), a microorganism (including the transformant) that produces the enzyme, or culture of the microorganism or transformant (R) -3-quinuclidinol or a salt thereof is efficiently produced by allowing the liquid or treated product to act on 3-quinuclidinone or a salt thereof. "3-Quinuclidinone or a salt thereof" means a compound that forms a salt with an organic acid, mineral acid, or the like at its nitrogen atom, and is also used for the asymmetric reduction of 3-quinuclidinone by the 3-quinuclidinone asymmetric reductase. I intend to be able to do it. Specific examples of the organic acid salt include aliphatic organic acid salts such as acetate, propionate, butyrate, fumaric acid, malonic acid, and oxalic acid, and aromatic organic acid salts such as benzoic acid. Examples of mineral acid salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate. Even in the case where only “3-quinuclidinone” is indicated in the following description, the salt of 3-quinuclidinone as described above can be used.

  In the method of the present invention, the 3-quinuclidinone asymmetric reductase of the present invention (including both the purified enzyme and the crudely purified enzyme), the microorganism producing the enzyme (including the transformant), or the microorganism Alternatively, 3-quinuclidinone or a salt thereof is asymmetrically reduced in a suitable reaction solution containing a culture solution or processed product of the transformant (hereinafter collectively referred to as “biological catalyst”). (R) -3-quinuclidinol is obtained as a product.

  In one embodiment of the method of the present invention, in the step of acting (hereinafter also referred to as “action step”), a microorganism that expresses 3-quinuclidinone reductase is cultured in the presence of 3-quinuclidinone. The amount of 3-quinuclidinone added to the culture medium is preferably 0.1 to 50% (w / v), more preferably 0.1% to 10% (w / v). The added 3-quinuclidinone may not necessarily be completely dissolved. If 3-quinuclidinone is not completely dissolved, for example, the solubility may be improved as a salt (e.g., hydrochloride), or an organic solvent such as ethanol will not substantially inhibit the reduction reaction (e.g., You may add to 5% (v / v)).

  In another embodiment of the method of the present invention, in the action step, microbial cells cultured in a suitable reaction solution (water or buffer) or a processed product thereof (for example, an acetone-treated product, an air-dried product, The lyophilized product) is contacted with 3-quinuclidinone. The amount of 3-quinuclidinone added to the reaction solution is preferably 0.1 to 30% (w / v), more preferably 0.1 to 20% (w / v). The added 3-quinuclidinone may not necessarily be completely dissolved. If 3-quinuclidinone is not completely dissolved, for example, the solubility may be improved as a salt (e.g., hydrochloride), or an organic solvent such as ethanol will not substantially inhibit the reduction reaction (e.g., 5% (v / v)). In particular, when a treated bacterial cell is used, it is preferable to add NADH as a coenzyme. The amount of coenzyme added is preferably 0.1 to 1000 mg / L, more preferably 2 to 200 mg / L.

  The reaction can be performed under a controlled pH. In order to maintain the optimum pH for the reaction, for example, a buffer solution such as a phosphate buffer is used as the reaction solution. The pH of the reaction solution is preferably 5-9, more preferably 6-8. Alternatively, the pH of the reaction solution is maintained and adjusted to an optimum pH by adding an acid (for example, sulfuric acid) and an alkali (for example, sodium hydroxide) while monitoring the pH that changes as the reaction proceeds. obtain.

  The amount of the biological catalyst is appropriately determined depending on its shape and activity, and is not particularly limited. About 0.1 to 10 g / mol substrate, preferably about 1 to 5 g / mol substrate relative to the substrate 3-quinuclidinone.

  The reaction temperature is preferably 10 ° C to 55 ° C, more preferably 25 ° C to 45 ° C.

  The reaction time varies depending on the amount of biological catalyst used, reaction temperature, reaction pH, etc., but is usually about 1 to 72 hours.

In the production method of the present invention, it is preferable to combine the above-mentioned coenzyme regeneration system with the action step. Playback from NAD ⁺ to NADH can be carried out by an enzyme to regenerate plants, microorganisms, and NADH from NAD ⁺ containing transformants. The enzyme that catalyzes the regeneration reaction of NADH may be a single enzyme or a multistage reaction composed of a plurality of enzymes. The NAD ⁺ reducing ability can be enhanced by adding sugars such as glucose and sucrose, organic acids, or alcohols such as ethanol and isopropanol to the reaction system. Enzymes useful for NADH regeneration include glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydration Elemental enzymes, and glycerol dehydrogenase. As these enzymes, not only purified enzymes but also microorganisms having the enzymes, processed products thereof, partially purified enzymes, and the like can be used. For example, in the case of glucose dehydrogenase, regeneration from NAD ⁺ to NADH is performed with the oxidation of glucose to gluconolactone.

  These components necessary for NADH regeneration may be added to the 3-quinuclidinone asymmetric reductase reaction system in the method of the present invention, or those in which these components are immobilized may be added. Alternatively, the reaction system can be contacted through a membrane capable of exchanging NADH.

  In the method for producing (R) -3-quinuclidinol, when a transformant transformed with a recombinant vector containing a polypeptide encoding the 3-quinuclidine asymmetric reductase of the present invention is used, NADH regeneration is performed. It is not necessary to add this reaction system. That is, by using an organism having a high NADH regeneration activity as a host, a reaction can be efficiently performed in a reduction reaction using a transformant without adding an NADH regeneration enzyme. Alternatively, a host in which the enzyme gene that can be used for NADH regeneration is introduced at the same time as the polynucleotide encoding the 3-quinuclidinone asymmetric reductase of the present invention can be used. As described above, the transformant expressing both enzymes expresses a vector containing a polynucleotide encoding 3-quinuclidinone asymmetric reductase and a vector containing an enzyme gene that can be used for NADH regeneration. A transformant that can be retained, and a transformant that can retain a vector containing a polynucleotide encoding 3-quinuclidinone asymmetric reductase and the enzyme gene that can be used for NADH regeneration are expressed. By using such a transformant, the expression of the 3-quinuclidinone asymmetric reductase and NADH regenerating enzyme of the present invention and the asymmetric reduction reaction of the substrate can be performed more efficiently.

When a coenzyme regeneration system is combined, an energy source such as coenzyme NAD ⁺ and / or glucose, sucrose, ethanol, methanol, etc. can be added to the culture solution or reaction solution. The addition amount of the coenzyme NAD ⁺ or the energy source can be appropriately determined based on the amount of the substrate 3-quinuclidinone and the biological catalyst. The reaction can be performed while controlling the reaction temperature and, if necessary, the pH of the reaction solution as described above. If necessary, the reaction may be continued by appropriately adding 3-quinuclidinone as a reaction substrate and / or the above-mentioned coenzyme and energy source during the reaction.

  The produced (R) -3-quinuclidinol can be separated utilizing the difference in chemical or physical properties of the substrate 3-quinuclidinone and the produced (R) -3-quinuclidinol. For example, after completion of the reaction, depending on the properties (for example, solubility, hydrophobicity), unreacted 3 using a separation operation usually used in the field such as a solvent extraction method, a crystal precipitation method, or a column chromatography method. -Separated from quinuclidinone. For example, in the solvent extraction method, unreacted 3-quinuclidinone is extracted and removed from the reaction solution with toluene under basic conditions, and then (R) -3-quinuclidinol is produced using an organic solvent such as chloroform or dichloromethane. It can be recovered.

  The resulting product can be further purified using the above means if necessary. In this case, the second means is preferably different from the first means (for example, when solvent extraction is used as the first means, the second means includes, for example, column chromatography).

  The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

(Measurement of conversion rate and optical purity of (R) -3-quinuclidinol)
The quantitative determination of 3-quinuclidinone in the reaction solution and the quantitative determination of 3-quinuclidinol produced by the reduction reaction and the measurement of optical purity were carried out by gas chromatography using the following analytical conditions.

Gas chromatography analysis conditions:
Column: gamma-DEX120 (length: 30 m, inner diameter: 0.25 mm ID; manufactured by Spelco)
Column initial temperature: 140 ° C. (10 minutes)
Injector temperature: 220 ° C
Detector temperature: 220 ° C
Carrier gas: helium (200 kPa)
Detector: FID

(Example 1: Preparation of dry cells of Agrobacterium tumefaciens NK1 strain)
Agrobacterium tumefaciens NK1 strain (strain deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology and given accession number FERM P-21144 on December 26, 2006) was isolated from soil Is a stock. This strain was preliminarily medium (glucose 20 g / L, yeast extract (Nippon Pharmaceutical) 3 g / L, polypeptone (Nippon Pharmaceutical) 5 g / L, render extract (Mikuni Chemical Industries) 3 g / L, ammonium sulfate 2 g / L, Pre-cultured at 28 ° C. with magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 1.0 g / L, pH 7.0).

  500 mL of the medium having the above composition was placed in a 3 L Erlenmeyer flask and autoclaved at 121 ° C. for 20 minutes. 5 mL of the culture solution of the Agrobacterium tumefaciens NK1 strain previously pre-cultured in the same medium was inoculated into the autoclave-sterilized medium, and cultured with shaking (80 rpm) on a rotary shaker at 28 ° C. for 3 days. The cells were collected from the obtained culture broth by centrifugation (11,300 × g, 20 minutes, 4 ° C.) and washed once with 300 mL of distilled water. The washed cells thus obtained were allowed to stand at 25 ° C. for 24 hours under reduced pressure conditions (50 mmHg) to obtain dry cells.

(Example 2: Reduction of 3-quinuclidinone using dry cells of Agrobacterium tumefaciens NK1 strain)
Put 0.2 mL of 0.1 M phosphate buffer (pH 7.0) in a 2 mL Eppendorf tube, and 3-quinuclidinone hydrochloride, NAD ⁺ , glucose dehydrogenase (GLUCDH Amano II, manufactured by Amano Enzyme), And glucose were added to final concentrations of 300 mM, 0.9 mM, 14 U / mL, and 450 mM, respectively. Further, 5.6 mg of the dried microbial cell of Example 1 was added, and the reaction was performed by inversion stirring at 25 ° C. for 5 hours.

  After completion of the reaction, 200 μL of a saturated aqueous potassium carbonate solution and 200 μL of chloroform were added to the reaction solution and stirred, and the resulting chloroform layer was analyzed by gas chromatography. As a result, the conversion rate was 5%, and the optical purity of the obtained (R) -3-quinuclidinol was 99% ee or higher.

(Example 3: Reduction of 3-quinuclidinone using dry cells of Agrobacterium tumefaciens strain NK1 under pH control)
In 5 mL of 0.1 M phosphate buffer (pH 7.0), 0.5 g of the dried cells of Example 1, 6 mg of NAD ⁺ , 1 mg of glucose dehydrogenase (GLUCDH Amano II, manufactured by Amano Enzyme), 0 The reaction was performed by adding 0.5 g of 3-quinuclidinone hydrochloride and 0.8 g of glucose and stirring with a stirrer at 25 ° C. During the reaction, 48% (w / v) KOH was added dropwise with a pH stat to neutralize the produced gluconic acid. After 19 hours of reaction, 100 μL of the reaction solution was sampled, 100 μL of saturated aqueous potassium carbonate solution and 200 μL of chloroform were added and stirred, and the resulting chloroform layer was analyzed by gas chromatography. As a result, the conversion rate was 99%, and the optical purity of the obtained (R) -3-quinuclidinol was 88.3% ee.

(Example 4: Purification of 3-quinuclidinone asymmetric reductase)
According to the following method, from the same Agrobacterium tumefaciens NK1 strain as in Example 1, an enzyme having the activity of asymmetrically reducing 3-quinuclidinone to produce (R) -3-quinuclidinol was electrophoretically Purified to single.

(A) Culture medium (glucose 1% (w / v), peptone 1.5% (w / v), yeast extract 0.1% (w / v), dipotassium hydrogen phosphate 0.3% (w / v) v), magnesium sulfate heptahydrate 0.02 (w / v)%, sodium chloride 0.2% (w / v), pH 7) 12,500 mL was prepared, and 500 mL was dispensed into a 2 L Sakaguchi flask. Poured and steam sterilized at 121 ° C. for 20 minutes. The same Agrobacterium tumefaciens NK1 strain as in Example 1 was pre-cultured in the same medium in advance. 10 mL of this microorganism culture solution was inoculated into the steam-sterilized medium, and cultured with shaking at 28 ° C. for 48 hours. The cells were collected from this culture by centrifugation (8,000 rpm, 20 minutes, 4 ° C.). The cells were washed twice with 0.85% aqueous sodium chloride solution and collected by centrifugation.

(B) Preparation of cell-free extract 84.5 g of wet cells were suspended in 170 mL of 10 mM potassium phosphate buffer (pH 7.0), and the cells were disrupted by sonication for 30 minutes. The cell residue was removed from the crushed cell by centrifugation (10,000 rpm, 20 minutes, 4 ° C.) to obtain 195 mL of a cell-free extract.

(C) Ammonium sulfate fraction Ammonium sulfate was added to the cell-free extract so as to be 60% (w / v) saturation and dissolved, and then the resulting precipitate was collected by centrifugation. This precipitate was dissolved in 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride, and dialyzed against the same buffer.

(D) DEAE-Sephacel Batch Chromatography DEAE-Sephacel (GE Healthcare Biosciences) was prepared by equilibrating the obtained crude enzyme solution in advance with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. (Made) It added to resin 600mL. After filtration, the resin was washed with the same buffer, and the active fraction was eluted with 10 mM potassium phosphate buffer (pH 7.0) containing 1 M sodium chloride.

(E) Phenyl Superose column chromatography The obtained active fractions were collected, desalted and concentrated by ultrafiltration (Amicon YM-10, manufactured by Millipore) and 10 mM potassium phosphate buffer (pH 7.0) containing 4 M sodium chloride. ). One-fifth of the enzyme solution was added to a Phenyl Superose HR 10/10 (GE) column equilibrated with the same buffer. The column was washed with the same buffer and the active fraction was eluted with a linear gradient of sodium chloride from 4M to 0M. The same operation was repeated 4 times for the remaining enzyme solutions.

(F) Superdex 200 column chromatography The obtained active fractions were collected and desalted and concentrated by ultrafiltration (Amicon YM-10, manufactured by Millipore). One-eighth volume of the above enzyme solution was added to a Superdex 200 HR 10/30 column (manufactured by GE) equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.2 M sodium chloride in advance, and the same buffer was used. The active fraction was eluted with the solution. The same operation was repeated 7 times for the remaining enzyme solutions.

(G) MonoQ column chromatography (first time)
The obtained active fractions were collected, desalted and concentrated by ultrafiltration (Amicon YM-10, manufactured by Millipore) and replaced with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. It was. Finally concentrated to 2 mL. Add half the amount of the above enzyme solution to a MonoQ HR 5/5 column (manufactured by GE Healthcare Biosciences) pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. did. The column was washed with the same buffer and the active fraction was eluted with a linear gradient of 0.1 M to 0.5 M sodium chloride. The same operation was repeated for the remaining enzyme solutions.

(H) MonoQ column chromatography (second time)
The obtained active fractions were collected, desalted and concentrated by ultrafiltration (Amicon YM-10, manufactured by Millipore) and replaced with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. It was. Finally concentrated to 2.5 mL. The concentrated solution obtained was added to a MonoQ HR 5/5 (GE Healthcare Bioscience) column pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. The column was washed with solution and the active fraction was eluted with a linear gradient of 0.1 M to 0.5 M sodium chloride.

(I) MiniQ column chromatography (first time)
The obtained active fractions were collected, desalted and concentrated by ultrafiltration (Amicon YM-10, manufactured by Millipore) and replaced with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. It was. Finally concentrated to 0.8 mL. Add 1/4 amount of the above enzyme solution to MiniQ PC 3.2 / 3 (GE Healthcare Biosciences) column pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. The column was washed with the same buffer, and the active fraction was eluted with a linear gradient of sodium chloride from 0.1 M to 0.25 M. The same operation was repeated three times for the remaining enzyme solutions.

(J) MiniQ column chromatography (second time)
The obtained active fractions were collected, desalted and concentrated by ultrafiltration (Amicon YM-10, manufactured by Millipore) and replaced with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. It was. Finally concentrated to 0.2 mL. The concentrated solution obtained was added to a MiniQ PC 3.2 / 3 (GE Healthcare Biosciences) column pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 M sodium chloride. The column was washed with solution and the active fraction was eluted with a linear gradient of sodium chloride from 0.1M to 0.25M. Active fractions were collected and analyzed by SDS-PAGE. As a result, it was confirmed that it was a single band. The specific activity of the purified enzyme with respect to 3-quinuclidinone was 132 U / mg.

  A summary of the purification is shown in Table 1 below.

(Example 5: Measurement of properties of 3-quinuclidinone asymmetric reductase)
The enzymatic properties of the purified enzyme obtained in Example 4 were examined. Standard reaction conditions for measurement of enzyme activity were as follows. A reaction solution containing the substrate 3-quinuclidinone hydrochloride 0.618 mM, the coenzyme NADH 0.32 mM, and an enzyme in 0.2 M potassium phosphate buffer (pH 7.0) was used. The reaction was initiated by incubating the reaction solution without substrate at 37 ° C. and adding the substrate after 2 minutes. The decrease in NADH accompanying the reaction was measured by the change in absorbance at a wavelength of 340 nm. The amount of enzyme that converts 1 micromole of NADH to NAD ⁺ per minute was defined as 1 unit (U).

  (1) Action: Using NADH as a coenzyme, it acted on 3-quinuclidinone to produce (R) -3-quinuclidinol with an optical purity of 99% ee or higher.

  (2) Substrate specificity: Reaction was carried out under the same conditions as for 3-quinuclidinone using various carbonyl compounds as substrates. The results are expressed as a relative ratio with the enzyme activity taken as 100 when 3-quinuclidinone is used as the substrate. Table 2 shows enzyme activities using 3-quinuclidinone and various carbonyl compounds as substrates. Furthermore, for comparison, the results of the enzyme described in Patent Document 18 (alcohol dehydrogenase derived from Microbacterium using NADH as a coenzyme) and TR-I described in Non-Patent Document 4 are also shown.

(3) Coenzyme: NADH-dependent, and NADPH is not used as a coenzyme. In addition, the oxidation reaction of 3-quinuclidinol using NAD ⁺ as a coenzyme was performed at 37 ° C. in Tris-HCl (pH 9.0) containing 6.18 mM (R) -3-quinuclidinol and 2 mM NAD ⁺ at 3.27 U / mg of oxidative activity.

  (4) Optimum pH of action: acetate buffer (pH 3.5 to 5.5), potassium phosphate buffer (pH 5.5 to 7.5), Tris-HCl buffer (pH 7.5 to 9) as buffer solutions 0.0) and glycine-sodium hydroxide buffer (pH 8.5 to 11.0), and the pH was changed to measure 3-quinuclidinone reducing activity. The optimum pH for the reaction was 7.0, showing an activity of 50% or more of the maximum activity over a wide range of pH from 5.5 to 8.0.

  (5) Optimum temperature of action: 3-quinuclidinone reducing activity was measured by changing only the temperature in the standard reaction conditions. The optimum temperature for the reaction was around 40-45 ° C.

  (6) Temperature stability: The purified enzyme was treated in 10 mM potassium phosphate buffer (pH 7.0) at various temperatures for 30 minutes, and the residual activity was measured under standard conditions. At 35 ° C. or lower, 90% or more of the activity before the treatment remained.

(7) Inhibitor: Various metal ions or inhibitors were added to the standard reaction solution to measure 3-quinuclidinone reduction activity. The results are shown in Tables 3 and 4 below as relative activities with the additive-free condition as 100. It was inhibited by Ag ⁺ , Ca ²⁺ , Mn ²⁺ , Be ²⁺ , Hg ²⁺ , Pb ²⁺ and NaIO ₄ .

  (8) Molecular weight: The molecular weight of the purified enzyme subunit was determined by SDS-PAGE (12.5% (w / v) polyacrylamide gel) and found to be about 28,500. Further, the molecular weight measured by gel filtration chromatography using a TSK G-3000SW column (manufactured by Tosoh Corporation) was about 102,000. From these results, the 3-quinuclidinone reductase of the present invention was predicted to be a homotetramer.

(9) K _m values: The reaction was conducted by changing the substrate concentration in the standard reaction solution was obtained by plotting the Lineweaver-Burk the Michaelis constant K _m. The resulting Michaelis constant K _m was 1.57 mM. Further, the substrate concentration of the standard reaction solution in the 20 mM, by changing the concentration of the coenzyme NADH, was similarly measured with respect to the Michaelis constant K _m. Michaelis constant _{K m} in this case was 0.109MM. When both the substrate and NADH concentrations were sufficiently higher than the K _m value, the V _max value was 794 U / mg.

(Example 6: Analysis of partial amino acid sequence of 3-quinuclidinone asymmetric reductase)
The purified enzyme obtained in Example 4 was denatured in the presence of 8M urea and digested with lysyl endopeptidase derived from Achromobacter (Wako Pure Chemical Industries). The obtained digest was added to a reverse phase column μRPC C2 / C8 SC2.1 / 10 (manufactured by GE) previously equilibrated with a 0.1% (v / v) trifluoroacetic acid solution, and a linear gradient of acetonitrile was obtained. (0 to 80%) to obtain peptide K27, peptide K31, and peptide K35. The purified enzyme and the collected three kinds of peptides were each subjected to sequence analysis with a protein sequencer (Applied Biosystems, model 491 HT). The amino acid sequences of the N-terminus of the purified enzyme, peptide K27, peptide K31, and peptide K35 are as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

(Example 7: Cloning of core region of 3-quinuclidinone asymmetric reductase gene)
Chromosomal DNA was extracted from the same cultured cells of Agrobacterium tumefaciens NK1 strain as in Example 1 according to the method described in Nucl. Acids Res., 1980, Vol. 8, pp. 4321-4325.

  Based on the N-terminus of the purified enzyme obtained in Example 6 and the amino acid sequence of peptide K35, sense primer S7 (SEQ ID NO: 7) and antisense primer A3 (SEQ ID NO: 8) were used as degenerate oligonucleotide primers for PCR. Designed. The PCR reaction solution composition is as follows: template chromosomal DNA 150 ng, 10 × Ex Taq buffer 5 μL, primers 150 nM, dNTP mixture 0.2 mM each, and Ex Taq polymerase 1.5 U, distilled water 50 μL in total. It added so that it might become. The PCR reaction conditions were as follows: Step 1; 94 ° C., 30 seconds; Step 2; 61 ° C., 30 seconds; Step 3; 72 ° C., 2 minutes; Step 1 to Step 3 were repeated 33 cycles; 72 ° C, 7 minutes; Step 5; 4 ° C, ∞. A specific amplification product of about 700 bp was obtained by PCR. The PCR reaction solution was subjected to agarose gel electrophoresis. The target band portion of about 700 bp was cut out and bound to the pCR4-TOPO vector using TOPO TA Cloning Kit for Sequencing (manufactured by Invitrogen). Escherichia coli TOP10 strain was transformed with the obtained vector. The transformed strain was cultured in LB medium (peptone 1% (w / v), yeast extract 0.5% (w / v), sodium chloride 1% (w / v), pH 7.0) containing ampicillin 50 μg / mL. Then, a plasmid for DNA sequencing was extracted and purified using QIAprep Spin Miniprep Kit (manufactured by Qiagen). Subsequently, the base sequence of the inserted fragment was determined by an automatic sequencer using the T7 primer and M13 reverse primer derived from the pCR4-TOPO vector.

  FIG. 1 shows the obtained base sequence and its deduced amino acid sequence. The underlined amino acid sequence in FIG. 1 matches the partial amino acid sequence of the purified enzyme of Example 4 obtained by the amino acid sequence analysis of Example 6. From the results of the sequence analysis shown in FIG. 1, it is clear that the PCR product consists of 694 bp nucleotides, and the deduced amino acid sequence matches the partial amino acid sequence of the natural 3-quinuclidinone asymmetric reductase purified in Example 4. became.

(Example 8: Cloning around the core region of 3-quinuclidinone asymmetric reductase gene)
In order to clarify the sequence of the peripheral region of the gene sequence determined in Example 7, it was divided into an upstream side and a downstream side, and each was amplified by PCR using a BD GenomeWalker kit (Clontech).

(A) Cloning on the upstream side Chromosomal DNA was completely digested with StuI, and the GenomeWalker Adapter attached to the kit was ligated to prepare a library. Using this as a template, amplification was performed by PCR using the adapter primer AP2 attached to the kit and the antisense primer NN (SEQ ID NO: 9) prepared based on the partial gene sequence of 3-quinuclidinone asymmetric reductase. The PCR reaction solution is the same as in Example 8. The PCR reaction conditions were as follows: Step 1; 94 ° C., 1 minute; Step 2; 94 ° C., 20 seconds; Step 3; 72 ° C., 4 minutes; Step 2 to Step 3 repeated 7 cycles; 94 ° C, 20 seconds; Step 5; 68 ° C, 4 minutes; Steps 4-5 are repeated 30 cycles; Step 6; 68 ° C, 7 minutes; Step 7; By this PCR, a specific amplification product of about 150 bp was obtained. This was subcloned into pCR4-TOPO vector and sequenced.

(B) Downstream Cloning Chromosomal DNA was completely digested with ApaI and PvuII to prepare a library. Southern analysis was performed using the nucleotide of about 700 bp obtained in Example 7 as a probe. As a result, a fragment of about 1.1 kbp was obtained by hybridization. This fragment was ligated to the GenomeWalker Adapter and amplified by PCR using the adapter primer AP2 and a sense primer CC (SEQ ID NO: 10) prepared based on the partial gene sequence of 3-quinuclidinone asymmetric reductase using this fragment as a template. . By this PCR, a specific amplification product of about 200 bp was obtained. This was subcloned into pCR4-TOPO vector and sequenced.

  The base sequence of the region containing the 3-quinuclidinone asymmetric reductase gene determined in Examples 7 and 8 is shown in FIG. Furthermore, regarding the structural gene portion of this base sequence, the amino acid sequence deduced from the base sequence is shown at the bottom of the base sequence in FIG. The result of the sequence analysis shown in FIG. 2 revealed that the structural gene encoding 3-quinuclidinone asymmetric reductase consists of 783 bp nucleotides and encodes 260 amino acids. The partial amino acid sequence of the purified enzyme of Example 4 obtained by the amino acid sequence analysis of Example 6 was found in the amino acid sequence deduced from the base sequence shown in FIG. 2 (indicated by the underline in FIG. 2). . Except for the deletion of the N-terminal methionine, there was a match at that part. The N-terminal methionine is considered to be removed by modification after protein synthesis.

  Using the sequence similarity search programs BLAST and FASTA, the deduced amino acid sequence of 3-quinuclidinone asymmetric reductase was compared with sequences in three types of protein sequence databases (PTR, PRF, and SWISS-PROT). As a result, it showed high similarity to proteins belonging to the short-chain dehydrogenase / reductase (SDR) family of Rhodosprillum rubrum ATCC11170 strain (81%).

(Example 9: Preparation of recombinant plasmid containing 3-quinuclidinone asymmetric reductase gene)
In order to express 3-quinuclidinone asymmetric reductase in E. coli, a recombinant plasmid used for transformation was prepared. First, based on the base sequence determined in Example 8, a sense primer (SEQ ID NO: 11) having an NdeI site added to the start codon portion of the structural gene of 3-quinuclidinone asymmetric reductase, and XhoI immediately after the stop codon. An antisense primer (SEQ ID NO: 12) to which was added was designed. Using these primers, PCR was performed using the chromosomal DNA of Example 7 as a template to obtain a specific amplification product of about 800 bp. This amplified fragment was subcloned into a plasmid pT7-Blue T-vector (Novagen), sequenced using M13 primer, and a clone free from errors due to PCR was selected to obtain pT7QR. The obtained pT7QR was double-digested with NdeI and XhoI, the structural gene of 3-quinuclidinone asymmetric reductase was taken out, and NdeI− located downstream of the T7 promoter in plasmid pET-21a (+) (Novagen) PETQR was created by inserting into the XhoI site.

(Example 10: Production of recombinant E. coli expressing 3-quinuclidinone asymmetric reductase and glucose dehydrogenase)
The recombinant plasmid pETQR prepared in Example 9 was used to transform E. coli BL21 (DE3) strain. Recombinant Escherichia coli BL21 (DE3) [pETQR] was made into competent cells based on a conventional method and a recombinant plasmid pACGD (Appl. Microbiol. Biotechnol., Containing the glucose dehydrogenase gene of Bacillus megaterium IWG3 strain). 1999, 51, pp.486-490) was introduced to produce recombinant E. coli BL21 (DE3) [pETQR & pACGD].

  The obtained recombinant Escherichia coli BL21 (DE3) [pETQR & pACGD] was cultured in 5 mL of LB medium supplemented with 50 μg / mL ampicillin and 25 μg / mL kanamycin at 37 ° C. for 3 hours at a final concentration of 0.1 mM. Then, IPTG was added so that the mixture was further shaken at 18 ° C. for 12 hours to induce protein expression. The cells were collected by centrifugation, washed once with distilled water, and lyophilized to prepare dry cells of recombinant Escherichia coli BL21 (DE3) [pETQR & pACGD].

(Example 11: 3-quinuclidinone reduction using recombinant E. coli)
Prepared in Example 10 in a 2 mL eppendorf tube with 100 μL 1M phosphate buffer (pH 7.0), 10 μL 60 mg / mL NAD ⁺ aqueous solution, 10 μL 50% (w / v) glucose aqueous solution, 250 μL 2 mg / mL The dried cell suspension in water, 620 μL of distilled water, and 100 μL of 10% (w / v) 3-quinuclidinone / hydrochloride aqueous solution were added, and the mixture was shaken at 37 ° C. for 24 hours. Subsequently, 200 μL of the reaction solution was sampled, 200 μL of a saturated aqueous potassium carbonate solution and 200 μL of chloroform were added and stirred, and the resulting chloroform layer was analyzed by gas chromatography. As a result, the conversion rate was 98%, and the optical purity of the obtained (R) -3-quinuclidinol was 99% ee or higher.

  According to the present invention, optically active 3-quinuclidinol, particularly (R) -3-quinuclidinol, can be efficiently produced with high optical purity. The resulting optically active 3-quinuclidinol is useful as a synthetic intermediate for producing therapeutic agents such as arteriosclerosis, bronchial asthma, or gastrointestinal motility inhibition.

A base sequence of a fragment obtained by PCR using sense primer S7 (SEQ ID NO: 7) and antisense primer A3 (SEQ ID NO: 8) using a chromosome DNA derived from Agrobacterium tumefaciens NK1 as a template, and its deduced amino acid sequence . The base sequence of the region containing the 3-quinuclidinone asymmetric reductase gene and the deduced amino acid sequence of the enzyme are shown.

Claims (17)

Enzymes having the biochemical properties shown in the following (1) to (3):
(1) Action: Using reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme, asymmetric reduction of 3-quinuclidinone or a salt thereof produces (R) -3-quinuclidinol;
(2) Substrate specificity: acting on 3-quinuclidinone, p-toluquinone and α-naphthoquinone, the relative activities of which are higher in the order of 3-quinuclidinone, p-toluquinone and α-naphthoquinone; and (3) optimum temperature: When the reaction is carried out at pH 7, the action is optimal at a temperature of 40 to 45 ° C.   The enzyme according to claim 1, wherein the molecular weight of the subunit when measured by SDS-PAGE is 28,500, and the molecular weight when measured by gel filtration chromatography is 102,000. Asymmetric reduction of 3-quinuclidinone or a salt thereof using reduced nicotinamide adenine dinucleotide (NADH) having any one of the following amino acid sequences (a) to (c) as a coenzyme: (R) -3 An enzyme that produces quinuclidinol:
(A) the amino acid sequence set forth in SEQ ID NO: 2;
(B) an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 2; or (c) 85% of the amino acid sequence described in SEQ ID NO: 2 Amino acid sequence having the above homology.   The enzyme according to any one of claims 1 to 3, wherein the enzyme is derived from Agrobacterium tumefaciens strain NK1 (FERM P-21144).   Agrobacterium tumefaciens NK1 strain (FERM P-21144).   A polynucleotide encoding the enzyme according to any one of claims 1 to 4. The polynucleotide according to claim 6, which is any of the following (i) to (iii):
(I) a polynucleotide having the base sequence set forth in SEQ ID NO: 1;
(Ii) hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions and asymmetrically reduces 3-quinuclidinone to produce (R) -3-quinuclidinol A polynucleotide encoding an enzyme having activity; or (iii) a base sequence having at least 60% sequence identity with the base sequence set forth in SEQ ID NO: 1, and asymmetrically reducing 3-quinuclidinone A polynucleotide encoding an enzyme having an activity to produce (R) -3-quinuclidinol.   A vector comprising the polynucleotide according to claim 6 or 7. The vector according to claim 8, further comprising a polynucleotide encoding an enzyme that regenerates oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) to NADH. 10. The vector according to claim 9, wherein the enzyme that regenerates NAD ⁺ to NADH is glucose dehydrogenase.   A transformant retaining the vector according to any one of claims 8 to 10 in an expressible manner. The transformant which respectively hold | maintains the vector containing the vector of Claim 8, and the vector which contains the polynucleotide which codes the enzyme which reproduce | regenerates NAD ⁺ to NADH so that expression is possible. The transformant according to claim 12, wherein the enzyme that regenerates NAD ⁺ to NADH is glucose dehydrogenase.   The transformant according to any one of claims 11 to 13, wherein the host is Escherichia coli.   A method for producing (R) -3-quinuclidinol or a salt thereof, comprising the enzyme according to any one of claims 1 to 4, the microorganism producing the enzyme, and the method according to any one of claims 11 to 14. A method comprising the step of allowing the transformant described above or the microorganism or a culture solution or a treated product of the transformant to act on 3-quinuclidinone or a salt thereof. The method according to claim 15, wherein NADH or NAD ⁺ is further added in the action step.   The method according to claim 15 or 16, wherein the microorganism is Agrobacterium tumefaciens strain NK1 (FERM P-21144).

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