JP2010187573A - Method for producing (r)-3-hydroxyisobutyric acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stereoselectively produce (R)-3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol using an enzyme. <P>SOLUTION: The method for stereoselectively producing (R)-3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol includes using lactoaldehyde reductase and lactoaldehyde dehydrogenase. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to diol oxidoreductase and aldehyde dehydrogenase that catalyze the oxidation reaction of 2-methyl-1,3-propanediol to (R) -3-hydroxyisobutyric acid, and (R) -3- The present invention relates to a method for producing hydroxyisobutyric acid.

  (R) -3-hydroxyisobutyric acid is known to be a useful compound as a pharmaceutical intermediate. (R) -3-Hydroxyisobutyric acid has one asymmetric carbon and has a stereoisomer, so that not only chemical purity but also stereochemical purity (optical purity) is high (R)- There is a need for 3-hydroxyisobutyric acid.

  Conventionally, as a method for producing (R) -3-hydroxyisobutyric acid, a method for producing isobutyric acid as a raw material using a microorganism is known. In Non-Patent Document 1, Candida rugosa IFO 0750 (Candida rugosa IFO 0750) was found as a yeast that produces (R) -3-hydroxyisobutyric acid, but its optical purity was as high as 93.3% ee. It was not a thing. Furthermore, in Non-Patent Document 2, it was shown that mutant MME 1027 derived from Candidalosa IFO 0750 as a parent strain has high stereoselectivity, but its production rate was 1.1 g / l / hour. It was not high enough in production.

  On the other hand, a method for producing 2-methyl-1,3-propanediol using a microorganism as a raw material is also known. In Non-Patent Document 3, Gluconobacter roseus IAM 1841 (Gluconobacter roseus IAM 1841) was found as a microorganism, but its optical purity was not as high as 83% ee. Furthermore, Non-Patent Document 4 discloses a method using bacteria belonging to the genus Acetobacter, and shows high production rates of 5 g / l and 2.5 g / l / hour or more within 2 hours. However, the optical purity of the produced (R) -3-hydroxyisobutyric acid was 97% ee, and the stereoselectivity was still unsatisfactory.

  Although microorganisms that produce (R) -3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol have been reported, enzymes that stereoselectively catalyze the oxidation reaction have been known so far. It was not done.

  Lactaldehyde reductase and lactaldehyde dehydrogenase are known as enzymes that catalyze the oxidation reaction of diols to optically active hydroxycarboxylic acids (Patent Document 1). However, even if these enzymes catalyze the oxidation reaction from 2-methyl-1,3-propanediol to 3-hydroxyisobutyric acid, or even if they catalyze, it is completely impossible to synthesize R-isomer stereoselectively. It is unknown.

WO2005 / 106005

J. et al. Ferment. Technol. 1981, Vol. 59, 203-208 J. et al. Biotechnol. 1999, Vol. 69,75-79 J. et al. Org. Chem. 1982, Vol. 47, 2400-2404 Tetrahedron: Asymmetry 2003, Vol. 14,2041-2043

  As described above, the stereoselectivity of the conventional method for producing (R) -3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol using a microorganism is not yet satisfactory, and its oxidation There is no known enzyme that stereoselectively catalyzes the reaction. The present invention has been made in view of the above circumstances, and an object thereof is to provide an enzyme that catalyzes an oxidation reaction from 2-methyl-1,3-propanediol to (R) -3-hydroxyisobutyric acid. The headline is to stereoselectively produce (R) -3-hydroxyisobutyric acid using the enzyme.

  In view of the above, the present inventors have evaluated the synthesis activity from 2-methyl-1,3-propanediol to (R) -3-hydroxyisobutyric acid for enzymes that catalyze various oxidation reactions. The present inventors have found that lactaldehyde reductase and lactaldehyde dehydrogenase have enzyme activities that catalyze the oxidation reaction of 2-methyl-1,3-propanediol to (R) -3-hydroxyisobutyric acid. And the method of producing (R) -3-hydroxyisobutyric acid stereoselectively using these enzymes was discovered, and it came to complete this invention.

That is, the present invention is as described in [1] to [3] below.
[1] A process for producing (R) -3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol, which comprises using diol oxidoreductase and aldehyde dehydrogenase.
[2] The method for producing (R) -3-hydroxyisobutyric acid according to claim 1, wherein the diol oxide reductase is lactaldehyde reductase, and the aldehyde dehydrogenase is lactaldehyde dehydrogenase.
[3] The lactaldehyde reductase is a protein having the amino acid sequence shown in SEQ ID NO: 2 or 3, and the lactaldehyde dehydrogenase is a protein having the amino acid sequence shown in SEQ ID NO: 5 or 6. The method for producing (R) -3-hydroxyisobutyric acid according to claim 2.

  According to the present invention, (R) -3-hydroxyisobutyric acid can be stereoselectively produced from 2-methyl-1,3-propanediol.

The result of having analyzed the soluble fraction of various transformants by SDS-polyacrylamide electrophoresis is shown. Lane 1 is a molecular weight marker, lane 2 is a soluble fraction of DH5α / pRed, and lane 3 is a soluble fraction of DH5α / pDeh. The result of having analyzed various purified enzyme liquids by SDS-polyacrylamide electrophoresis is shown. Lane 1 is a recombinant lactaldehyde reductase, Lane 2 is a recombinant lactaldehyde dehydrogenase, Lane 3 is a molecular weight marker.

The present invention is described in detail below.
In the present invention, the diol oxidoreductase and aldehyde dehydrogenase that catalyze the oxidation reaction of 2-methyl-1,3-propanediol to (R) -3-hydroxyisobutyric acid are 2-methyl-1,3-propanediol. A diol oxidoreductase capable of producing 3-hydroxy-2-methylpropanal using as a reaction substrate, and an aldehyde dehydrogenase capable of producing (R) -3-hydroxyisobutyric acid using 3-hydroxy-2-methylpropanal as a reaction substrate If it is, there will be no restriction | limiting in particular, All the enzymes like those are pointed out. For example, lactaldehyde reductase is mentioned as a diol oxidoreductase. Moreover, lactaldehyde dehydrogenase is mentioned as an aldehyde dehydrogenase.

  In this specification, in the synthesis or production of (R) -3-hydroxyisobutyric acid, “stereoselective” means that the optical purity of the target product (R) -3-hydroxyisobutyric acid is 97% ee or more. However, it is desirably 98% ee or more, and most desirably 99% ee or more.

  The lactaldehyde reductase in the present invention is classified into enzyme number 1.1.1.77 based on the report of the International Biochemical Union (I.U.B.) enzyme committee. A generic term for enzymes that reversibly catalyze the reaction of producing lactaldehyde in the presence of oxidized nicotinamide adenine dinucleotide, which is an enzyme. Also, lactaldehyde reductase is sometimes called L-1,2-propanediol oxidoreductase.

  The lactaldehyde reductase used in the present invention is derived from, for example, Escherichia bacteria, Shigella bacteria, Citrobacter bacteria, Salmonella bacteria, or Klebsiella bacteria Are preferably used.

  The lactaldehyde reductase in the present invention may be a protein having the amino acid sequence represented by SEQ ID NO: 2 or 3. Further, the amino acid sequence represented by SEQ ID NO: 2 or 3 may have an amino acid sequence in which one to a plurality of amino acids are substituted, deleted, inserted or added.

  In this specification, the term “plurality” varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein, but specifically 2 to 30, preferably 2 to 15, more preferably 2 to 2. 10 pieces.

  The protein having the amino acid sequence represented by SEQ ID NO: 2 is L-1,2-propanediol oxidoreductase (Genbank accession number: BAE76871) derived from Escherichia coli W3110 (Escherichia coli W3110), and the amino acid represented by SEQ ID NO: 3 The protein having the sequence has a sequence in which 6 histidine residues are added to the C-terminus of the amino acid sequence represented by SEQ ID NO: 2.

The lactaldehyde dehydrogenase in the present invention is classified into enzyme number 1.2.1.22 in accordance with the report of the International Biochemical Union (I.U.B.) Enzyme Committee, and is oxidized from lactaldehyde as a coenzyme. This refers to the generic name of enzymes that catalyze the reaction of producing lactic acid in the presence of type nicotinamide adenine dinucleotide, and enzyme number 1.2. Based on the report of the International Biochemical Union (IUB) enzyme committee. The generic name of the enzyme glycol aldehyde dehydrogenase which is classified into 1.21 and catalyzes a reaction for producing glycolic acid from glycol aldehyde in the presence of oxidized nicotinamide adenine dinucleotide as a coenzyme is also shown. This is because lactaldehyde aldehyde dehydrogenase and glycolaldehyde dehydrogenase are reported to be the same enzyme in the prior literature using Escherichia coli. (J. Biol. Chem. 1983, Vol. 258, 7788-7792).

  The lactaldehyde dehydrogenase used in the present invention is, for example, Escherichia bacterium, Shigella genus bacteria, Enterobacter genus bacteria, Citrobacter genus bacteria, or Klebsiella genus bacteria. The origin is preferably used.

  The lactaldehyde dehydrogenase in the present invention may be a protein having the amino acid sequence represented by SEQ ID NO: 5 or 6. Further, the amino acid sequence represented by SEQ ID NO: 5 or 6 may have an amino acid sequence in which one to a plurality of amino acids are substituted, deleted, inserted or added.

  The protein having the amino acid sequence represented by SEQ ID NO: 5 is lactaldehyde dehydrogenase (Genbank accession number: BAA15032) derived from Escherichia coli W3110, and the protein having the amino acid sequence represented by SEQ ID NO: 6 It has a sequence in which 6 histidine residues are added to the N-terminus of the amino acid sequence shown by No. 5.

The lactaldehyde reductase or lactaldehyde dehydrogenase according to the present invention can be obtained from organisms that produce the respective enzymes. As an organism that produces each enzyme, Escherichia coli W3110 (Escherichia coli)
An example is W3110).

  Alternatively, a host cell may be transformed with DNA encoding lactaldehyde reductase or lactaldehyde dehydrogenase to express and isolate each enzyme. According to this method, lactaldehyde reductase or lactaldehyde dehydrogenase can be easily and efficiently obtained. Furthermore, in order to make purification more convenient, 6 to 10 histidine residues can be added to the N-terminus or C-terminus of each protein.

  The DNA encoding lactaldehyde reductase or lactaldehyde dehydrogenase used for the transformant can be used as a recombinant DNA incorporated into a vector.

  The DNA encoding lactaldehyde reductase in the present invention may be a DNA having the base sequence represented by SEQ ID NO: 1. In addition, it hybridizes with DNA having the base sequence represented by SEQ ID NO: 1 under stringent conditions and oxidizes 2-methyl-1,3-propanediol to 3-hydroxy-2-methylpropanal. It may be DNA encoding a protein having an enzymatic activity to catalyze.

  The DNA encoding lactaldehyde dehydrogenase in the present invention may be a DNA having the base sequence represented by SEQ ID NO: 4. Further, it hybridizes with a DNA having the base sequence represented by SEQ ID NO: 4 under stringent conditions and catalyzes an oxidation reaction from 3-hydroxy-2-methylpropanal to (R) -3-hydroxyisobutyric acid. It may be a DNA encoding a protein having an enzyme activity.

“Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNA with high homology, ie 50% or more, preferably 70% or more, more desirably 80% or more, most desirably 90%
Examples include conditions in which complementary strands of DNA having the above homology hybridize and complementary strands of nucleic acids having lower homology do not hybridize.

More specifically, the hybridization under the stringent conditions can be exemplified by conditions commonly used by those skilled in the art to detect a specific hybridization signal. For example, Molecular Cloning: Cold Spring Harbor Laboratory Press, Current Protocols in
It can be carried out by the method described in Molecular Biology; Wiley Interscience, and a commercially available system includes Gene Image system (Amarsham). Specifically, hybridization can be performed by the following operation. The membrane to which the DNA or RNA to be tested is transferred is hybridized with a labeled probe in a hybridization buffer specified in the protocol according to the product protocol. The composition of the hybridization buffer consists of 0.1 wt% SDS, 5 wt% dextran sulfate, 1/20 of the total volume of blocking reagent attached to the kit, and 5 x SSC. The blocking reagent is, for example, 100 × Denhardt's solution (2% (weight / volume) Bovine serum albumin, 2% (weight / volume) Ficoll ^™ 400, 2% (weight / volume) polyvinylpyrrolidone). / 20 can be used after diluting. The hybridization temperature is in the range of 40 ° C. or higher and 80 ° C. or lower, more preferably 50 ° C. or higher and 70 ° C. or lower. After incubation for several hours to one day, the plate is washed with a washing buffer. The washing temperature is preferably room temperature, more preferably the temperature during hybridization. The composition of the washing buffer is 6 × SSC + 0.1 wt% SDS solution, more preferably 4 × SSC + 0.1 wt% SDS solution, and further preferably 1 × SSC + 0.1 wt% SDS solution. The membrane can be washed with such a washing buffer, and the DNA or RNA hybridized with the probe can be identified using the label used for the probe.

  Any host organism can be used as a transformant as long as the recombinant DNA can be stably and autonomously propagated and can express a trait of the foreign DNA. Examples of host organisms include Escherichia coli, but are not limited to Escherichia coli, and bacteria such as Escherichia bacteria, Bacillus subtilis bacteria, Pseudomonas bacteria, Saccharomyces Yeasts such as Genus, Pichia and Candida, and filamentous fungi such as Aspergillus can be used.

  As a method for transferring the recombinant DNA into the host organism, for example, when the host cell is Escherichia coli, a competent cell method using calcium treatment, an electroporation method, or the like can be used. The transformant thus obtained can be stably cultured to produce a large amount of lactaldehyde reductase or lactaldehyde dehydrogenase.

  Recombinant DNA carrying DNA encoding lactaldehyde reductase or lactaldehyde dehydrogenase can be removed from the transformant and can be transferred to other microorganisms. In addition, a recombinant DNA having a DNA encoding lactaldehyde reductase or lactaldehyde dehydrogenase is amplified using PCR to a DNA fragment encoding the enzyme, treated with a restriction enzyme, etc. They can also be easily combined and transferred to a host organism.

  As the medium, any of a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance and other nutrients. Culturing can be performed in a liquid medium containing the above-described culture components by using a normal culture method such as shaking culture, aeration-agitation culture, continuous culture, or fed-batch culture.

    The culture conditions may be appropriately selected depending on the type of culture medium and the culture method, and are not particularly limited as long as the strain can grow and produce lactaldehyde reductase or lactaldehyde dehydrogenase. For example, when the culture is carried out under an aerobic or microaerobic condition with a pH of 6 or more and 8 or less and a temperature of 25 ° C. or more and 40 ° C. or less while appropriately controlling the pH and temperature, the time required for cultivation is 48. Within hours.

  The lactaldehyde reductase or lactaldehyde dehydrogenase produced by the transformant can also be used by collecting the culture solution containing the transformant in the culture as it is. In addition, when lactaldehyde reductase or lactaldehyde dehydrogenase is present in the culture solution, the solution containing each enzyme and the transformant can be separated by filtration, centrifugation, or the like. When lactaldehyde reductase or lactaldehyde dehydrogenase is present in the transformant, the transformant can also be collected from the obtained culture by means of filtration or centrifugation and used. In addition, the collected transformant is destroyed by a mechanical method or an enzymatic method such as lysozyme, and a chelating agent such as EDTA and / or a surfactant is added as necessary to allow lactaldehyde reductase or lactaldehyde dehydrogenase. It can be solubilized and separated and collected as a solution.

  The solution containing lactaldehyde reductase or lactaldehyde dehydrogenase thus obtained is concentrated, for example, under reduced pressure, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or a hydrophilic organic solvent such as methanol, ethanol or acetone. It can be precipitated by fractional precipitation. Heat treatment and isoelectric point treatment are also effective purification means. Subsequently, purified lactaldehyde reductase or lactaldehyde dehydrogenase can be obtained by performing gel filtration with an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography. In addition, when 6 to 10 histidine residues are added to the N-terminus or C-terminus of lactaldehyde reductase or lactaldehyde dehydrogenase, purification using interaction with nickel ions can also be performed.

  The method for producing (R) -3-hydroxyisobutyric acid in the present invention synthesizes (R) -3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol using lactaldehyde reductase and lactaldehyde dehydrogenase. It is a manufacturing method including the process to do.

  Lactaldehyde reductase and lactaldehyde dehydrogenase are oxidized nicotinamide adenine dinucleotide-dependent enzymes. In the production of (R) -3-hydroxyisobutyric acid, when an organism producing lactaldehyde reductase or lactaldehyde dehydrogenase is used, oxidized nicotinamide adenine dinucleotide produced by the organism can be used. When purified lactaldehyde reductase or lactaldehyde dehydrogenase is used, oxidized nicotinamide adenine dinucleotide can be added to the reaction solution.

  The reaction conditions are not particularly limited, but it is preferable to carry out the reaction while appropriately controlling pH and temperature. For example, the pH is 7 or more and 11 or less, preferably 8 or more and 10 or less. The temperature is in the range of 4 ° C. or higher and 60 ° C. or lower, and the reaction can be carried out under shaking, stirring or standing conditions.

As a medium for the reaction liquid, water, an aqueous medium, an organic solvent, or a mixed liquid of water or an aqueous medium and an organic solvent is used. As the aqueous medium, for example, a buffer solution such as a phosphate buffer solution or a glycine-sodium hydroxide buffer solution is used. Any organic solvent may be used as long as it does not inhibit the reaction.

  Separation and purification of (R) -3-hydroxyisobutyric acid from the reaction solution is performed by a method used in ordinary organic synthetic chemistry, for example, extraction with an organic solvent, crystallization, thin layer chromatography, high performance liquid chromatography, etc. It can be carried out.

(Analysis conditions)
(R)-and (S) -3-hydroxyisobutyric acid were quantified by high performance liquid chromatography. The analysis conditions are as follows.
Column; SUMICHILAR OA-6100 (5 μm) 4.6 mmI. D. × 150mm (manufactured by Sumika Chemical Analysis Service)
Column temperature: 40 ° C
Pump flow rate; 1.0 ml / min
Eluent: 2 mmol / l copper sulfate detection; UV 254 nm

(Reference Example 1)
Preparation of (R) -and (S) -3-hydroxyisobutyric acid (R)-and (S) -3-hydroxyisobutyric acid are described in J. Am. Biol. Chem. 1988, Vol. It was prepared according to the method described in 263,327-331. (R)-and (S) -3-hydroxyisobutyric acid methyl used as raw materials were purchased from Tokyo Chemical Industry Co., Ltd.

  Using the prepared (R)-and (S) -3-hydroxyisobutyric acid as a preparation for high performance liquid chromatography, the optical purity of 3-hydroxyisobutyric acid produced by an enzymatic reaction was calculated.

Example 1
Construction of Lactaldehyde Reductase Expression Plasmid With reference to the nucleotide sequence of the lactaldehyde reductase gene (fucO) registered in Genbank (Accession number: AP009048), the oligonucleotide primers 5′-agtgaattcgtaaagagatagagatagagtggatagagtgattagattagat-3 5'-gtcgatccttaatgatgatgatgatgcccaggcggtatgggtaaagctctac-3 '(SEQ ID NO: 8) was synthesized. Using each primer, PCR was performed using Escherichia coli W3110 genomic DNA as a template to obtain a DNA fragment of about 1.2 kbp. This DNA fragment contains the base sequence of the fucO gene, and the base sequences derived from the oligonucleotide primers shown in SEQ ID NOs: 7 and 8 are added to the 5 ′ end and 3 ′ end, respectively. The obtained DNA fragment and plasmid pUC18 were digested with restriction enzymes EcoRI and BamHI and ligated using Ligation High (manufactured by Toyobo Co., Ltd.), and then Escherichia coli DH5α (Toyobo) was obtained using the obtained recombinant plasmid. Spinning). The transformant was cultured in an LB agar medium containing 100 μg / ml of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), and was Am-resistant and white colony. The resulting transformant was obtained. A plasmid was extracted from the transformant thus obtained. This plasmid was named pRed.

  By culturing E. coli transformed with pRed, a recombinant lactaldehyde reductase in which 6 histidine residues are added to the C-terminal amino acid sequence of lactaldehyde reductase can be produced.

  Escherichia coli W3110 can be obtained from the American Type Culture Collection.

(Example 2)
Construction of Lactaldehyde Dehydrogenase Expression Plasmid With reference to the nucleotide sequence of the lactaldehyde dehydrogenase gene (aldA) (Accession number: AP009048) registered in Genbank, the oligonucleotide primers 5'-ggaattcataaaaagagatagacatatacatcatcatcatcatccatcatcatccatcatcatccatcatgat 5′-tctaagcttttaagactgtataataac-3 ′ (SEQ ID NO: 10) was synthesized. Using each primer, PCR was performed using Escherichia coli W3110 genomic DNA as a template to obtain a DNA fragment of about 1.5 kbp. This DNA fragment contains the base sequence of the aldA gene, and the base sequences derived from the oligonucleotide primers shown in SEQ ID NOs: 9 and 10 are added to the 5 ′ end and 3 ′ end, respectively. The obtained DNA fragment and plasmid pUC18 were digested with restriction enzymes EcoRI and HindIII and ligated using ligation high, and Escherichia coli DH5α was transformed with the obtained recombinant plasmid. The transformant was cultured on an LB agar medium containing 100 μg / ml of Am and X-Gal to obtain a transformant that was Am resistant and became a white colony. A plasmid was extracted from the transformant thus obtained. This plasmid was named pDeh.

  By culturing Escherichia coli transformed with pDeh, a recombinant lactaldehyde dehydrogenase in which 6 histidine residues are added to the N-terminal amino acid sequence of lactaldehyde dehydrogenase can be produced.

(Example 3)
Production and expression of transformants Escherichia coli DH5α was transformed with pRed and pDeh in the usual manner, and the resulting transformants were named DH5α / pRed and DH5α / pDeh, respectively.

  DH5α / pRed was inoculated into 80 ml of LB medium containing 100 μg / ml of Am in a 500 ml Erlenmeyer flask, shake-cultured at 30 ° C. until OD (660 nm) became 0.5, and then IPTG (isopropyl-β-thiogalacto) Pyranoside) was added to 1 mmol / l, and the mixture was further cultured with shaking for 20 hours. The culture solution was centrifuged at 8000 rpm for 20 minutes, and the obtained bacterial cells were suspended in 10 mmol / l phosphate buffer (pH 8.0).

  DH5α / pDeh was inoculated into 80 ml of LB medium containing 100 μg / ml of Am in a 500 ml Erlenmeyer flask with baffle, and after shaking culture at 30 ° C. until OD (660 nm) was 0.5, IPTG was 1 mmol / l. Then, the mixture was further cultured with shaking for 16 hours. The culture solution was centrifuged at 8000 rpm for 20 minutes, and the obtained bacterial cells were suspended in 10 mmol / l phosphate buffer (pH 8.0).

Example 4
Confirmation of Expression by SDS-Polyacrylamide Gel Electrophoresis 1.0 ml of the suspension of the two transformants prepared in Example 3 was crushed in ice water for 5 minutes using a biopluter (Olympus). did. The processed product of the obtained transformant was centrifuged, and the obtained supernatant was used as a soluble fraction. FIG. 1 shows the result of analyzing each soluble fraction by SDS-polyacrylamide electrophoresis. Lane 1 is an electrophoresis of molecular weight markers. Lanes 2 and 3 are obtained by electrophoresis of soluble fractions of DH5α / pRed and DH5α / pDeh, respectively. In lane 2, a band of recombinant lactaldehyde reductase was detected at a position between the molecular weight markers of 39 kDa and 51 kDa. In Lane 3, a recombinant lactaldehyde dehydrogenase band was detected in the vicinity of the 51 kDa molecular weight marker.

(Example 5)
Purification of recombinant enzyme The above-mentioned recombinant lactaldehyde reductase and recombinant lactaldehyde dehydrogenase have six histidine residues added to the C-terminal and N-terminal amino acid sequences, respectively. The purification used was performed.

[1] Purification of recombinant lactaldehyde reductase DH5α / pRed cells obtained in the same manner as in Example 3 were added to 1 ml of Buffer 1 (50 mmol / l monosodium phosphate, 300 mmol / l sodium chloride, 20 mmol / l). The suspension was suspended in imidazole (pH 8.0) and crushed at 0 ° C. for 3 minutes and 30 seconds using a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.). The disrupted solution was centrifuged, and the obtained 0.6 ml supernatant was applied to a Ni-NTA spin column (QIAGEN) equilibrated with buffer solution 1, and centrifuged at 2000 rpm for 2 minutes to obtain Ni-NTA resin. The adsorbed recombinant lactaldehyde reductase. Next, 0.6 ml of Buffer 2 (50 mmol / l monosodium phosphate, 300 mmol / l sodium chloride, 30 mmol / l imidazole, pH 8.0) is poured onto the Ni-NTA spin column and centrifuged at 2000 rpm for 2 minutes. Thus, the Ni-NTA resin was washed. The washing operation was repeated 4 times. Furthermore, 0.5 ml of Buffer 3 (50 mmol / l monosodium phosphate, 300 mmol / l sodium chloride, 250 mmol / l imidazole, pH 8.0) is poured onto the Ni-NTA spin column and centrifuged at 2000 rpm for 2 minutes. Then, the recombinant lactaldehyde reductase was eluted. The elution operation was repeated 5 times. The obtained 2.5 ml eluate was desalted using PD-10 Desalting column (manufactured by GE Healthcare). A MOPS buffer (pH 7.8) was used as an eluent for desalting to obtain 3.5 ml of an eluate. This eluate was used as a purified enzyme solution for recombinant lactaldehyde reductase.

[2] Purification of recombinant lactaldehyde dehydrogenase 1 ml of buffer 4 (50 mmol / l monosodium phosphate, 300 mmol / l sodium chloride, 10 mmol / l) was obtained from DH5α / pDeh obtained in the same manner as in Example 3. (Imidazole, pH 8.0) and suspended using a multi-bead shocker at 0 ° C. for 3 minutes and 30 seconds. The disrupted solution is centrifuged, and 0.6 ml of the obtained supernatant is applied to a Ni-NTA spin column equilibrated with buffer solution 4 and centrifuged at 2000 rpm for 2 minutes, whereby recombinant lactaldehyde is added to Ni-NTA resin. Dehydrogenase was adsorbed. Next, the Ni-NTA resin was washed by pouring 0.6 ml of Buffer 1 onto the Ni-NTA spin column and centrifuging at 2000 rpm for 2 minutes. The washing operation was repeated 4 times. Furthermore, pour 0.5 ml of buffer 5 (50 mmol / l monosodium phosphate, 300 mmol / l sodium chloride, 400 mmol / l imidazole, pH 8.0) onto the Ni-NTA spin column and centrifuge at 2000 rpm for 2 minutes. To elute the recombinant lactaldehyde dehydrogenase. The elution operation was repeated 5 times. The obtained 2.5 ml eluate was desalted using PD-10 Desalting column. A MOPS buffer (pH 7.8) was used as an eluent for desalting to obtain 3.5 ml of an eluate. This eluate was used as a purified enzyme solution for recombinant lactaldehyde dehydrogenase.

  FIG. 2 shows the results of SDS-polyacrylamide electrophoresis analysis of purified enzyme solutions of recombinant lactaldehyde reductase and recombinant lactaldehyde dehydrogenase. As a result of the analysis, each purified enzyme solution was almost a single band.

(Example 6)
(R) -3-Hydroxyisobutyric acid production method The reaction solution (1.0 ml) was 300 mmol / l 2-methyl-1,3-propanediol (manufactured by Junsei Kagaku), 50 mmol / l glycine-hydroxylated. Sodium buffer (pH 9.0), 1 mmol / l oxidized nicotinamide adenine dinucleotide (manufactured by SIGMA), and purified lactaldehyde reductase and recombinant lactaldehyde dehydrogenase purified by the same method as in Example 5 The solution was contained at 0.2 ml and reacted at 25 ° C. for 16 hours. After completion of the reaction, the reaction solution was adjusted to pH 2 or lower with 2 mol / l hydrochloric acid, extracted three times with 1.0 ml of ethyl acetate, and distilled under reduced pressure to obtain (R) -3-hydroxyisobutyric acid. It was. This (R) -3-hydroxyisobutyric acid was dissolved in 0.4 ml of 50 mmol / l sodium hydroxide aqueous solution and analyzed under the above (analysis conditions). As a result, (S) -3-hydroxyisobutyric acid was below the detection limit. The optical purity of (R) -3-hydroxyisobutyric acid was 99% ee or higher.

  As described above, according to the present invention, (R) -3-hydroxyisobutyric acid can be stereoselectively produced from 2-methyl-1,3-propanediol.

  Although the configuration of the present invention has been described above, the present invention is not limited to this and includes various modes.

Claims (3)

A method for producing (R) -3-hydroxyisobutyric acid from 2-methyl-1,3-propanediol, which comprises using diol oxidoreductase and aldehyde dehydrogenase. The method for producing (R) -3-hydroxyisobutyric acid according to claim 1, wherein the diol oxidoreductase is lactaldehyde reductase, and the aldehyde dehydrogenase is lactaldehyde dehydrogenase. The lactaldehyde reductase is a protein having the amino acid sequence shown in SEQ ID NO: 2 or 3, and the lactaldehyde dehydrogenase is a protein having the amino acid sequence shown in SEQ ID NO: 5 or 6. Item 3. A method for producing (R) -3-hydroxyisobutyric acid according to Item 2.