WO2010098505A1

WO2010098505A1 - Process for producing novel optically active mandelic acid and derivative thereof

Info

Publication number: WO2010098505A1
Application number: PCT/JP2010/053597
Authority: WO
Inventors: 宮本憲二
Original assignee: 学校法人慶應義塾
Priority date: 2009-02-27
Filing date: 2010-02-26
Publication date: 2010-09-02
Also published as: JPWO2010098505A1

Abstract

Disclosed is a deracemization reaction of a mandelic acid derivative utilizing a biocatalyst, which is a process for producing an optically active mandelic acid derivative. Specifically disclosed is a process for producing optically active mandelic acid or a derivative thereof by deracemizing racemic mandelic acid or a derivative thereof, which comprises allowing a transformed microorganism having a gene encoding mandelate oxidase, a gene encoding (R)-mandelate dehydrogenase and a gene encoding a coenzyme-regenerating enzyme introduced therein to act on racemic mandelic acid or a derivative thereof in the presence of a substrate for the coenzyme-regenerating enzyme and an oxidized coenzyme to deracemize the racemic mandelic acid or the derivative thereof.

Description

Process for producing novel optically active mandelic acid and derivatives thereof

TECHNICAL FIELD The present invention relates to a method for producing optically active mandelic acid and its derivatives important as pharmaceuticals, and a method for producing optically active mandelic acid and its derivatives using a microorganism into which a gene involved in the production of optically active mandelic acid and its derivatives is introduced. About.

Optically active substances are becoming more important year by year, mainly for pharmaceuticals. There are three methods for synthesizing optically active substances: (1) using optically active raw materials (chiral pools), (2) synthesizing racemates and optically resolving them, and (3) asymmetric synthesis. It is used properly by making use of.
Recently, the number of drugs with complicated chemical structures has increased, and there are not only one asymmetric center, and many have 4 to 5 or more. Therefore, in order to synthesize these compounds, the synthesis is not carried out sequentially from one starting material to the final product, but the final compound is divided into several components, each synthesized separately, and finally combined into a product. It is efficient to adopt the method. As a result, the demand for optically active pharmaceutical intermediates is increasing worldwide.
Catalysts that perform synthetic reactions using biochemical reactions are collectively referred to as biocatalysts. In recent years, biocatalysts have been used as catalysts for synthesizing optically active substances. For the synthesis of useful compounds, a single enzyme to a plurality of enzyme systems, microorganism cells, animal and plant cultured cells, and the like are used as biocatalysts.
In addition to conventional biocatalysts, many new biocatalysts have recently appeared. Enzymes that could only be obtained by isolation from various organisms have recently been genetically modified. An oxidoreductase that requires recycling of the coenzyme is not an isolated enzyme, but if a microbial cell is used, it can be regenerated by an enzyme system in the microbial cell and is practical. However, since there are many enzymes in the cells, stereoselectivity often deteriorates. In such a case, the yield and stereoselectivity can be improved by introducing genes of enzymes of other bacterial cells and expressing them in large quantities. If the coenzyme regeneration system is weak, the enzyme can also be introduced to increase the reaction rate.
Optically active mandelic acid derivatives are useful as pharmaceutical intermediates. For example, the R form of mandelic acid is used as a side chain modifier of the cephalosporin antibiotic “cefamandol”. The R form of o-chloromandelic acid is a raw material for the antiplatelet agent “clopidogrel” and antifungal agents.
As a method for producing an optically active mandelic acid derivative, the following method is known.
(A) Optical resolution method by fractionated crystal of racemic body (see Patent Document 1)
(B) Chromatographic optical resolution method (see Non-Patent Document 1)
(C) A method of obtaining an optically active substance by oxidizing one of the racemates (see Patent Document 2)
(D) Method using nitrilase (see Patent Documents 3 and 4)
(E) Method using hydroxyl nitrile lyase (see Patent Document 5)
(F) Reduction method of benzoylformic acid derivative (see Patent Documents 6 and 7)
(G) Method using microorganisms (see Patent Document 8)
While the above methods (a), (b) and (c) recover the target enantiomer, the unnecessary enantiomer cannot be used, resulting in the loss of half of the raw material. That is, since the raw material cannot be used effectively, it causes a cost increase. A method of recovering unnecessary enantiomers and reusing them as racemates by racemization has been reported, but the operation is complicated. The method (d) above requires a mandelonitrile derivative as a raw material. Sodium cyanide is required for the synthesis of the mandelonitrile derivative, and the sodium cyanide inhibits nitrilase, so that it is necessary to control the sodium cyanide concentration, and is not suitable for industrial production. The method (e) uses benzaldehyde and hydrogen cyanide as raw materials. Since hydrogen cyanide is highly toxic to humans and highly toxic to enzymes, it is not suitable for industrial production. The method (f) is a process that does not costly because the benzoylformic acid derivative is unstable and more expensive than the optically active mandelic acid derivative, although the mandelic acid derivative can be obtained with high optical purity and yield. . Furthermore, in the method (g), one kind of naturally-derived microorganism is used, which requires long-term culture, and a high-concentration substrate cannot be used from the viewpoint of enzyme activity. A large amount of optically active mandelic acid could not be produced efficiently. Furthermore, 100% optically active purity could not be achieved. Thus, each of the conventional techniques has problems.

JP 2001-72644 A JP-A-6-165695 Japanese Patent Laid-Open No. 4-99496 JP-A-6-237789 JP 2001-354616 A JP 2004-65049 A JP 2003-199595 A JP-A-6-7196

An object of the present invention is to provide a deracemization reaction of a mandelic acid derivative using a biocatalyst as a method for producing an optically active mandelic acid derivative.
The present inventors have intensively studied to solve the problems of the conventional methods for producing optically active mandelic acid and its derivatives and to develop new methods for producing optically active mandelic acid and its derivatives.
The present inventors have combined the three enzyme reactions using Escherichia coli in which three enzyme genes involved in the synthesis of optically active mandelic acid and its derivatives have been introduced and co-expressed as biocatalysts. It was thought that mandelic acid and its derivatives could be deracemized by a hydration reaction (a reaction that converts a racemate into one enantiomer in 100% yield). As three types of enzymes, only the S form of the racemic mandelic acid derivative is stereoselectively oxidized to produce the benzoylformic acid derivative, mandelate oxidase (MOX), and the coenzyme NADH is consumed to stereoselect the benzoylformic acid derivative. (R) -mandelate dehydrogenase ((R) -MDH) that produces only the R form of the mandelic acid derivative and glucose dehydrogenase that oxidizes glucose to produce gluconic acid and regenerates the coenzyme NADH ( GDH) and other coenzyme regeneration system enzymes were used to perform deracemization of mandelic acid and its derivatives efficiently by using recombinant Escherichia coli that had been studied and introduced genes encoding the above three types of enzymes. It has been found that optically active mandelic acid and its derivatives can be produced. The inventors of the present invention introduced and co-expressed E. coli introduced with genes encoding MOX and (R) -MDH and GDH, called a “deracemization factory” and constructed it in three stages. First, as the first stage, an “oxidation factory”, which is Escherichia coli in which a gene encoding MOX was introduced and expressed, was constructed. Next, as a second stage, a “reduction factory”, which is Escherichia coli in which a gene encoding (R) -MDH and a gene encoding GDH were introduced and co-expressed, was constructed. Finally, as a third stage, a deracemization factory was constructed by combining the above oxidation factory and reduction factory, and the present invention was completed.
That is, the present invention is as follows.
[1] A method for producing optically active mandelic acid or a derivative thereof by deracemizing racemic mandelic acid or a derivative thereof, which encodes mandelate oxidase, (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme An optically active mandelic acid or a derivative thereof, comprising detransformation of a transformed microorganism into which a gene has been introduced by acting on a racemic mandelic acid or a derivative thereof in the presence of a substrate of a coenzyme regeneration system enzyme and an oxidized coenzyme A transformed microorganism in which mandelic acid or a derivative thereof is represented by the following formula (I):

(In the formula, X represents a hydrogen atom or an alkali or alkaline earth metal, R means that one or a plurality of ortho, meta or para positions are substituted, and the substituent is a hydrogen atom, a halogen atom, A hydroxyl group, an alkyl group having 1 to 3 carbon atoms, an alkoxy group or a thioalkyl group, an amino group, a nitro group, a mercapto group, a phenyl group, or a phenoxy group).
[2] The optically active mandelic acid or derivative thereof according to [1], wherein the enzyme regeneration system enzyme is selected from the group consisting of glucose dehydrogenase, hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase and glucose-6-phosphate dehydrogenase. How to manufacture.
[3] Two types of expression vectors including an expression vector into which a transformed microorganism has inserted a gene encoding mandelate oxidase and a 2-gene expression vector into which a gene encoding (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme has been inserted A method for producing the optically active mandelic acid or derivative thereof according to [1] or [2], wherein
[4] A method for producing the optically active mandelic acid or derivative thereof according to any one of [1] to [3], wherein the mandelate oxidase, (R) -mandelate dehydrogenase and the coenzyme regeneration system enzyme are derived from a microorganism.
[5] A method for producing the optically active mandelic acid or derivative thereof according to any one of [1] to [4], wherein the coenzyme regeneration system enzyme is glucose dehydrogenase.
[6] Mandelate oxidase is derived from Pseudomonas microorganisms, (R) -Mandelate dehydrogenase is derived from Enterococcus microorganisms, and a coenzyme regeneration system enzyme is derived from Bacillus microorganisms [1] A method for producing the optically active mandelic acid or derivative thereof according to any of [1] to [5].
[7] A method for producing the optically active mandelic acid or derivative thereof according to any one of [1] to [6], wherein the transformed microorganism is Escherichia coli.
[8] A gene encoding mandelate oxidase, (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme for producing optically active mandelic acid or a derivative thereof by deracemizing racemic mandelic acid or a derivative thereof. An introduced transformed microorganism, wherein mandelic acid or a derivative thereof is represented by the following formula (I):

(In the formula, X represents a hydrogen atom or an alkali or alkaline earth metal, R means that one or a plurality of ortho, meta or para positions are substituted, and the substituent is a hydrogen atom, a halogen atom, A hydroxyl group, an alkyl group having 1 to 3 carbon atoms, an alkoxy group or a thioalkyl group, an amino group, a nitro group, a mercapto group, a phenyl group, or a phenoxy group).
[9] To produce an optically active mandelic acid or a derivative thereof, wherein the coenzyme regeneration system enzyme is selected from the group consisting of glucose dehydrogenase, hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase and glucose-6-phosphate dehydrogenase. The transformed microorganism of [8].
[10] Two types of expression vectors: an expression vector into which a transformed microorganism has inserted a gene encoding mandelate oxidase and a 2-gene expression vector into which a gene encoding (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme has been inserted The transformed microorganism of [8] or [9] for producing an optically active mandelic acid or a derivative thereof transformed with
[11] Any one of [8] to [10] for producing optically active mandelic acid or a derivative thereof, wherein mandelate oxidase, (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme are derived from a microorganism. Transformed microorganisms.
[12] The transformed microorganism of any one of [8] to [11] for producing optically active mandelic acid or a derivative thereof, wherein the coenzyme regeneration system enzyme is glucose dehydrogenase.
[13] Mandelate oxidase is derived from Pseudomonas microorganisms, (R) -Mandelate dehydrogenase is derived from Enterococcus microorganisms, and a coenzyme regeneration system enzyme is derived from Bacillus microorganisms The transformed microorganism of any one of [8] to [12] for producing optically active mandelic acid or a derivative thereof.
[14] The transformed microorganism of any one of [8] to [13] for producing optically active mandelic acid or a derivative thereof, which is Escherichia coli.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2009-046995 which is the basis of the priority of the present application.

FIG. 1 is a diagram showing a deracemization reaction of a mandelic acid derivative by a deracemization factory.
FIG. 2 is a diagram showing a reaction in an oxidation factory.
FIG. 3 is a diagram showing a reaction in a reduction factory.
FIG. 4 is a diagram showing the MOX reaction.
FIG. 5 is a diagram showing a reaction of (R) -MDH.
FIG. 6 is a diagram showing the reaction of GDH.
FIG. 7 shows the structure of pASA1 containing the mox gene.
FIG. 8 is a diagram showing the expression of MOX in E. coli transformed with pASA1.
FIG. 9 is a diagram showing a product from an oxidation factory.
FIG. 10 is a diagram showing the structure of pET3aRMDH containing the (R) -mdh gene.
FIG. 11 is a figure which shows the result of colony PCR of pET3aRMDH.
FIG. 12 is a diagram showing codon correction in the (R) -mdh gene.
FIG. 13 shows (R) -MDH expression in E. coli transformed with pET3aRMDH.
FIG. 14 is a diagram showing the structure of pACYCGDH containing the gdh gene.
FIG. 15 shows the results of pACYCGDH colony PCR.
FIG. 16 shows the expression of (R) -MDH in E. coli transformed with pACYCGDH.
FIG. 17 is a diagram showing two types of co-expression of (R) -MDH and GDH. FIG. 17A is a diagram showing co-expression with two types of expression plasmids, and FIG. 17B is a diagram showing co-expression with one type of expression plasmid.
FIG. 18 shows the results of co-expression with two types of expression plasmids.
FIG. 19 shows the structure of pACYCCMG containing the (R) -mdh and gdh genes.
FIG. 20 is a diagram showing the base sequence of the deletion mutation introduction site.
FIG. 21 is a diagram showing the results of colony PCR of pACYCCMG.
FIG. 22 shows the results of co-expression with one type of expression plasmid.
FIG. 23 is a diagram showing a reaction by CFE.
FIG. 24 is a diagram showing a product produced by a reduction factory.
FIG. 25 is a diagram showing the co-expression of MOX, (r) -MDH and GDH.
FIG. 26 is a diagram showing the results of co-expression of MOX, (r) -MDH and GDH.
FIG. 27 is a diagram showing a product produced by a deracemization factory.
FIG. 28 is a diagram showing the change over time of the mandelic acid deracemization reaction (0.5 g wet cell, 10 mM substrate concentration).
FIG. 29 is a diagram showing the change over time of the mandelic acid deracemization reaction (0.5 g wet cell, 50 mM substrate concentration).
FIG. 30 is a diagram showing the change over time of the mandelic acid deracemization reaction (0.5 g wet cells, substrate concentration 100 mM).
FIG. 31 is a diagram showing the results of confirmation of MOX solubilization.
FIG. 32 is a diagram showing the time course of mandelic acid deracemization reaction (wet bacterial cell 0.5 g, substrate concentration 500 mM).
FIG. 33 is a diagram showing the time course of mandelic acid deracemization reaction (2.5 g wet cells, substrate concentration 500 mM).
FIG. 34 is a diagram showing the change over time of the mandelic acid deracemization reaction (5 g wet cells, substrate concentration 500 mM).
FIG. 35 is a diagram showing the change over time of the mandelic acid deracemization reaction (10 g of wet cells, substrate concentration of 385 mM).
FIG. 36 is a diagram showing the change over time in the deracemization reaction of o-chloromandelic acid (0.5 g wet cell, 10 mM substrate concentration).
FIG. 37 is a diagram showing the change over time in the deracemization reaction of o-chloromandelic acid (0.5 g wet cells, substrate concentration 100 mM).
FIG. 38 is a diagram showing a change with time of the enantiomeric excess in the deracemization reaction.

Hereinafter, the present invention will be described in detail.
In the present invention, the object of the deracemization reaction is mandelic acid or a derivative thereof. Mandelic acid derivatives include compounds of the following formula I:

(In the formula, X represents a hydrogen atom or an alkali or alkaline earth metal, R means that one or a plurality of ortho, meta or para positions are substituted, and the substituent is a hydrogen atom, a halogen atom, A hydroxyl group, an alkyl group having 1 to 3 carbon atoms, an alkoxy group or a thioalkyl group, an amino group, a nitro group, a mercapto group, a phenyl group, or a phenoxy group)
In the compound represented by the following formula II, a compound in which R at the ortho position is H is mandelic acid, and a compound in which R at the ortho position is Cl is 0-chloromandelic acid.

The “optically active mandelic acid or a derivative thereof” in the present invention refers to a mandelic acid derivative in which a certain optical isomer is contained more than another optical isomer. In the present invention, a preferable optically active mandelic acid derivative has an optical purity of 50% ee or higher, preferably 80% ee or higher, more preferably 90% ee or higher, still more preferably 95% ee or higher, particularly preferably 97% ee or higher. (Enantiomericexcess; ee). The optical purity of the optically active mandelic acid or its derivative can be confirmed using, for example, an optical resolution column. The “optical isomer” of the present invention may be generally referred to as “optically active form” and “enantiomer”.
In the present invention, genes encoding three types of enzymes involved in deracemization of mandelic acid or its derivatives are introduced into microorganisms, and the three types of enzymes are co-expressed.
The three types of enzymes used are mandelate oxidase (MOX), (R) -mandelate dehydrogenase, and a coenzyme regeneration system enzyme.
Mandelic acid oxidase stereoselectively oxidizes racemic mandelic acid or its derivative S form to produce a benzoylformic acid derivative (FIG. 2).
The benzoylformic acid derivative is represented by the following formula (II) with respect to mandelic acid represented by the above formula (I) or a derivative thereof.

(Wherein X represents a hydrogen atom or an alkali or alkaline earth metal, R means that one or more of ortho, meta or para positions are substituted, and the substituent is hydrogen, halogen atom, hydroxyl, Group, an alkyl group having 1 to 3 carbon atoms, an alkoxy group or a thioalkyl group, an amino group, a nitro group, a mercapto group, a phenyl group, or a phenoxy group)
Mandelic acid oxidase may be derived from microorganisms such as Pseudomonas genus, Candida genus, Saccharomyces genus, Enterococcus genus, Rhodococcus genus, Corynebacterium, Cornebactrium, Enterobacter, Lactobacillus, Micrococcus, Cryptococcus, Hansenula, i, Ogataa, Ogataa Rhodosporidium (Rhodosporid um), Rhodotorula genus, Trichosporon genus, Yamadazyma genus, Amycolatopsis genus, Alcaligenes genus, Althrobacter genus Examples include microorganisms belonging to the genus, the genus Comamonas, the genus Leuconostoc, the genus Microbacterium, the genus Proteus and the like. Preferably, those derived from Pseudomonas microorganisms are used, and those derived from Pseudomonas putida are used. For example, those derived from Pseudomonas putida ATCC 12633 can be used. For the mandelate oxidase derived from Pseudomonas putida ATCC12633, see Asterian R. et al. et al. , Biochemistry, 2004, 43, 183-1890, Isabelle E. et al. Lehoux et al, Biochemistry, 1999, 38, 5836-5848, Amy Y. et al. Tsuou et al, Biochemistry, 1990, 29, 9856-9862, Bharati Mitra et al. Biochemistry, 1993, 32, 12959-12967, Yang Xu et al. Biochemistry, 1999, 38 (38), 12367-12376, Narayanasami Sukumar et al. Biochemistry, 2001, 40 (33), 9870-9878, and the like.
Mandelic acid oxidase activity is caused by, for example, reacting a reaction solution containing 50 mM Tris-HCl buffer pH 7.5, 1 mM DCPIP (oxidized type), 1 mM mandelic acid and enzyme, and increasing the absorbance at 600 nm due to an increase in DCPIP (reduced type). It can be measured by measuring. 1 U of mandelic acid oxidase is the amount of enzyme that converts 1 μmol of mandelic acid into a benzoylformic acid derivative per minute.
(R) -Mandelic acid dehydrogenase sterically oxidizes a benzoylformic acid derivative using the coenzyme NADH to produce only the R form of mandelic acid or a mandelic acid derivative (FIG. 3). Mandelic acid oxidase may be derived from microorganisms, for example, Enterococcus, Pseudomonas, Candida, Saccharomyces, Rhodococcus, Cornebactrium, Enterobacter, Lactobacillus, Micrococcus, Cryptococcus, Hansenula, i, Ogataa, Ogataa Rhodosporidium (Rhodosporid um), Rhodotorula genus, Trichosporon genus, Yamadazyma genus, Amycolatopsis genus, Alcaligenes genus, Althrobacter genus Examples include microorganisms belonging to the genus, the genus Comamonas, the genus Leuconostoc, the genus Microbacterium, the genus Proteus and the like. Preferably, those derived from microorganisms of the genus Enterococcus are used, and those derived from Enterococcus faesalis are used. For example, those derived from Enterococcus faesalis IAM10071 can be used. Regarding (R) -mandelate dehydrogenase derived from Enterococcus faesalis IAM10071, JP 2004-65049, Yusuke Tamura et al. , Applied and Environmental Microbiology, 2002, 68 (2), 947-951, Yusuke Wada, et al. Biosci. Biotechnol. Biochem. 2008, 72 (4), 1087-1094, and the like.
(R) -Mandelate dehydrogenase activity is, for example, 50 mM Tris-HCl buffer pH 7.5, 1 mM NAD ⁺ It can be measured by reacting a reaction solution containing 1 mM mandelic acid and an enzyme and measuring an increase in absorbance at 340 nm due to an increase in NADH. 1 U is defined as the amount of enzyme that converts 1 μmol of mandelic acid into a benzoylformic acid derivative per minute.
A coenzyme regeneration system enzyme is an NAD that is converted to an oxidized form as the enzyme reaction progresses in an enzyme reaction that requires a reduced coenzyme such as NADH or HADPH. ⁺ An enzyme having the ability to convert a coenzyme such as NADP into a reduced form (referred to as coenzyme regeneration ability). Examples of the coenzyme regeneration system enzyme include glucose dehydrogenase (GDH) (FIG. 3), hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, and the like. Among these, glucose dehydrogenase is preferably used.
As the coenzyme regeneration system enzyme, those derived from microorganisms can be used, for example, Bacillus genus, Thiobacillus genus, Pseudomonas genus, Candida genus, Kloeckera genus, Pichia. Examples include microorganisms belonging to the genus (Pichia), the genus Lipomyces, the genus Moraxella, the genus Hyphomicrobium, the genus Paracoccus, the Ancylobacter, and the like. Preferably, those derived from microorganisms belonging to the genus Bacillus are used, and those derived from Bacillus megaterium are used. For example, glucose dehydrogenase derived from Bacillus megaterium IAM13481 can be used. For glucose dehydrogenase derived from Bacillus megaterium IAM13481, see S. et al. -H. Baik et al. , Appl. Microbiol. Biotechnol. , 2003, 61, 329-335, Tadashi Ema et al. Tetrahedron: Asymmetry, 2005, 16, 1075-1078, Tadashi Ema et al. , Tetrahedron, 2006, 62, 6143-6149, and the like.
Glucose dehydrogenase activity is, for example, 50 mM Tris-HCl buffer pH 7.5, 1 mM NAD ⁺ It can be measured by reacting a reaction solution containing 1 mM D-glucose and an enzyme and measuring an increase in absorbance at 340 nm due to an increase in NADH. 1 U is defined as the amount of enzyme that converts 1 μmol of glucose into gluconic acid per minute. The coenzyme regeneration 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.
Genes encoding these enzymes can be prepared based on known sequence information from the above-mentioned microorganisms using known methods such as a method using amplification means such as PCR, a method of chemically synthesizing, and the like. .
The sequence and amino acid sequence of DNA encoding mandelate oxidase derived from Pseudomonas putida ATCC12633 are respectively represented by SEQ ID NOs: 14 and 15, and the sequence and amino acid sequence of DNA encoding (R) -mandelate dehydrogenase derived from Enterococcus faesalis IAM10071 are sequenced, respectively. Nos. 16 and 17 show the DNA sequence and amino acid sequence encoding glucose dehydrogenase derived from Bacillus megaterium IAM13481, respectively, in SEQ ID Nos. 18 and 19, respectively.
In addition, it encodes a protein having a mandelate oxidase activity, which is capable of hybridizing with a DNA comprising a sequence complementary to the DNA comprising the nucleotide sequence represented by SEQ ID NO: 14 under the following stringent conditions: (R) -mandelate dehydrogenase activity which is capable of hybridizing with DNA comprising a sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 16 under the following stringent conditions Glucose dehydrogenase activity, which is capable of hybridizing under the following stringent conditions with a DNA encoding a protein having a DNA and a DNA complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 18 DNA encoding a protein having It is possible to have. Specifically, hybridization was performed at 68 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA was immobilized, and then a 0.1 to 2 fold concentration of SSC solution (with 1 fold concentration of SSC) was obtained. Is a condition that can be identified by washing at 68 ° C. using 150 mM NaCl and 15 mM sodium citrate). Alternatively, after transferring and immobilizing DNA on a nitrocellulose membrane by Southern blotting, hybridization buffer [50% formamide, 4 × SSC, 50 mM HEPES (pH 7.0), 10 × Denhardt's solution, 100 μg / ml salmon sperm DNA], which can form a hybrid by reacting overnight at 42 ° C. Furthermore, when calculated using BLAST or the like (for example, using default or initial setting parameters), the base sequence represented by SEQ ID NO: 14 is at least 85% or more, preferably 90% or more, more preferably 95 At least 85%, preferably 90% or more of the DNA encoding the protein having mandelate oxidase activity and the base sequence represented by SEQ ID NO: 16. More preferably 95% or more, particularly preferably 97% or more of the homologous DNA encoding a protein having (R) -mandelate dehydrogenase activity and at least the base sequence represented by SEQ ID NO: 18 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97 Has a higher homology can be used also DNA encoding a protein having a glucose dehydrogenase activity.
In the present invention, the genes encoding the above three types of enzymes are introduced into one type of microorganism to produce a recombinant microorganism containing the genes encoding the three types of enzymes.
A recombinant microorganism may be obtained by inserting a gene encoding the above enzyme into an expression vector and transforming the microorganism using the expression vector.
Combinations for inserting genes encoding three types of enzymes into expression vectors include the following combinations, and any combination can be taken.
(1) Each of the genes encoding the three types of enzymes is separately inserted into three expression vectors.
(2) Of the genes encoding the three types of enzymes, two types of genes are simultaneously inserted into one expression vector (two gene expression vectors), and the remaining one type of gene is inserted into another expression vector. In this case, the combination of two types of genes inserted simultaneously into one expression vector can be a combination of (R) -mdh gene and gdh gene, mox gene and gdh gene, and (R) -mdh gene and mox gene. . It is desirable that the activity of (R) -MDH, which requires a coenzyme, and GDH to regenerate the coenzyme are approximately equal. Therefore, preferably, the (R) -mdh gene and the gdh gene that regenerates the coenzyme are co-expressed in one expression vector, and inserted into one expression vector.
(3) A gene encoding three kinds of enzymes is inserted into one expression vector (three gene expression vector).
When inserting a plurality of genes into one expression vector, the expression may be controlled by separate promoters, or a plurality of genes may be linked downstream of one promoter and a plurality of genes may be expressed by controlling one promoter. You may let them. Preferably, the expression of each gene is controlled by different promoters. At this time, an expression vector containing a plurality of multiple cloning sites may be used.
The expression vector for inserting the gene encoding the enzyme is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA.
Plasmid DNA includes plasmids derived from E. coli (for example, pUC vectors such as pUC19, pUC18, pUC118, and pUC119, pET vectors such as pET3a to 3d, pET9a to 9d, pET11a to 11d, and pETpBR322, pBR vectors such as pACYC vector and pBR325) ), Plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), plasmids derived from yeast (eg, YEp13, YEp24, YCp50, etc.), and the like. Phage DNA includes λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, DNA viruses or RNA viruses such as detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, poxviruses, polioviruses, synbisviruses, Sendai viruses, SV40, immunodeficiency viruses (HIV), etc. Insect virus vectors such as animal viruses such as pCI-neo, pcDNA3, and pZeoSV, and baculovirus can also be used.
To insert an enzyme-encoding gene into an expression vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and ligated to the expression vector. Method is adopted. In the case where genes encoding two or three enzymes are simultaneously inserted into one expression vector for co-expression, an expression vector containing a plurality of multiple cloning sites may be used. Examples of the expression vector containing a plurality of multicloning sites include pACYC Duet-1.
A gene encoding an enzyme needs to be incorporated into an expression vector so that the function of the gene is exhibited. Therefore, the expression vector to be used includes a promoter, a gene encoding the above enzyme, a cis element such as an enhancer, a splice donor site present on the 5 ′ end side of the intron, and a splice present on the 3 ′ end side of the intron. Those containing a splicing signal consisting of a receptor site, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like can be operably linked. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
A transformant can be obtained by introducing a recombinant vector into which a gene encoding an enzyme is inserted into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. Examples include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, and the genus Pseudomonas such as Pseudomonas putida, and Saccharomyces c. Examples thereof include yeast such as Schizosaccharomyces pombe, animal cells such as COS cells and CHO cells, and insect cells such as S121.
When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and at the same time is composed of a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Moreover, the gene which controls a promoter may be contained.
Examples of Escherichia coli include Escherichia coli DH1 and BL21. Examples of Bacillus subtilis include, but are not limited to, Bacillus subtilis.
Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example trp promoter, lac promoter, P _L Promoter, P _R A promoter derived from E. coli or phage, such as a promoter, is used. An artificially designed and modified promoter such as a tac promoter may be used.
The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions [Cohen, S .; N. et al. : Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)], electroporation method and the like.
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like. Can be used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, the electroporation method [Becker, D. et al. M.M. et al. : Methods. Enzymol. 194: 182 (1990)], spheroplast method [Hinnen, A. et al. et al. : Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method [Itoh, H. et al. : J. Bacteriol. , 153: 163 (1983)].
When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like may be used, and human cytomegalovirus early gene promoter and the like may be used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
When insect cells are used as hosts, S121 cells and the like are used. Examples of the method for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.
When bacteria such as E. coli are used as a host, the codons may be corrected in consideration of the codon usage frequency and GC content of the bacteria.
By culturing under conditions that allow expression of the gene encoding the enzyme into which the transformed host has been introduced, three types of enzymes are expressed in the host. By adding mandelic acid or a derivative thereof as a substrate, a substrate of a coenzyme regeneration system enzyme and a coenzyme, a catalytic reaction of each enzyme occurs, a deracemization reaction occurs, and mandelic acid and a derivative thereof are generated. At this time, after sufficient expression of the enzyme, mandelic acid or a derivative thereof, a substrate of a coenzyme regeneration system enzyme and a coenzyme may be added, or when culturing a host containing a gene encoding the enzyme, mandel An acid or a derivative thereof, a substrate of a coenzyme regeneration system enzyme, and a coenzyme may be added to cause the enzyme expression and the enzyme reaction to be performed simultaneously.
A method for culturing a transformant host containing a gene encoding an enzyme is performed according to a usual method used for culturing a host.
As a medium for culturing transformants obtained using microorganisms such as E. coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and the transformants are efficiently cultured. As long as the medium can be used, either a natural medium or a synthetic medium may be used. For example, when culturing Escherichia coli, LB medium, 2 × YT medium or the like may be used.
Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like.
Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the Lac promoter, a microorganism transformed with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like with an expression vector using the trp promoter is cultured. Sometimes indole acetic acid (IAA) or the like may be added to the medium.
As a medium for culturing a transformant obtained using animal cells as a host, a generally used RPMI 1640 medium, DMEM medium, a medium obtained by adding fetal calf serum or the like to these mediums, or the like is used.
Also, optically active mandelic acid and its derivatives can be produced by reacting a culture of a transformant host containing three kinds of enzymes with a substrate. Here, the “culture” means either a culture supernatant, a cultured cell or a disrupted cell.
Cultivation and enzyme reaction are usually carried out at a temperature of 20 to 40 ° C. and a pH of 6.0 to 9.0 for several hours to several days under aerobic conditions such as shaking culture or aeration stirring culture. The pH of the medium may be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.
For example, by adding 0.5 g of a microorganism containing three kinds of enzymes by wet weight and adding 10 mM to 100 mM of mandelic acid or a derivative thereof, all the substrates react by culturing for several to 24 hours, An optically active mandelic acid or derivative thereof having an optical purity of 99% ee or higher can be obtained in a yield of 100%.
A person skilled in the art can determine the addition amount of the microorganism and the substrate as appropriate, and produce optically active mandelic acid at a desired scale.
Furthermore, an optically active mandelic acid can be produced by immobilizing microorganisms and performing a deracemization reaction.
Usually, holding a biocatalyst such as an enzyme, a microorganism, or an animal or plant cell on a suitable insoluble carrier is called immobilization, and the immobilized one is called an immobilized biocatalyst. The greatest advantage of immobilizing the biocatalyst is that it can be used repeatedly or continuously. It is also possible to enhance the stability of the biocatalyst by appropriate immobilization. Furthermore, the following various effects can be expected by enzyme immobilization.
(I) Improvement of optimum reaction temperature and thermal stability
(Ii) Substrate affinity (K _m Value) and maximum reaction rate (V _max Value)
(Iii) Expansion of optimal pH range and improved pH stability
(Iv) Stability improvement and activity expression in organic solvents
(V) Changes in substrate specificity, reaction specificity, reaction position specificity, stereospecificity
(Vi) Changes in cofactor requirement and allosteric regulation
(Vii) Improved resistance to degradation by proteolytic enzymes (proteases)
In particular, the present invention uses a transformed microorganism into which three kinds of enzymes have been introduced at the same time, so that a product can be obtained efficiently. Regarding the specific effect of (ii), the reaction is completed in 3 to 22 hours of culture, for example, 3 hours when the substrate concentration is 50 mM, 18 hours when the substrate concentration is 100 mM, and 22 hours when the substrate concentration is 385 mM. In addition, an optically active mandelic acid or derivative thereof having an optical purity of 99% ee or higher can be obtained in a yield of 100%. Moreover, the reaction at a high concentration of 385 mM is possible, and a yield: 99% and an optical purity: 100% can be realized. In the conventional method for producing optically active mandelic acid using one kind of microorganism (Japanese Patent Laid-Open No. 6-7196), a long culture period of 40 hours is required, and the substrate concentration cannot be as high as that of the present invention. . Also, it was impossible to achieve an optical purity of 100%. The present invention makes it possible to produce optically active mandelic acid more efficiently than conventional methods using microorganisms.
The immobilization of microorganisms is roughly classified into a case where a stationary microorganism without growth is immobilized, and a case where a microorganism showing proliferation in a living state is immobilized. They are referred to as an immobilized resting cell and an immobilized growing cell, respectively. Immobilized static cells are applied when a specific intracellular enzyme is used. In the industrialization of bioreactors that use microorganisms as biocatalytic elements, immobilized stationary cells are often used. This is because, when an enzyme is used, not only a complicated operation of crushing and removing the microbial cells is required, but also the stability of the enzyme is reduced by taking it out from the microbial cells. The carrier binding method and the entrapment method are used for immobilization of stationary cells. In the carrier binding method, a method of holding the carrier directly or via a prepolymer or polymer has been proposed. In the inclusion method, a method of holding in a polymer gel is used.
The produced optically active mandelic acid or derivative thereof can be isolated from the reaction solution by a general separation and purification method. For example, insoluble substances such as host cells are removed from the reaction solution by centrifugation, the pH of the reaction solution is adjusted to acidity, extracted with a solvent such as ethyl acetate, dehydrated, vacuum-dried or recrystallized. It can be isolated and purified by performing.
The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
enzyme
The enzymes used in the following examples are shown in Table 1.

Primer
Table 2 shows the base sequences of primers used for PCR in the following examples.

E. coli transformation
In the following examples, E. coli BL21-Gold (DE3) was transformed by the heat shock method, and E. coli JM109 was transformed by the electroporation method. When using two types of expression plasmids, a competent cell for electroporation of E. coli transformed with one type of expression plasmid was prepared, and further transformed with another type of expression plasmid. .
Measurement of enzyme activity
Furthermore, the enzyme activity of MOX, (R) -MDH and GDH was measured by the following method.
MOX activity measurement
1 U was defined as the amount of enzyme that converted 1 μmol of mandelic acid or o-chloromandelic acid into a benzoylformic acid derivative per minute. In a 1 cm cell with an optical path length, 1 mM (RS) -mandelic acid or o-chloromandelic acid, 1 mM DCPIP (oxidized form), a sample of the solubilized fraction, 50 mM Tris-HCl buffer (pH 7.5) and distilled water, The mixture was mixed to 1 ml, and the decrease in absorbance at 600 nm where DCPIP (oxidized type) had absorption was measured (FIG. 4).
(R) -MDH activity measurement
1 U was defined as the amount of enzyme that converted 1 μmol of mandelic acid or o-chloromandelic acid into a benzoylformic acid derivative per minute. 1 mM (RS) -mandelic acid or o-chloromandelic acid, 1 mM NAD in a 1 cm optical path length cell ⁺ A sample of the soluble fraction, 50 mM Tris-HCl buffer (pH 7.5) and distilled water were mixed so that the total amount was 1 ml, and the increase in absorbance at 340 nm where NADH had absorption was measured (FIG. 5).
GDH activity measurement
1 U was defined as the amount of enzyme that converts 1 μmol of glucose into gluconic acid per minute. 1mM D-glucose, 1mM NAD in 1cm cell ⁺ The sample of the soluble fraction, 50 mM Tris-HCl buffer (pH 7.5) and distilled water were mixed so that the total amount became 1 ml, and the increase in absorbance at 340 nm where NADH had absorption was measured (FIG. 6).
In the following expression study, the culture solution was collected by centrifuging at 8,000 rpm for 10 minutes, and the wet cells after removing the supernatant were suspended in 50 mM Tris-HCl buffer (pH 7.5). Washed by centrifuging at 1,000 rpm for 10 minutes. The supernatant was again removed, suspended in 50 mM Tris-HCl buffer (pH 7.5) so that the wet cell mass was 10%, and sonicated twice for 5 minutes. Then, the crushed liquid was centrifuged at 12,000 rpm for 30 minutes to obtain a soluble fraction (CFE: cell-free extract) as a supernatant and an insoluble fraction as a precipitate. These samples and the crushing liquid were used for SDS-PAGE. Moreover, the sample of the soluble fraction was used for the enzyme activity measurement of (R) -MDH and GDH. Further, the precipitate obtained as an insoluble fraction was suspended in 50 mM Tris-HCl buffer (pH 7.5) and washed by centrifugation at 12,000 rpm for 10 minutes. The supernatant was removed, and the precipitate obtained as an insoluble fraction was solubilized by suspending in a solubilization buffer so that the precipitate was 10%. And the insoluble matter was removed by centrifuging the suspension at 12,000 rpm for 30 minutes to obtain a solubilized fraction. This sample was used for measuring the enzyme activity of MOX.

Construction of Oxidation Factory In this example, the oxidation factory refers to E. coli into which the MOX gene has been introduced and expressed. An oxidation factory was constructed as the first stage of deracemization factory construction, and an S selective oxidation reaction of mandelic acid was attempted (FIG. 2).
(1) Expression of MOX The Pseudomonas putida ATCC12633-derived mox gene was cloned into the EcoR I site of the vector pUC19 to construct pASA1 (FIG. 7), a plasmid for MOX expression.
Escherichia coli JM109 was transformed with this pASA1, and the expression of MOX was induced by culturing with the addition of IPTG. Moreover, the same experiment was conducted using the vector pUC19 as a control. After completion of the culture, protein expression was confirmed by SDS-PAGE, and the activity of MOX against mandelic acid was measured by the above method. At this time, since MOX is an insoluble membrane protein, the fraction obtained by solubilizing the insoluble fraction with a surfactant was used for the activity measurement. As a result, about 40 kDa MOX was expressed in the insoluble fraction by pASA1 (FIG. 8), and the enzyme activity was 24 U / g wet cell (control: 0 U). In this way, MOX was successfully expressed. Hereinafter, the oxidation factory refers to E. coli that has induced the expression of MOX under the above-described conditions.
(2) Reaction by oxidation factory Next, using an oxidation factory, 10 mM (RS) -mandelic acid, wet bacterial cell (oxidation factory) 0.5 g, 50 mM Tris-HCl buffer (pH 7.5), distilled water, The mixture was mixed so that the total amount became 50 ml, put into a 500-ml Erlenmeyer flask, swirled at 30 ° C. and 180 rpm, and subjected to S selective oxidation of mandelic acid. Then, the progress of the reaction was analyzed by TLC. At this time, since mandelic acid as a substrate and benzoylformic acid as a product are difficult to separate, a methyl esterified sample was used for analysis in order to facilitate the separation. As a result, it was suggested that some mandelic acid was converted to benzoylformic acid by the oxidation factory 17 hours after the start of the reaction (FIG. 9). Therefore, methyl benzoylformate and methyl mandelate were fractionated by PLC, and the structure of each was measured by ¹ H-NMR, and the optical purity of methyl mandelate was measured by HPLC. As a result, it was found that the product was benzoylformic acid and the residual substrate was 100% ee (R) -mandelic acid. In this way, the oxidation factory succeeded in proceeding the S selective oxidation reaction of the target mandelic acid.

Construction of Reduction Factory The reduction factory in this example refers to Escherichia coli in which (R) -MDH and GDH genes have been introduced and co-expressed. A reduction factory was constructed as the second stage of deracemization factory construction, and an R-selective reduction reaction of benzoylformic acid was attempted (FIG. 3).
(1) Construction of pET3aRMDH pET3aRMDH (FIG. 10) was constructed as a plasmid for expression of (R) -MDH. pET3aRMDH was constructed by cloning the (R) -mdh gene region derived from Enterococcus faecalis IAM 10071 into the Nde I and Bam HI sites of the vector pET-3a.
First, PCR was performed under the conditions shown in Table 3 to amplify from the 5 ′ end of the (R) -mdh gene to 120 bases downstream of the 3 ′ end. In addition, primer1 is obtained by adding an adapter sequence containing a restriction enzyme NdeI site to a sequence specific to the 5 ′ end of the (R) -mdh gene with a corrected codon. Primer 2 is obtained by adding an adapter sequence containing a restriction enzyme BamHI site to a sequence specific to the downstream region at the 3 ′ end of the (R) -mdh gene.

Next, the obtained PCR product (insert) and vector pET-3a were digested with restriction enzymes Nde I and BamH I under the conditions shown in Table 4.

Subsequently, a ligation reaction was performed on the obtained two kinds of DNA fragments under the conditions shown in Table 5.

After the ligation reaction, E. coli TOP10 was transformed using the reaction solution, and colony PCR was performed on the grown colonies as follows. First, a colony on a plate was suspended in 30 μl of sterile distilled water using a toothpick, heated at 98 ° C. for 5 minutes, and centrifuged at 12,000 rpm for 1 minute to obtain a template DNA solution. Next, PCR was performed under the conditions shown in Table 6 using this template DNA solution.

Then, a plasmid was extracted from a colony (FIG. 11) in which a DNA fragment of the desired size was amplified, and the nucleotide sequence was analyzed by sequencing. In sequencing, a T7 promoter primer or a T7 terminator primer specific to the vector pET-3a was used.
At this time, in order to optimize the expression of the target protein, correction was made for codons and GC contents which are infrequently used. The sequence of 24 bases at the 5 ′ end of the (R) -mdh gene was analyzed. As a result, it was found that there was one codon that was infrequently used and one codon that could be replaced with a codon with a low GC content (FIG. 12). Therefore, these codons were corrected to codons that are frequently used in E. coli and have a low GC content.
Successful construction of the plasmid was confirmed by colony PCR (FIG. 11) and sequencing.
(2) Cloning and Expression of (R) -MDH The constructed pET3aRMDH was used to transform E. coli BL21-Gold (DE3), and expression of (R) -MDH was induced by culturing with addition of IPTG. In addition, a similar experiment was performed using the vector pET-3a as a control. After completion of the culture, protein expression was confirmed by SDS-PAGE, and the activity of (R) -MDH against mandelic acid was measured by the above method. As a result, about 35 kDa (R) -MDH was expressed by pET3aRMDH (FIG. 13), and the enzyme activity was 74 U / g wet cell (control: 0 U). In this way, (R) -MDH was successfully expressed.
(3) Construction of pACYCGDH First, pACYCGDH (FIG. 14) was constructed as a GDH expression plasmid. pACYCGDH was constructed by cloning the Bacillus megaterium IAM 13418-derived gdh gene into the Nde I and Kpn I sites of MCS (Multiple Cloning Sites) 2 of the vector pACYCDuet-1.
First, PCR was performed under the conditions shown in Table 7 to amplify from the 5 ′ end to the 3 ′ end of the gdh gene. Primer 3 is obtained by adding an adapter sequence containing a restriction enzyme Nde I site to a sequence specific to the 5 ′ end of the gdh gene. Primer 4 is obtained by adding an adapter sequence containing a restriction enzyme Kpn I site to a sequence specific to the 3 ′ end of the gdh gene.

Next, the obtained PCR product (insert) and the vector pACYCDuet-1 were digested with the restriction enzymes NdeI and then KpnI under the conditions shown in Table 8.

Subsequently, a ligation reaction was performed on the obtained two types of DNA fragments under the conditions shown in Table 9.

After the ligation reaction, E. coli TOP10 was transformed using the reaction solution, and colony PCR was performed on the grown colonies as follows. First, a template DNA solution was prepared as in the previous section. Next, PCR was performed using this template DNA solution under the conditions shown in Table 10.

Then, a plasmid was extracted from a colony (FIG. 15) in which a DNA fragment of the desired size was amplified, and the nucleotide sequence was analyzed by sequencing. In sequencing, a DuetUP2 primer or a T7 terminator primer specific for MCS2 of the vector pACYCDuet-1 was used.
Successful construction of the plasmid was confirmed by colony PCR (FIG. 15) and sequencing.
(4) Cloning and expression of GDH Escherichia coli BL21-Gold (DE3) was transformed with pACYCGDH, and expression of GDH was induced by culturing with addition of IPTG. Moreover, the same experiment was performed using the vector pACYCDuet-1 as a control. After completion of the culture, protein expression was confirmed by SDS-PAGE, and the activity of GDH was measured by the above method. As a result, about 30 kDa GDH was expressed by pACYCGDH (FIG. 16), and the enzyme activity was 6.9 U / g wet cell (control: 0 U). In this way, GDH expression was successful.

(R) Co-expression study of MDH and GDH (R) -MDH was successfully expressed with the expression plasmid pET3aRMDH, and GDH was successfully expressed with the expression plasmid pACYCGDH. Then, next, co-expression examination of these two kinds of proteins was performed. As the method, co-expression with two types of expression plasmids and co-expression with one type of expression plasmid were tried (FIG. 17), and the results were compared.
(1) Co-expression with two types of expression plasmids (FIG. 17A)
For co-expression with two types of expression plasmids, the (R) -MDH expression plasmid pET3aRMDH and the GDH expression plasmid pACYCGDH constructed so far were used. E. coli BL21-Gold (DE3) was transformed with these plasmids, and (R) -MDH and GDH co-expression was induced by culturing with the addition of IPTG. In addition, similar experiments were performed using vectors pET-3a and pACYCDuet-1 as controls. After completion of the culture, protein expression was confirmed by SDS-PAGE, and (R) -MDH and GDH activities were measured by the above methods. As a result, about 35 kDa (R) -MDH was expressed by pET3aRMDH, and the enzyme activity against mandelic acid was 250 U (control: 0 U) per 1 g of wet cells. Moreover, about 30 kDa GDH was expressed by pACYCGDH, and the enzyme activity was 2.8 U / mg (control: 0 U) per 1 g of wet cells (FIG. 18).
(2) Co-expression with one expression plasmid (FIG. 17B)
(I) Construction of pACYCMG For co-expression with two types of expression plasmids, pACYCMG was newly constructed as a plasmid for co-expression of (R) -MDH and GDH (FIG. 19). pACYCGDH was constructed by cloning the (R) -mdh gene region into the Nco I and Not I sites of MCS1 of the vector pACYCDuet-1 and the gdh gene into the Nde I and Kpn I sites of MCS2. The plasmid pACYCMG for co-expression of (R) -MDH and GDH was constructed as follows.
First, PCR was performed under the conditions shown in Table 11 to amplify from the 5 ′ end of the (R) -mdh gene to 120 bases downstream of the 3 ′ end. In addition, primer5 is obtained by adding an adapter sequence containing a restriction enzyme NcoI site to a sequence specific to the 5 ′ end of the (R) -mdh gene with a corrected codon. Primer 6 is obtained by adding an adapter sequence containing a restriction enzyme Not I site to a sequence specific to the downstream region at the 3 ′ end of the (R) -mdh gene.

Next, the obtained PCR product (insert) and the GDH expression plasmid pACYCGDH (vector) constructed in the previous section were digested with the restriction enzymes Nco I and Not I under the conditions shown in Table 12.

Subsequently, a ligation reaction was performed on the obtained two kinds of DNA fragments under the conditions shown in Table 13.

After the ligation reaction, E. coli TOP10 was transformed using the reaction solution, and colony PCR was performed on the grown colonies as follows. First, a template DNA solution was prepared as in the previous section. Next, PCR was performed using this template DNA solution under the conditions shown in Table 14.

Then, a plasmid was extracted from a colony (FIG. 19) in which a DNA fragment of the desired size was amplified, and the nucleotide sequence was analyzed by sequencing. In sequencing, an ACYCDuetUP1 primer or DuetDOWN1 primer specific for MCS1 of the vector pACYCDuet-1 was used.
However, if the plasmid constructed in this way is used for expression as it is, the translation start position from mRNA to protein is not the start codon (ATG) of the (R) -mdh gene but the start codon contained in the Nco I site. , The frame of the codon will shift. Therefore, a 4-base deletion mutation was introduced by the method shown in FIG.
First, the entire plasmid was amplified with the reaction solution composition and reaction conditions shown in Table 15, and a deletion mutation was introduced. In addition, primer7 is obtained by adding 11 bases before and 23 bases after the deletion mutation at the center. Primer8 is a complementary strand of primer7.
Successful construction of the plasmid was confirmed by colony PCR (FIG. 21) and sequencing.

Next, restriction enzyme Dpn I was added to the reaction solution and incubated at 37 ° C. for 1 hour to cleave the template DNA. Dpn I is a restriction enzyme that recognizes and cleaves the 4-base sequence GATC in which A is methylated. The template DNA grows in E. coli and is cleaved because it is methylated, but the PCR product is not cleaved because it is not methylated.
Finally, E. coli TOP10 was transformed, a plasmid was extracted from the grown colonies, and the nucleotide sequence was analyzed by sequencing. In the sequencing, the ACYCDuetUP1 primer or DuetDOWN1 primer was used as described above.
(Ii) Co-expression with one type of expression plasmid Escherichia coli BL21-Gold (DE3) was transformed with pACYCMG, and co-expression of (R) -MDH and GDH was induced by culturing with the addition of IPTG. Moreover, the same experiment was performed using the vector pACYCDuet-1 as a control. After completion of the culture, protein expression was confirmed by SDS-PAGE, and (R) -MDH and GDH activities were measured by the above methods. As a result, about 35 kDa (R) -MDH was expressed by pACYCMG, and the enzyme activity against mandelic acid was 67 U per 1 g of wet cells (control: 0 U). Moreover, about 30 kDa GDH was also expressed, and the enzyme activity was 6.7 U (control: 0 U) per 1 g of wet cells (FIG. 22).
(3) Comparison of the results of (1) and (2) (1) The results of co-expression with two types of plasmids and (2) co-expression with one type of plasmids are summarized in Table 16.

In order to efficiently rotate the coenzyme and advance the reaction in the reduction factory, it is desirable that the (R) -MDH activity and the GDH activity are comparable. Table 16 shows that GDH activity is lower than (R) -MDH activity in either method, but the difference is smaller in (2) than in (1). Moreover, as a result of actually trying the R selective reduction reaction of benzoylformic acid using the soluble fraction (CFE: cell-free extract) prepared in (1) and (2) and analyzing the progress of the reaction by TLC, 15 hours after the start of the reaction, the substrate remained in (1), but it was suggested that the reaction was sufficiently advanced in (2) (FIG. 23). From these results, the method (2) was adopted for co-expression of (R) -MDH and GDH. Hereinafter, the reduction factory refers to E. coli in which the co-expression of (R) -MDH and GDH is induced under the above conditions by using one type of expression plasmid pACYCMG.
Here, the difference in the expression level will be considered. From Table 16, it can be seen that in (1) there is a considerable difference in the expression levels of (R) -MDH and GDH, but in (2) there is little difference. This is considered to be related to the copy number of the vector used for the expression plasmid. First, in (1), (R) -MDH and GDH have different expression plasmids, and the copy number of the vector used for the expression plasmid is higher than that of GDH pACYCDuet-1 (R) -MDH pET-3a. There are more (Table 16). Therefore, (R) -MDH is preferentially expressed over GDH, and it is considered that the expression level is considerably different. In (2), the expression plasmids for (R) -MDH and GDH are the same. Therefore, it is considered that there was almost no difference in the expression level.

Reaction by reduction factory Using a reduction factory, 10 mM benzoylformic acid, 20 mM D-glucose, 0.1 mM NADH, wet cell (reduction factory) 0.5 g, 50 mM Tris-HCl buffer (pH 7.5) and distilled water, The mixture was mixed to a total volume of 50 ml, placed in a 500-ml Erlenmeyer flask, and swirled at 30 ° C. and 180 rpm to attempt R-selective reduction of benzoylformic acid. At this time, the reaction is considered to proceed by NADH in Escherichia coli. However, in order to promote the reaction, the literature (Tadashi Ema et al, Tetrahedron: 2005, 16, 1075-1078: Tadashi Ema et al., Tetrahedron, 2006). 62, 6143-6149), it was decided to add a catalytic amount of NADH. In the study described in this document, asymmetric reduction of ketones by E. coli co-expressed by introducing carbonyl reductase and GDH genes was achieved, but the reaction was promoted by addition of a catalytic amount of coenzyme. It is shown. As a result of analyzing the progress of the reaction by TLC, it was suggested that all the benzoylformic acid was converted to mandelic acid by the reduction plant 17 hours after the start of the reaction (FIG. 24). Therefore, methyl mandelate was fractionated by PLC, the structure was measured by ¹ H-NMR, and the optical purity was measured by HPLC. As a result, the product was found to be 100% ee (R) -mandelic acid. In this way, the reduction factory succeeded in proceeding the R selective reduction reaction of the target benzoylformic acid.

Construction of Deracemization Factory The deracemization factory refers to E. coli that has been introduced and co-expressed with MOX, (R) -MDH, and GDH genes. As the third stage of deracemization factory construction, a deracemization factory was constructed by combining an oxidation factory and a reduction factory, and a deracemization reaction of mandelic acid was attempted (Fig. 1).
(1) Co-expression of MOX with (R) -MDH and GDH MOX was successfully expressed with the expression plasmid pASA1. (R) -MDH and GDH were successfully co-expressed with the co-expression plasmid pACYCMG. Then, next, combining these, examination of co-expression of three types of proteins using two types of expression plasmids was performed (FIG. 25).
E. coli BL21-Gold (DE3) was transformed with the MOX expression plasmid pASA1 constructed so far and the (R) -MDH and GDH co-expression plasmid pACYCMG, and IPTG was added to the MOX ( R) -MDH and GDH co-expression was induced. In addition, similar experiments were performed using vectors pET-3a and pACYCDuet-1 as controls. After completion of the culture, protein expression was confirmed by SDS-PAGE, and the activities of MOX, (R) -MDH, and GDH were measured by the above methods. As a result, about 40 kDa MOX was expressed in the insoluble fraction by pASA1, and the enzyme activity was 9.9 U / g wet cell (control: 0 U). Furthermore, about 35 kDa (R) -MDH was expressed by pACYCMG, and the enzyme activity against mandelic acid was 290 U per 1 g of wet cells (control: 0 U / mg). Moreover, about 30 kDa GDH was also expressed, and the enzyme activity was 100 U (control: 0 U) per 1 g of wet cells (FIG. 26). Thus, the co-expression of MOX, (R) -MDH, and GDH was successful. Hereinafter, the deracemization plant refers to E. coli that has induced the co-expression of MOX, (R) -MDH, and GDH under the above conditions.
(2) Reaction by the deracemization factory Using the deracemization factory, 10 mM (RS) -mandelic acid, 20 mM D-glucose, 0.1 mM NADH, wet cells (deracemization factory) 0.5 g, 50 mM Tris-HCl buffer (PH 7.5) and distilled water were mixed so that the total amount would be 50 ml, put into a 500-ml Erlenmeyer flask, and swirled at 30 ° C. and 180 rpm to try a deracemization reaction of mandelic acid. As a result of analyzing the progress of the reaction by TLC, only mandelic acid was detected and benzoylformic acid was not detected 17 hours after the start of the reaction (FIG. 27). Mandelic acid is both a substrate and a product, and benzoylformic acid is an intermediate, suggesting two possibilities. One is the possibility that the reaction did not proceed at all. The other is the possibility that all the benzoylformic acid produced in the first stage oxidation reaction was converted to mandelic acid in the second stage reduction reaction. In order to verify this, methyl mandelate was fractionated by PLC, the structure was measured by ¹ H-NMR, and the optical purity was measured by HPLC. As a result, the product was found to be 100% ee (control: 0% ee) (R) -mandelic acid. That is, all (S) -mandelic acid in (RS) -mandelic acid was converted to (R) -mandelic acid. In this way, the deracemization plant succeeded in proceeding the deracemization reaction of the target mandelic acid.
(3) Time-dependent change of reaction by deracemization factory The time-dependent change of the deracemization reaction of mandelic acid by the deracemization factory was measured. The reaction solution having the composition shown in Table 17 was placed in a 500-ml Erlenmeyer flask and swirled at 30 ° C. and 180 rpm.

The reaction was attempted using 0.5 g of wet cells at substrate concentrations of 10 mM, 50 mM, and 100 mM, and the yield of mandelic acid and benzoylformic acid during the reaction and the optical purity of mandelic acid were measured by HPLC (FIGS. 28 to 30). ). In addition, 1 g of wet cells corresponds to 200 ml culture. 28 to 30, the reaction was completed in about 3 hours at substrate concentrations of 10 mM and 50 mM, and about 18 hours at 100 mM, and the yield of mandelic acid reached 99% or more and the optical purity reached 100% ee at all substrate concentrations. You can see that However, the number of data is insufficient to show changes with time at substrate concentrations of 10 mM and 50 mM. Therefore, the change with time at 100 mM with a sufficient number of data will be considered. In the first 3 hours, the yield of mandelic acid was 69% of the minimum value, the yield of benzoylformic acid was 28% of the maximum value, and the optical purity of mandelic acid was 100% ee of the maximum value. In the subsequent 15 hours, the yield of mandelic acid was 100%, the yield of benzoylformic acid was 1%, and the optical purity of mandelic acid remained 100% ee. From this, the first three hours of oxidation reaction proceeded at a faster rate than the second stage reduction reaction, and benzoylformic acid accumulated, and the subsequent 15 hours, only the reduction reaction proceeded. I understand. Therefore, it can be said that the reduction reaction is a rate-limiting step. As the cause, it is considered that the activity of MOX related to the oxidation reaction is higher than the activities of (R) -MDH and GDH related to the reduction reaction in the wet cells. However, as described above, the enzyme activity per 1 g of wet cells in the deracemization plant is 9.9 U for MOX, 290 U for (R) -MDH, and 100 U for GDH, which seems to contradict this idea. Here, considering the sample used for the activity measurement, the following can be considered. First, with respect to MOX, which is a membrane protein, since a sample of a solubilized fraction was used, the activity may be lower than that in wet cells for two reasons. The first reason is a decrease in the amount of enzyme. When the solubilization of MOX was confirmed by SDS-PAGE, it was found that it was contained not only in the solubilized fraction but also in the precipitate, and not all was solubilized (FIG. 31). The second reason is a decrease in enzyme activity. Since MOX solubilization was performed by denaturation using a surfactant, it is possible that not only the membrane binding site but also the conformation of the active site was changed to reduce the activity. Thus, it is difficult to accurately measure the activity of membrane proteins in wet cells. On the other hand, (R) -MDH and GDH, which are cytoplasmic proteins, are considered to have almost the same activity as in wet cells because samples of the soluble fraction were used. From these facts, it is highly likely that the activity of MOX related to the oxidation reaction is actually higher than that of (R) -MDH and GDH related to the reduction reaction in the wet cells. Furthermore, when the activities of (R) -MDH and GDH are compared, the rate-limiting enzyme of the reduction reaction is considered to be GDH.

Utilization of deracemization factory In order to make the deracemization factory constructed according to the above examples more valuable, the reaction at a high substrate concentration, the reaction with immobilized stationary cells, the deracemization of o-chloromandelic acid, I tried to use it for the chemical reaction.
(1) Reaction at High Substrate Concentration The goal was to sufficiently proceed the mandelic acid deracemization reaction at the deracemization plant at a substrate concentration of 500 mM. For this purpose, first, a reaction was attempted using 0.5 g of wet bacterial cells in the same manner as in the above example, and the change with time was measured (FIG. 32).
The reaction solution having the composition shown in Table 18 was placed in a 500-ml Erlenmeyer flask and swirled at 30 ° C. and 180 rpm.

As a result, the reaction stopped after about 30 hours, the yield of mandelic acid was 72%, and the optical purity remained at 55% ee. Then, in order to increase the reaction rate, it was decided to increase the amount of wet cells. The reaction was attempted with a wet cell mass of 2.5 g (5 times) and 5 g (10 times), and changes over time were measured (FIGS. 33 and 34). As a result, when the amount of wet cells was increased 5 times, the yield of mandelic acid increased to 76% and the optical purity increased to 77%, but even if the amount of wet cells was increased further, the yield and optical purity of mandelic acid were increased. Did not rise. This is thought to be due to the nature of general enzymes. When the substrate concentration is kept in a large excess, the reaction rate is proportional to the enzyme concentration while the enzyme concentration is low, but as the enzyme concentration increases, the reaction rate gradually approaches a constant value. In addition, it is considered that the cause of the reaction being stopped in the middle is that the effects of substrate inhibition, product inhibition, and deactivation of each of the three types of enzymes are intertwined in a complicated manner. Therefore, these matters need to be analyzed in detail for further consideration. Thus, the reaction could not proceed sufficiently at a substrate concentration of 500 mM. Therefore, finally, a reaction was attempted using 10 g of wet cells at a substrate concentration of 385 mM, and the change with time was measured (FIG. 35). As a result, the reaction was completed in about 22 hours, the yield of mandelic acid reached 99% or more, the optical purity reached 100% ee, and the yield of benzoylformic acid remained at 5%. In this way, the deracemization reaction of mandelic acid by the deracemization factory was successfully advanced at a substrate concentration of 385 mM. The substrate concentration of 385 mM corresponds to 59 g / l.
(2) Deracemization reaction of o-chloromandelic acid The time-dependent change of the deracemization reaction of o-chloromandelic acid by the deracemization factory was measured. The reaction was attempted at substrate concentrations of 10 mM and 100 mM using 0.5 g of wet cells, and the yield and optical purity of o-chloromandelic acid during the reaction were measured by HPLC (FIGS. 36 and 37). As a result, the reaction was completed in about 4 hours at a substrate concentration of 10 mM and about 21 hours at 100 mM, and the yield of o-chloromandelic acid reached 100% and the optical purity reached 99% ee or higher at both substrate concentrations. After completion of the reaction, o-chloromandelic acid was fractionated by PLC, the structure was measured by ¹ H-NMR, and the optical purity was measured by HPLC. As a result, it was found that the product was indeed 99% ee or higher o-chloro- (R) -mandelic acid. In this way, the deracemization reaction of o-chloromandelic acid by the deracemization factory was successfully advanced at a substrate concentration of 100 mM. A substrate concentration of 100 mM corresponds to 19 g / l.

Mandelic acid deracemization reaction by two kinds of E. coli (oxidation factory and reduction factory) Two kinds of E. coli (oxidation factory and reduction factory) were prepared by the above method. Next, 10 mM (RS) -mandelic acid, 20 mM D-glucose, 0.1 mM NADH, 50 mM Tris-HCl buffer (pH 7.5), wet cells (oxidation factory and reduction factory) 0.5 g each and distilled water The mixture was mixed so that the total amount became 50 ml, and a reaction solution was prepared in a 500 ml Erlenmeyer flask. And it swirled and reacted at 30 degreeC and 180 rpm, and the time-dependent change of the enantiomeric excess of mandelic acid was measured by the said method.
The results are shown in FIG. As shown in FIG. 38, the enantiomeric excess reached 100% ee 4 hours after the start of the reaction. However, although not quantified, unlike the case of the deracemization reaction by one type of E. coli (deracemization plant), a trace amount of the intermediate benzoylformic acid remains even after 8 hours, which is 22 hours. It did not change even after the passage.
As described above, pACYCMG was constructed as a plasmid for co-expression of Enterococcus faecalis IAM 10071-derived (R) -mandelate dehydrogenase and Bacillus megaterium IAM 13418-derived glucose dehydrogenase. Escherichia coli BL21-Gold (DE3) was transformed with pASA, which is a plasmid for expression of mandelate oxidase derived from pACYCMG and Pseudomonas putida ATCC 12633, and three types of enzymes were co-expressed. Using this Escherichia coli, a mandelic acid derivative was deracemized. As a result, when the substrate was mandelic acid, an R-isomer with a substrate concentration of 59 g / l and a yield of 92% and an optical purity of 100% ee was obtained. Further, when the substrate was o-chloromandelic acid, an R-isomer with an optical purity of 100% ee was obtained at a yield of 100% at a substrate concentration of 19 g / l. In this way, a deracemization reaction of a mandelic acid derivative was successfully developed.

The method of the present invention has two features that are epoch-making compared to the prior art. The first feature is that the cost is low. In the deracemization reaction, since an inexpensive racemate is used as a raw material and all unnecessary enantiomers are converted into the desired enantiomers, the raw materials can be used effectively. Moreover, since the coenzyme regeneration system is introduced, expensive coenzyme NADH can be recycled. The second feature is a safety point because a biocatalyst is used.

SEQ ID NOs: 1 to 13 Primers All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A method for deracemizing racemic mandelic acid or a derivative thereof to produce optically active mandelic acid or a derivative thereof, wherein a gene encoding mandelate oxidase, (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme is introduced. Producing an optically active mandelic acid or a derivative thereof, comprising subjecting the transformed microorganism to deracemization by acting on a racemic mandelic acid or a derivative thereof in the presence of a substrate of a coenzyme regeneration system enzyme and an oxidized coenzyme A method wherein mandelic acid or a derivative thereof is represented by the following formula (I):

(In the formula, X represents a hydrogen atom or an alkali or alkaline earth metal, R means that one or a plurality of ortho, meta or para positions are substituted, and the substituent is a hydrogen atom, a halogen atom, A hydroxyl group, an alkyl group having 1 to 3 carbon atoms, an alkoxy group or a thioalkyl group, an amino group, a nitro group, a mercapto group, a phenyl group, or a phenoxy group).
The optically active mandelic acid or a derivative thereof according to claim 1, wherein the coenzyme regeneration system enzyme is selected from the group consisting of glucose dehydrogenase, hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase and glucose-6-phosphate dehydrogenase. how to.
The transformed microorganism is transformed using two types of vectors: an expression vector in which a gene encoding mandelate oxidase is inserted and a two-gene expression vector in which a gene encoding (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme is inserted. The method for producing an optically active mandelic acid or a derivative thereof according to claim 1, which is converted.
The method for producing an optically active mandelic acid or a derivative thereof according to any one of claims 1 to 3, wherein the mandelate oxidase, (R) -mandelate dehydrogenase and the coenzyme regeneration system enzyme are derived from a microorganism.
The method for producing an optically active mandelic acid or a derivative thereof according to any one of claims 1 to 4, wherein the coenzyme regeneration system enzyme is glucose dehydrogenase.
The mandelate oxidase is derived from a Pseudomonas microorganism, the (R) -mandelate dehydrogenase is derived from an Enterococcus microorganism, and the coenzyme regeneration system enzyme is derived from a Bacillus microorganism. 6. A process for producing the optically active mandelic acid or derivative thereof according to any one of 1 to 5.
The method for producing an optically active mandelic acid or a derivative thereof according to any one of claims 1 to 6, wherein the transformed microorganism is Escherichia coli.
A trait introduced with a gene encoding mandelate oxidase, (R) -mandelate dehydrogenase, and a coenzyme regeneration system enzyme for deracemizing racemic mandelic acid or its derivative to produce optically active mandelic acid or its derivative A transformed microorganism, wherein mandelic acid or a derivative thereof is represented by the following formula (I):

(In the formula, X represents a hydrogen atom or an alkali or alkaline earth metal, R means that one or a plurality of ortho, meta or para positions are substituted, and the substituent is a hydrogen atom, a halogen atom, A hydroxyl group, an alkyl group having 1 to 3 carbon atoms, an alkoxy group or a thioalkyl group, an amino group, a nitro group, a mercapto group, a phenyl group, or a phenoxy group).
Claims for producing an optically active mandelic acid or derivative thereof, wherein the coenzyme regeneration system enzyme is selected from the group consisting of glucose dehydrogenase, hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase and glucose-6-phosphate dehydrogenase Item 9. A transformed microorganism according to Item 8.
The transformed microorganism is transformed using two types of vectors: an expression vector in which a gene encoding mandelate oxidase is inserted and a two-gene expression vector in which a gene encoding (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme is inserted. The transformed microorganism according to claim 8 or 9, for producing a converted optically active mandelic acid or a derivative thereof.
The trait according to any one of claims 8 to 10, for producing optically active mandelic acid or a derivative thereof, wherein mandelate oxidase, (R) -mandelate dehydrogenase and a coenzyme regeneration system enzyme are derived from a microorganism. Convert microorganisms.
The transformed microorganism according to any one of claims 8 to 11, for producing an optically active mandelic acid or a derivative thereof, wherein the coenzyme regeneration system enzyme is glucose dehydrogenase.
Optical activity wherein mandelate oxidase is derived from a microorganism belonging to the genus Pseudomonas, (R) -mandelate dehydrogenase is derived from a microorganism belonging to the genus Enterococcus, and a coenzyme regeneration enzyme is derived from a microorganism belonging to the genus Bacillus. The transformed microorganism according to any one of claims 8 to 12, for producing mandelic acid or a derivative thereof.
The transformed microorganism according to any one of claims 8 to 13, for producing optically active mandelic acid or a derivative thereof, which is Escherichia coli.