JP2003061668A - New glycerol dehydrogenase and method for utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a glycerol dehydrogenase which has the optimal pH value of 7.5 to 8.0 in an oxidation reaction and is suitable for industrial utilizations, to provide a DNA encoding the glycerol dehydrogenase, to provide a transformant containing the DNA, and to provide a method for producing an optically active alcohol in a high yield with the transformant. SOLUTION: A glycerol dehydrogenase-producing bacterium is searched from various microorganisms to provide the enzyme having a property suitable for the above-described industrial utilizations. A DNA containing a gene for the enzyme, a DNA recombined with a vector, and a transformant due to the vector are provided. The enzyme or the transformant is used to produce an optically active alcohol.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel glycerol dehydrogenase, a gene thereof, a vector containing the gene, a transformant containing the vector, and a method for producing an optically active alcohol using the transformant. .

Glycerol dehydrogenase is composed of glycerol and oxidized nicotine adenine dinucleotide (hereinafter referred to as NA).
D) or oxidized nicotine adenine dinucleotide phosphate (hereinafter NADP), and then dihydroxyacetone and reduced nicotine adenine dinucleotide (hereinafter NAD).
H) or reduced nicotinamide dinucleotide phosphate (hereinafter NADPH) is an enzyme that catalyzes the reaction. Glycerol dehydrogenase can be used for quantification of glycerol and the like, and also stereoselectively reduces the carbonyl group of α-hydroxyketones to produce optically active 1,2-diols useful as a synthetic raw material for drugs and the like. It is an industrially useful enzyme that can also be used as a catalyst in such cases.

[0003]

BACKGROUND OF THE INVENTION Glycerol dehydrogenase is known to exist in green algae, yeasts, molds, bacteria, actinomycetes and the like, and yeast is one whose enzyme has been purified and its properties have been clarified. Candida Barida ( C
andida valida : J. Gen. Micro
biol. , 130, p. 3225, 1984), Schizosacch
aromys pombe : J. Gen. Micr
obiol. , 131, 1581, 1985)
Or the mold Aspergillus niger ( Asperg
illus niger : J. Gen. Microbi
ol. , 136, 1043, 1990), Aspergillus n.
Idulans : J. Gen. Microbiol. ，
136, pp. 1043, pp., 1990), Mucor Jabanikasu (Mucor javanicus: Eur.
J. Biochem. , 75, 423, 1977
Year), Neurospor
a crassa : J. Gen. Microbio
l. , 107, 289, 1978) and the bacteria Cellulomonas sp. NT3060 ( Cellulom
onas sp. NT3060: Agric. Bio
l. Chem. , 48, 1603, 1982),
Geotorikamu Candy dam (Geotrichum
candidum : Agric. Biol. Che
m. , 46, 3029, 1982), Bacillus
Stearothermophilus ( Bacillus stea)
othermophilus : Biochim. Bi
ochem. Acta, 994, 270, 1989.
Year), Citrobaca Freundi
cter frendii : J. Bacterio
l. , 177, 4392, 1995), Klebsiella pne.
umoniae : J. Bacteriol. , 164
Vol. 479, 1985), Bacillus megaterium : Hopp.
e-Seyler's Z. Physiol. Che
m. , 364, 911, 1983), Escherichia coli : J.
Bacteriol. , 140, 182, 1979
Year), Acetobacter suboxidance ( Acetob
acter subboxydans : J. Biol. C
hem. , 235, 1820, 1960), Lactobacillus fermentum.
us fermentum : JP-A-10-11317
2), Erwinia
aroideae : Chem. Pharm. Bul
l. , 26, 716, 1978).

However, each of these enzymes
The optimum pH in the glycerol oxidation reaction is as high as 9.0 or higher, and there is a problem in industrial use such that sufficient oxidation activity cannot be obtained in the oxidation reaction at a pH near neutral.

[0005]

DISCLOSURE OF THE INVENTION The present invention has the above-mentioned problems of the conventionally known glycerol dehydrogenase,
That is, the problem that the optimum pH of the oxidation reaction is high is solved, and the optimum pH of the oxidation reaction is close to the industrially available neutral.
And a method for producing the same.

[0006]

In view of the above problems, the present inventors searched for microorganisms having a wide range of glycerol dehydrogenase activity, and found that the glycerol dehydrogenase showing the maximum glycerol oxidative activity in the neutral range was Serratia. ( Serra
tia ) newly found in bacteria. Then, the glycerol dehydrogenase was isolated and purified from the microorganism, the glycerol dehydrogenase gene was isolated, and the expression in the host microorganism was achieved, thus completing the present invention.

That is, it has glycerol dehydrogenation activity, and the optimum pH in the glycerol oxidation reaction is 7.5 to 8.0.
Is a protein.

Further, the present invention is the above protein (glycerol dehydrogenase) having the physicochemical properties shown in the following (1) and (2). (1) Optimum temperature of action: 55 ° C to 60 ° C (2) Molecular weight: about 330,000 by gel filtration analysis, S
About 40 in DS polyacrylamide electrophoresis analysis,
000.

The present invention also provides the following (a) or (b):
Is a protein. (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing (b) 1 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing
Alternatively, a protein having an amino acid sequence in which several amino acids are deleted, substituted, inserted or added, and having glycerol dehydrogenase activity.

The present invention is also a DNA encoding the above protein (glycerol dehydrogenase).

The present invention is also the following DNA (c) or (d): (C) DNA consisting of the nucleotide sequence shown in SEQ ID No. 2 in the Sequence Listing (d) consisting of a nucleotide sequence in which one or several nucleotides in the nucleotide sequence shown in SEQ ID No. 2 are deleted, substituted, inserted or added, A DNA encoding a protein having glycerol dehydrogenase activity.

Further, the present invention is a vector containing the above DNA.

The present invention is also a transformant obtained by transforming a host microorganism with the above DNA or vector.

The present invention also provides a method for producing an optically active alcohol, which comprises reacting the above-mentioned protein or transformant and / or a treated product thereof with a carbonyl compound.

Further, in the present invention, the above protein or transformant and / or its treated product is allowed to act on a racemic alcohol to oxidize an alcohol having one steric form and leave an alcohol having the other steric form. It is also a method for producing an optically active alcohol, which is characterized in that

The term "having glycerol dehydrogenase activity" means that glycerol and oxidized nicotine adenine dinucleotide (hereinafter, NAD) or oxidized nicotine adenine dinucleotide phosphate (hereinafter, NADP)
It represents catalyzing the reaction that produces dihydroxyacetone and reduced nicotine adenine dinucleotide (hereinafter NADH) or reduced nicotinamide dinucleotide phosphate (hereinafter NADPH).

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION First, the protein of the present invention will be described. The protein of the present invention is characterized by having glycerol dehydrogenase activity and having an optimum pH in the glycerol oxidation reaction of 7.5 to 8.0.

The protein of the present invention is, in addition to the above,
It is characterized by having the physicochemical properties shown in the following (1) and (2). (1) Optimum temperature: 55 ° C to 60 ° C (2) Molecular weight: about 330,00 in gel filtration analysis
0, about 40,000 in SDS polyacrylamide electrophoresis analysis.

In the present invention, the glycerol oxidation activity is, for example, 100 mM phosphate buffer (pH 8.0).
Glycerin 100 mM, coenzyme NAD 1 mM and enzyme solution are added to the above, and the increase in absorbance at a wavelength of 340 nm is measured at 30 ° C.

The optimum pH or optimum temperature is determined by measuring the activity while changing the temperature or pH of the above activity measuring conditions.

The molecular weight can be measured, for example, by TSK-G300.
It can be determined by gel filtration analysis using a 0SW (7.8 mm ID × 30 cm) (manufactured by Tosoh Corporation) column and by calculating from the relative elution time of the standard protein. The molecular weight of the subunit can be determined by 20% SDS-polyacrylamide gel electrophoresis and calculating from the relative mobility of the standard protein.

The protein of the present invention can be obtained from a microorganism having glycerol dehydrogenase activity. The microorganism which is a source of the protein of the present invention is not particularly limited, but bacteria and the like are preferable, and for example, Serratia ( Se)
A microorganism belonging to the genus Rratia is used, and preferably Serratia marcescens ( Serratia mar)
cesces ), more preferably Serratia marcescens IF
The O12648 strain can be mentioned.

The protein can be obtained as follows. For example, a microorganism having glycerol dehydrogenase activity is cultivated under suitable conditions, and after completion of the culturing, cells are collected by centrifugation or the like, and the cells are crushed by a means such as ultrasonication to obtain a crude enzyme solution. obtain. The protein of the present invention can be obtained by purifying this crude enzyme solution by a standard method such as a salting-out method or a column chromatography method.

The protein of the present invention may be a natural enzyme or a recombinant enzyme in which one or several amino acids in the amino acid sequence are deleted, substituted, inserted or added. Such a recombinant enzyme can be obtained by deleting, substituting, inserting or adding an amino acid by a method well known to those skilled in the art such as a partial directed mutagenesis method. Specifically, Nucleic Acid Re
s. Vol. 10, p. 6487, 1982, Method
s in Enzymology, 100 volumes, 448
Page, 1983, etc.

Next, the DNA of the present invention will be described.
The DNA of the present invention may be any DNA that encodes the above protein. D shown in SEQ ID NO: 2 in the sequence listing
NA may be used, or DNA having a base sequence in which one or several bases have been substituted, inserted, deleted or added in the base sequence shown in SEQ ID NO: 2 may be used.
Here, "a nucleotide sequence in which one or several bases have been substituted, inserted, deleted or added" means "protein nucleic acid enzyme extra number gene amplification PCR method" (1990) or Henry.
A. Errich ed. Kato Ikunoshinkan Translated by a method known to those skilled in the art as described in PCR Technology (1990) and the like, in which a sufficient number of bases are replaced, inserted, deleted or added, It means the following base sequence.

The following is an example of the method for obtaining the DNA of the present invention, but the present invention is not limited to this.

First, the purified glycerol dehydrogenase was digested with an appropriate endopeptidase, and the cleaved fragment was purified by reverse phase HPLC using a YMC-Pack SIL-06 (YMC) column and then used as a protein sequencer. To determine a part of the partial amino acid sequence.
Then, based on the obtained partial amino acid sequence, PC
R (Polymerase Chain Reacti)
on) synthesize the primer.

Next, from the microorganism which is the origin of the DNA,
Conventional DNA isolation methods such as the Hereford method (Ce
11, 18: 1261, 1979), the chromosomal DNA of the microorganism is prepared. Using this chromosomal DNA as a template, PCR is carried out using the above-mentioned PCR primers, a part of the DNA encoding the enzyme is amplified (core sequence), and the nucleotide sequence is determined. The base sequence is dideoxy
It can be determined by the chain termination method or the like. For example, ABI 373A DNA Sequencer
(Manufactured by Applied Biosystems) or the like.

Next, in order to clarify the base sequence of the peripheral region of the core sequence, the chromosomal DNA of the microorganism is digested with a restriction enzyme whose recognition sequence is not present in the core sequence,
Inverse PCR (Nucleic Acids) was performed by subjecting the generated DNA fragment to self-circularization with T4 ligase.
Res. , 16, page 8186, 1988).

Next, based on the core sequence, a primer that becomes the starting point of DNA synthesis toward the outside of the core sequence is synthesized, and the peripheral region of the core sequence is amplified by inverse PCR. By clarifying the base sequence of the DNA thus obtained, the base sequence of the entire coding region of the target enzyme can be read.

Any vector DNA can be used as a vector DNA for introducing and expressing the DNA encoding the glycerol dehydrogenase of the present invention into a host microorganism as long as it can express the enzyme gene in a suitable host microorganism. Momo can be used.

Examples of such vector DNA include plasmid vector, phage vector, cosmid vector and the like. Also, a shuttle vector capable of gene exchange with another host strain may be used.
Such a vector includes operably linked promoters (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp
It can be suitably used as an expression vector containing an expression unit operably linked to the DNA of the present invention, which contains regulatory factors such as a promoter, tufB promoter, recA promoter, pL promoter and the like. For example, pUCNT
(WO94 / 03613) and the like can be preferably used.

The term "regulatory factor" as used herein refers to a nucleotide sequence having a functional promoter and any related transcription elements (eg enhancer, CCAAT box, TATA box, SPI site, etc.).

The term "operably linked" used in the present specification means that a gene and various regulatory elements such as a promoter and an enhancer that regulate its expression are operated in a host cell so that the gene is expressed. It means that they are connected in the state of gaining. It is well known to those skilled in the art that the type and kind of the regulatory factor can vary depending on the host.

Host cells into which the vector having the DNA of the present invention is introduced include bacteria, yeasts, filamentous fungi, plant cells,
Animal cells and the like can be mentioned, but Escherichia coli is particularly preferable.
The DNA of the present invention can be introduced into a host cell by a conventional method. When Escherichia coli is used as the host cell, the DNA of the present invention can be introduced by, for example, the calcium chloride method.

The transformant obtained in the present invention comprises a carbon source,
It can be cultured in a normal medium containing nutrients such as a nitrogen source and inorganic salts. Addition of organic micronutrients such as vitamins and amino acids to this often gives favorable results.

As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols and the like are appropriately used. As the nitrogen source, ammonium salt, aqueous ammonia, ammonia gas, urea, yeast extract, peptone, corn steep liquor and the like are used. As inorganic salts, phosphate, magnesium salt, potassium salt, sodium salt, calcium salt, iron salt,
Sulfates, chlorides and the like are used.

Culturing can be carried out in the temperature range of 25 to 40 ° C.,
25-37 degreeC is especially preferable. Further, the culture can be carried out at a pH of 4 to 8, but it is preferably 5 to 7.5. Also, batch type,
Any continuous culture method may be used.

Next, the method for producing an optically active alcohol by reacting the protein or transformant of the present invention with a carbonyl compound will be described in detail.

When the protein or transformant of the present invention is used to asymmetrically reduce a carbonyl compound to obtain an optically active alcohol, NADH is required as a coenzyme. Although it can be carried out by adding NADH to the reaction system in a necessary amount, the oxidized coenzyme (NAD)
By combining an enzyme having the ability to convert saccharin to a reduced form (NADH) (hereinafter referred to as coenzyme regeneration ability) and its substrate, that is, a coenzyme regeneration system, with the protein or transformant of the present invention, The amount of expensive coenzyme used can be significantly reduced.

As the enzyme having the ability to regenerate coenzyme, hydrogenase, formate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase and the like can be used. Glucose dehydrogenase and formate dehydrogenase are preferably used. Such an enzyme having a coenzyme-regenerating ability may be added separately in the asymmetric reduction reaction system, but the DNA encoding the protein of the present invention and the enzyme having a coenzyme-regenerating ability (for example, glucose dehydrogenase) When a transformant transformed with both of the DNAs encoding) is used as a catalyst, it is not necessary to separately prepare an enzyme having a coenzyme-regenerating ability and add it to the reaction system, and to carry out the reaction efficiently. You can Such a transformant is a DN encoding the protein of the present invention.
DNA encoding A and an enzyme capable of regenerating coenzyme
Are introduced into the same vector and introduced into a host cell, and these two DNAs are introduced into two vectors having different incompatibility groups and introduced into the same host cell. It can also be obtained.

When the protein or transformant of the present invention has the ability to regenerate coenzyme, NADH regeneration reaction can be carried out simultaneously by adding a substrate for coenzyme regeneration to the reaction system. Therefore, the amount of expensive coenzyme used can be significantly reduced without adding another enzyme having a coenzyme regenerating ability to the reaction system. For example, the protein or transformant of the present invention has glycerin oxidation activity, and it may be possible to regenerate NADH by adding glycerin to the reduction reaction system.

Next, the glycerol dehydrogenase or transformant of the present invention is allowed to act on racemic alcohol,
A method for producing an optically active alcohol by preferentially oxidizing an alcohol having one of the three steric groups and leaving the alcohol having the other steric group to remain will be described in detail.

NAD is required as a coenzyme in order to allow the glycerol dehydrogenase or the transformant of the present invention to act on the racemic alcohol and preferentially oxidize the alcohol having one steric structure. NAD in the reaction system
Can be carried out by adding a necessary amount of the enzyme, but an enzyme having the ability to convert the reduced coenzyme to an oxidized form and its substrate are combined with the protein or transformant of the present invention to carry out the reaction. By doing so, the amount of expensive coenzyme used can be significantly reduced.

As the enzyme having the ability to convert the reduced coenzyme to the oxidized form, NADH oxidase, NADH dehydrogenase and the like can be used. These may be used as a purified enzyme, or a microorganism having the enzyme activity or a treated product thereof may be used.

When the protein or transformant of the present invention has the ability to regenerate NAD, the regeneration reaction of NAD can be carried out simultaneously by adding a substrate for the regeneration to the reaction system. . In this case, another NAD
The amount of expensive coenzyme used can be significantly reduced without separately adding an enzyme having a regenerating ability. For example, the protein or transformant of the present invention has dihydroxyacetone reducing activity, and it may be possible to regenerate NAD by adding dihydroxyacetone to the reaction system.

When the transformant of the present invention is used, the reaction proceeds with NAD present in the cells, and NADH produced by the reduction of NAD is also reoxidized in the cells, so that coenzymes and It can be carried out without the addition of an enzyme capable of regenerating NAD.

In the above-mentioned method for producing an optically active alcohol, the transformant may be not only a cultured bacterial cell but also a treated product thereof. Here, the "treated product" represents, for example, a crude enzyme solution, freeze-dried bacterial cells, acetone-dried bacterial cells, a ground product thereof, or a mixture thereof.

The collection of the optically active alcohol compound obtained by any of the above methods is not particularly limited, but may be carried out directly from the reaction solution or after separating the bacterial cells and the like, and then ethyl acetate,
A highly pure optically active alcohol compound can be easily obtained by extraction with a solvent such as toluene, t-butyl methyl ether, hexane, etc., dehydration, and purification by distillation, crystallization, silica gel column chromatography, or the like.

[0050]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following description, "%" means "% by weight" unless otherwise specified.

Example 1 Purification of Enzyme After that, glycerol oxidation activity was measured by using 100 mM phosphate buffer (pH 8.0) and the substrate glycerin 100 mM.
2. NAD 1 mM and enzyme solution 0.05 ml 3.
React in 0 ml of reaction solution at 30 ° C for 3 minutes, and
This was done by measuring the increase in absorbance at 0 nm. This was used as a standard reaction condition for measuring oxidation activity. Under this reaction condition, the enzyme activity of reducing 1 μmol of NAD to NADH in 1 minute was defined as 1 unit.

Glycerin 2 g, diammonium hydrogen phosphate 13 g, potassium dihydrogen phosphate 7 g, magnesium sulfate heptahydrate 0.8 g, zinc sulfate heptahydrate 70 mg, iron sulfate heptahydrate 90 mg, copper sulfate 5 water 50 ml of a liquid medium having a composition of 5 mg of a hydrate, 10 mg of manganese sulfate tetrahydrate, and 100 mg of sodium chloride (all per 1 L),
One drop of ADECANOL (LG-10) was placed in a 500 ml Sakaguchi flask for sterilization, and Serratia marcescens ( Serra) was pre-cultured in the same medium.
tia marcescens) the 1ml culture solution of IFO 12648 was aseptically inoculated, 24 hours by shaking culture at 30 ° C., which was used as a Tanehaha.

A 5 L jar fermenter was added to 3 L of liquid medium having the above composition, and Adecanol (LG-10).
Sterilize by adding 5 drops, and aseptically inoculate 50 ml of the seed mother into it. Under the conditions of culture temperature of 30 ° C., stirring number of 450 rpm, and aeration rate of 1.5 L / min, 60 g of glycerin was added at 16 and 20 hours, respectively. It was added and cultured for 32 hours. During the culture, if the pH was lower than 6.0, the pH was adjusted to 6.0 with a 5N aqueous sodium hydroxide solution.

With respect to 10 L of the culture broth obtained by the above culturing method, the bacterial cells were collected by centrifugation and washed twice with 5 L of physiological saline, and the wet bacterial cells were added with 0.1 mM phenylmethanesulfonyl fluoride. 100 mM phosphate buffer (p
H7.0) was suspended in 1200 ml, and the cells were crushed with an ultrasonic crusher (manufactured by BRANSON). The cell residue was removed by centrifugation to obtain 1300 ml of cell-free extract.

Protamine sulfate 2.5 was added to this cell-free extract.
After adding g and stirring at 4 ° C. overnight, the generated precipitate was removed by centrifugation. Ammonium sulfate was added to the supernatant to 20% saturation, the mixture was stirred at 4 ° C. for 30 minutes, the resulting precipitate was collected by centrifugation, and 100 mM phosphate buffer (pH
It was suspended in 500 ml of 7.0) and dialyzed against 25 L of 10 mM phosphate buffer (pH 7.0).

A 10 mM phosphate buffer solution (pH 7.
0) DEAE-TOYOPEA previously equilibrated with
RL 650M (manufactured by Tosoh Corporation) column (340m
1), the enzyme was adsorbed, and the active fraction was eluted with a linear gradient of sodium chloride concentration from 0 mM to 500 mM. Ammonium sulfate was added to this active fraction so as to be 6.25% saturated, and a 10 mM phosphate buffer solution (pH was added) containing 6.25% saturated ammonium sulfate.
Phenyl-TOY pre-equilibrated with 7.0)
It was applied to an OPEARL 650M (Tosoh Corporation) column (74 ml) to adsorb the enzyme, and after washing the column with the same buffer, the active fraction was eluted with a linear gradient of 6.25% to 0% ammonium sulfate. . This active fraction was mixed with 10 mM phosphate buffer (pH 7.
0) SuperQ-TOYOP pre-equilibrated with
After applying to an EARL (manufactured by Tosoh Corporation) column (20 ml) to adsorb the enzyme and washing the column with the same buffer,
The active fraction was eluted with a linear gradient of sodium chloride concentration from 0 mM to 500 mM. The active fractions were collected to electrophoretically obtain a single purified enzyme preparation. SD
The molecular weight of the band in S-PAGE is about 40,000.
It was 0. In addition, TSK-G3000SW (7.8 mmID × 3) using 100 mM phosphate buffer (pH 7.0) containing 0.1 M sodium sulfate as an eluent.
It was about 330,000 when the gel filtration analysis using the 0 cm) (made by Tosoh Corporation) column was performed. Hereinafter, this enzyme is referred to as RSG.

Example 2 Action of RSG Optimal pH Under standard reaction conditions for measuring glycerol oxidation activity, buffers were imidazole-hydrochloric acid buffer, phosphate buffer, Tris-hydrochloric acid buffer, diethanolamine-hydrochloric acid buffer (all were The activity was measured by using 50 mM ammonium chloride and 50 mM potassium chloride) in the range of pH 6.5 to 10.0. As a result, the optimum pH was 7.5 to 8.0.

Example 3 Optimum temperature of action of RSG Under the standard reaction conditions for glycerol oxidation activity measurement, the reaction temperature was changed in the range of 25 to 75 ° C. and the activity was measured. As a result, the optimum temperature was 55-60 ° C.

Example 4 Measurement of Km value Under the standard reaction conditions for measuring glycerol oxidation activity, the glycerol concentration and the NAD concentration were changed, and the Km value for each was determined. As a result, the Km value for glycerol was 5.4 mM and the Km value for NAD was 80 μM.

Example 5 Cloning of RSG gene (obtaining chromosomal DNA) Serratia marcescens IF
From the culture solution of O12648, the Hereford (Cel
Chromosomal DNA was extracted according to the method described in No. 1, Vol. 18, p. 1261, 1979).

(Cloning of RSG gene by PCR method) 8M of purified RSG obtained as in Example 1
After denaturing in the presence of urea, it was digested with lysyl endopeptidase derived from Achromobacter (manufactured by Wako Pure Chemical Industries, Ltd.), and the sequence of the obtained peptide fragment was determined by the Edman method. Considering the DNA sequence predicted from this amino acid sequence, two PCR primers (primer-
1: Sequence listing SEQ ID NO: 3, primer-2: Sequence listing SEQ ID NO: 4) were synthesized.

100 pmol each of the above two kinds of primers,
Chromosomal DNA 260 ng, dNTP 20 nmol each, E
ExT including 2.5U of xTaq (Takara Shuzo Co., Ltd.)
Prepare 100 μl of aq buffer and heat denaturate (96 ° C, 1
Min), annealing (50 ° C, 1 min), extension reaction (65
After 30 cycles of 2 ° C.) and cooling to 4 ° C., the amplified DNA was confirmed by agarose gel electrophoresis. (Subcloning of DNA fragment amplified by PCR method) The amplified DNA was cloned into pT7Blue Vector (No.
(manufactured by Vagen) and the base sequence was determined. As a result, the amplified DNA was composed of 795 bases including the primer sequence, and had the base sequence from 31st to 825th of the base sequence shown in SEQ ID NO: 2 in Sequence Listing. Hereinafter, this sequence is referred to as "core sequence". (Cloning of sequences around core sequence by inverse PCR method)
Complementary sequence CGGCGA near the 5'-side of the core sequence
GGGATTTGGCGTAT (primer-3: SEQ ID NO: 5 in the Sequence Listing) and the sequence CGC of the portion near the 3'-side
ACGCCATTCCAACAGGT (primer-
4: Sequence listing SEQ ID NO: 6) was prepared.

As a template for inverse PCR, first, the chromosomal DNA of Serratia marcescens IFO12648 was digested with the restriction enzyme BamHI, and the digest was self-closed using T4 DNA ligase. 80 ng of this self-cyclized product, 2 kinds of primers (primer-3 and primer-
4) 100 pmol each, dNTP each 20 nmol, Ex
ExTa including 2.5U of Taq (Takara Shuzo Co., Ltd.)
Prepare 100 μl of q buffer and heat denature (95 ° C, 1
Min), annealing (60 ° C, 1 min), extension reaction (72
After 30 cycles of 1 ° C.) and cooling to 4 ° C., the amplified DNA was confirmed by agarose gel electrophoresis.

Amplified DNA was added to pT7Blue Vector
r (manufactured by Novagen) was subcloned and its nucleotide sequence was determined. From this result and the result of the core array,
The entire nucleotide sequence of the DNA encoding RSG was determined. The entire base sequence and the deduced amino acid sequence encoded by the DNA are shown in SEQ ID NO: 2 in the Sequence Listing.

Example 6 Recombinant vector containing RSG gene
To express RSG in Preparation E. coli coater, to prepare a recombinant vector used for transformation. First, a double-stranded DN in which an NdeI site was added to the start codon of the RSG structural gene and a SacI site was added immediately after the stop codon.
A was obtained by the following method. Based on the nucleotide sequence determined in Example 5, primer-5 (SEQ ID NO: 7 in the Sequence Listing) in which an NdeI site was added to the start codon of the RSG structural gene, and a SacI site immediately after the termination codon of the RSG structural gene were added. The added primer-6 (SEQ ID NO: 8 in Sequence Listing) was synthesized.

Two kinds of primers (primer-5 and p
rimer-6) 100 pmol each, 35 ng, d of Serratia marcescens IFO 12648 chromosomal DNA
100 μl of ExTaq buffer containing 20 nmol each of NTP and 2.5 U of ExTaq (manufactured by Takara Shuzo Co., Ltd.) was prepared and subjected to heat denaturation (95 ° C., 1 minute) and annealing (56).
C., 1 minute) and extension reaction (72.degree. C., 1 minute) for 30 cycles. After cooling to 4.degree. C., amplified DNA was confirmed by agarose gel electrophoresis. This amplified fragment was NdeI and S
Digested with acI and plasmid pUCNT (WO94 / 0
3613) NdeI, S downstream of the lac promoter
By inserting into the acI site, the recombinant vector pN
I got TSG. The manufacturing method and structure of pNTSG are shown in FIG.

Example 7 RSG gene and glucose desorption
Construction of recombinant vector containing both hydrogen enzyme genes at the same time
Made of Bacillus megaterium
terium) glucose-dehydrogenase (hereinafter referred to as GDH) gene derived from the strain IAM1030 is located 5 bases upstream from the start codon of the gene for Escherichia coli Shin-Dalg.
Arno sequence (9 nucleotides) immediately before A
A vaI breakpoint was also created immediately after the stop codon and HindI
The double-stranded DNA to which the II cutting point was added was obtained by the following method. Based on the nucleotide sequence information of the GDH gene, GD
Shin-Dalgarno sequence of Escherichia coli (9 nucleotides) 5 bases upstream from the start codon of the H structural gene
And a prim with an AvaI breakpoint added immediately before
er-7 (SEQ ID NO: 9 in the Sequence Listing) and p added with a HindIII site immediately after the termination codon of the GDH structural gene.
Primer-8 (SEQ ID NO: 10 in Sequence Listing) was synthesized according to a conventional method. Using these two primers, the plasmid pGDK1 (Eur. J. Biochem., 186
Vol., 389, 1989) was used as a template to synthesize double-stranded DNA by PCR. The obtained DNA fragment is Ava
A recombinant vector pNTSGG1 which was digested with I and HindIII and inserted into the AvaI and HindIII sites of pNTSG constructed in Example 6 was obtained. pNTSG
The manufacturing method and structure of G1 are shown in FIG.

Example 8 Production of recombinant Escherichia coli The recombinant vector pNTSG obtained in Example 6 and Example 7
The recombinant vector pNTSGG1 obtained in
B101 (Takara Shuzo Co., Ltd.) was transformed and recombinant E. coli HB101 (pNTCP) and HB101 were obtained from each.
(PNTSGG1) was obtained. The transformants E. coli HB101 (pNTSG) and HB10 thus obtained
1 (pNTSGG1) is the accession number FERM
P-18448 and FERM P-18449 have been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology.

Example 9: RSG in recombinant E. coli
Expression Recombinant Escherichia coli HB101 (pNTSG) obtained in Example 8
2 × YT medium containing 120 μg / ml of ampicillin (Bacto tryptone 1.6% (w / v), Bacto yeast extract 1.0% (w / v), NaCl 0.5%.
(W / v), pH 7.0), and after harvesting the cells, 100m
The cells were suspended in M phosphate buffer (pH 7) and ultrasonically disrupted to obtain a cell-free extract. The glycerol oxidation activity of this cell-free extract was measured by the method described in Example 1.

[0070]

[Table 1] In E. coli HB101 (pNTSG), E. coli HB101 (pUCG), which is a transformant containing only the vector plasmid,
There was a clear increase in glycerol oxidative activity compared to NT).

Example 10 RSG in recombinant E. coli
And GDH co-expression Recombinant E. coli HB101 (pNTSGG obtained in Example 8)
The glycerol oxidation activity and GDH activity of the cell-free extract obtained by treating 1) in the same manner as in Example 9 were measured. GDH activity was measured by adding 1M Tris-HCl buffer (pH 8.0), substrate glucose 0.1M, coenzyme N.
AD2mM and enzyme were added, and the wavelength was 340mM at 25 ° C.
It was carried out by measuring the increase in the absorbance of. Under these reaction conditions, 1 μmol of NAD was added to NADH in 1 minute.
The enzyme activity of reducing to 1 was defined as 1 unit. The glycerol oxidation activity was also measured in the same manner as in Example 1. The glycerol oxidation activity and GDH activity of the cell-free extract thus measured were compared and expressed.

[0072]

[Table 2] In E. coli HB101 (pNTSGG1), E. coli HB101 (pNTSGG1), which is a transformant containing only the vector plasmid,
Glycerol oxidation and G as compared to UCNT)
An increase in DH activity was seen.

Example 11 RSG gene-introduced recombination
E. The combination of (S) -1,2-propanediol with E. coli
Recombinant E. coli HB101 obtained in adult Example 8 (pNTSG)
Was inoculated into 50 ml of 2 × YT medium that had been sterilized in a 500 ml Sakaguchi flask, and cultured with shaking at 37 ° C. for 18 hours. 10 ml of the obtained culture solution was adjusted to pH 6.5, 10 mg of racemic 1,2-propanediol was added thereto, and the mixture was stirred at 37 ° C. for 24 hours. After the conversion reaction, the reaction solution was saturated with ammonium sulfate, extracted with ethyl acetate, and the remaining 1,2-propanediol and hydroxyacetone produced were subjected to gas chromatography (column: PEG-20M 1 manufactured by GL Sciences Inc.).
0% Chromsorb WAW DMCS 80/10
0, detection: FID, column temperature: 130 ° C.). Further, the optical purity of the obtained 1,2-propanediol was determined by tosylating the hydroxyl group of 1,2-propanediol,
High performance liquid chromatography (column: Chiralpak AD manufactured by Daicel Chemical Industries, Ltd., eluent: hexane / isopropanol = 9/1, flow rate:
0.8 ml / min, detection: 235 nm, column temperature:
Room temperature, elution time: R-form 22 minutes, S-form 26 minutes). As a result, 4.3 mg of hydroxyacetone was produced, and the remaining 1,2-propanediol was 5.2 mg,
Its optical purity is (S) 93.4% e. e. Met.

Example 12 Co-expression of RSG and GDH
(R) -1,2-propanedi by recombinant E. coli
Synthesis of all Recombinant E. coli HB101 (pNTSGG obtained in Example 8)
50m sterilized 1) in a 500 ml Sakaguchi flask
1 × 2 × YT medium was inoculated and shake-cultured at 37 ° C. for 18 hours. 1 ml of the obtained culture solution was adjusted to pH 6.5,
Add 10mg of hydroxyacetone to this,
It was stirred for 4 hours. After the conversion reaction, the reaction solution was saturated with ammonium sulfate, extracted with ethyl acetate, and the production amount and optical purity of 1,2-propanediol as a product were measured in the same manner as in Example 11. 9.9 mg, optical purity (R) 100% e. e. Met.

[0075]

In the present invention, the optimum p in the oxidation reaction
A glycerol dehydrogenase glycerol dehydrogenase suitable for industrial use, in which H is 7.5 to 8.0 is provided. In addition, DNA containing the gene for the present glycerol dehydrogenase,
Recombinant DNA with a vector and a transformant with this vector are provided. Further, by using the enzyme and the transformant, it is possible to efficiently produce an optically active alcohol.

[0076]

[Sequence list] SEQUENCE LISTING <110> Kanegafuchi Chemical Industry Co., Ltd. <120> Novel glycerol dehydrogenase and method of using the same <130> TKS-4564 <160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 365 <212> PRT <213> Serratia marcescens <400> 1 Met Leu Arg Ile Ile Gln Ser Pro Gly Lys Tyr Ile Gln Gly Ala Asn   1 5 10 15 Ala Leu Ala Ala Val Gly Glu Tyr Ala Lys Ser Leu Ala Asp His Tyr              20 25 30 Phe Val Ile Ala Asp Asp Phe Val Met Leu Leu Ala Gly Asp Thr Leu          35 40 45 Met Gly Ser Leu Arg Gln His Gly Val Lys His His Ala Ala Arg Phe      50 55 60 Asn Gly Glu Cys Cys Arg Lys Glu Ile Asp Arg Leu Gly Cys Glu Leu  65 70 75 80 Gln Thr His Gly Cys Arg Gly Val Ile Gly Val Gly Gly Gly Lys Thr                  85 90 95 Leu Asp Thr Ala Lys Ala Val Ala His Tyr Gln Arg Leu Pro Val Val             100 105 110 Leu Ile Pro Thr Ile Ala Ser Thr Asp Ala Pro Thr Ser Ala Leu Ser         115 120 125 Val Ile Tyr Thr Glu Gln Gly Glu Phe Ala Glu Tyr Leu Ile Tyr Pro     130 135 140 Arg Asn Pro Asp Met Val Met Asp Ser Ala Ile Ile Ala Asn Ala Pro 145 150 155 160 Val Arg Leu Leu Val Ala Gly Met Gly Asp Ala Leu Ser Thr Tyr Phe                 165 170 175 Glu Ala Gln Ala Cys Phe Asp Ala Gln Ala Thr Ser Met Ala Gly Gly             180 185 190 Lys Ser Thr Leu Ala Ala Leu Ser Leu Ala Arg Leu Cys Tyr Asp Thr         195 200 205 Leu Leu Ala Glu Gly Val Lys Ala Lys Leu Ala Val Glu Ala Gly Val     210 215 220 Val Thr Glu Ala Val Glu Arg Ile Ile Glu Ala Asn Thr Tyr Leu Ser 225 230 235 240 Gly Ile Gly Phe Glu Ser Ser Gly Leu Ala Ala Ala His Ala Ile His                 245 250 255 Asn Gly Phe Thr Val Leu Glu Glu Cys His His Leu Tyr His Gly Glu             260 265 270 Lys Val Ala Phe Gly Thr Leu Ala Gln Leu Met Leu Gln Asn Ser Pro         275 280 285 Met Ala Gln Ile Glu Thr Val Leu Ala Phe Cys His Asp Ile Gly Leu     290 295 300 Pro Ile Thr Leu Ala Gln Met Gly Val Thr Gly Asp Ala Ala Ala Lys 305 310 315 320 Met Met Ala Val Ala Glu Ala Ser Cys Ala Ala Gly Glu Thr Ile His                 325 330 335 Asn Met Pro Phe Lys Val Thr Pro Ala Gly Val Gln Ala Ala Ile Leu             340 345 350 Thr Ala Asp Arg Leu Gly Ala Ala Trp Leu Gln Arg His         355 360 365 <210> 2 <211> 1098 <212> DNA <213> Serratia marcescens <400> 2 atg ttg aga atc atc cag tca cca ggc aaa tac att cag ggc gcc aat 48 Met Leu Arg Ile Ile Gln Ser Pro Gly Lys Tyr Ile Gln Gly Ala Asn   1 5 10 15 gcg ctg gcc gcc gtc ggc gaa tac gcc aaa tcc ctc gcc gac cat tat 96 Ala Leu Ala Ala Val Gly Glu Tyr Ala Lys Ser Leu Ala Asp His Tyr              20 25 30 ttc gtg atc gcc gac gac ttc gtg atg ctg ctg gcg ggc gac acc ctg 144 Phe Val Ile Ala Asp Asp Phe Val Met Leu Leu Ala Gly Asp Thr Leu          35 40 45 atg ggc agc ctg cgg cag cac ggc gtg aaa cat cat gcg gcc cgc ttc 192 Met Gly Ser Leu Arg Gln His Gly Val Lys His His Ala Ala Arg Phe      50 55 60 aac ggc gag tgc tgc cgt aaa gag ata gac cgg ctt ggc tgc gag ctg 240 Asn Gly Glu Cys Cys Arg Lys Glu Ile Asp Arg Leu Gly Cys Glu Leu  65 70 75 80 cag acc cat ggc tgc cgc ggg gtg atc ggc gtc ggc ggc ggc aaa acg 288 Gln Thr His Gly Cys Arg Gly Val Ile Gly Val Gly Gly Gly Lys Thr                  85 90 95 ctc gac acc gcc aag gcc gtc gcc cac tac caa cgg ctg ccg gtg gtg 336 Leu Asp Thr Ala Lys Ala Val Ala His Tyr Gln Arg Leu Pro Val Val             100 105 110 ctc att ccc acc atc gcc tcc acc gat gcg ccg acc agc gcg ctg tcg 384 Leu Ile Pro Thr Ile Ala Ser Thr Asp Ala Pro Thr Ser Ala Leu Ser         115 120 125 gtt atc tac acc gaa cag ggc gaa ttc gcc gaa tac ctg atc tac ccg 432 Val Ile Tyr Thr Glu Gln Gly Glu Phe Ala Glu Tyr Leu Ile Tyr Pro     130 135 140 cgc aac ccg gac atg gtg atg gac agc gcc atc atc gcc aac gcg ccg 480 Arg Asn Pro Asp Met Val Met Asp Ser Ala Ile Ile Ala Asn Ala Pro 145 150 155 160 gtg cgc ctg ctg gtg gcc ggc atg ggc gac gcg ctc tcc acc tat ttc 528 Val Arg Leu Leu Val Ala Gly Met Gly Asp Ala Leu Ser Thr Tyr Phe                 165 170 175 gaa gcg cag gcc tgt ttc gac gcc cag gcc acc agc atg gcc ggc ggc 576 Glu Ala Gln Ala Cys Phe Asp Ala Gln Ala Thr Ser Met Ala Gly Gly             180 185 190 aaa tcg aca ctg gcg gcg ctc agt ctg gcg cgg ctg tgc tac gac acg 624 Lys Ser Thr Leu Ala Ala Leu Ser Leu Ala Arg Leu Cys Tyr Asp Thr         195 200 205 ctg ctg gcc gaa ggg gtg aag gcc aag ctg gcg gtc gag gcc ggc gtg 672 Leu Leu Ala Glu Gly Val Lys Ala Lys Leu Ala Val Glu Ala Gly Val     210 215 220 gtg acg gaa gcg gtg gaa cgc atc att gag gcc aat acg tat ctg agc 720 Val Thr Glu Ala Val Glu Arg Ile Ile Glu Ala Asn Thr Tyr Leu Ser 225 230 235 240 ggc atc ggc ttc gag agc agc ggg ctg gcg gcg gcg cac gcc att cac 768 Gly Ile Gly Phe Glu Ser Ser Gly Leu Ala Ala Ala His Ala Ile His                 245 250 255 aac ggt ttc acc gtg ctg gag gag tgt cac cac ctg tat cac ggc gag 816 Asn Gly Phe Thr Val Leu Glu Glu Cys His His Leu Tyr His Gly Glu             260 265 270 aaa gtg gcg ttc ggc acc ctg gcg caa ctg atg ctg cag aac agc ccg 864 Lys Val Ala Phe Gly Thr Leu Ala Gln Leu Met Leu Gln Asn Ser Pro         275 280 285 atg gcg cag att gaa acc gtg ctg gcg ttt tgt cat gac atc ggc ctg 912 Met Ala Gln Ile Glu Thr Val Leu Ala Phe Cys His Asp Ile Gly Leu     290 295 300 ccg atc acg ttg gcg cag atg ggc gtc acc ggc gat gcg gcg gcg aag 960 Pro Ile Thr Leu Ala Gln Met Gly Val Thr Gly Asp Ala Ala Ala Lys 305 310 315 320 atg atg gcg gtg gcc gag gcc agc tgc gcc gcg ggt gaa acc att cac 1008 Met Met Ala Val Ala Glu Ala Ser Cys Ala Ala Gly Glu Thr Ile His                 325 330 335 aac atg ccg ttc aag gtc acc cct gcg ggc gtg cag gcg gcg atc ctg 1056 Asn Met Pro Phe Lys Val Thr Pro Ala Gly Val Gln Ala Ala Ile Leu             340 345 350 acg gcg gat agg ctg ggc gcc gcc tgg ttg cag cgt cat tga 1098 Thr Ala Asp Arg Leu Gly Ala Ala Trp Leu Gln Arg His         355 360 365 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-1 <400> 3 tayathcarg gngcnaaygc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-2 <400> 4 gcnacyttyt cnccrtgrta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-3 <400> 5 cggcgaggga tttggcgtat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-4 <400> 6 cgcacgccat tcacaacggt 20 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-5 <400> 7 tccgcatatg ttgagaatca tccagtc 27 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-6 <400> 8 cccagagctc tcaatgacgc tgcaaccag 29 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-7 <400> 9 ataacccggg taaggaggtt aacaattata aag 33 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer-8 <400> 10 gcagaagctt ttatccgcgt cctgcttgga 30

[Brief description of drawings]

FIG. 1 Recombinant vectors pNTSG and pNTSGG1
It is a figure which shows the creation method of.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA03 BA08 CA05 DA06                 4B050 CC03 DD02 FF01 FF03 FF05                       FF11 FF12 LL05                 4B064 AC05 CA19 CB18 CC24 CD05                       DA16                 4B065 AA01Y AA26X AB01 BB07                       CA28

Claims (19)

[Claims] 1. A protein having glycerol dehydrogenase activity and having an optimum pH of glycerol oxidation reaction of 7.5 to 8.0. 2. The protein according to claim 1, which has the following physicochemical properties (1) and (2). (1) Optimum temperature for glycerin oxidation: 55 ° C to 60 ° C (2) Molecular weight: Approximately 330000 by gel filtration analysis, SD
About 4000 in S polyacrylamide electrophoresis analysis
0. 3. The following protein (a) or (b): (A) A protein having the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing (b) 1 in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing
Alternatively, a protein having an amino acid sequence in which several amino acids are deleted, substituted, inserted or added, and having glycerol dehydrogenase activity. 4. The protein is Serratia ( Serra).
The protein according to any one of claims 1 to 3, which is a protein obtained from a microorganism belonging to the genus tia ). 5. The microorganism belonging to the genus Serratia is Serratia.
Marcense ( Serratia marcescen
s ) is a protein according to claim 4. 6. The microorganism belonging to the genus Serratia is Serratia.
Marcense ( Serratia marcescen
s ) IFO12648 strain, The protein according to claim 4. 7. A DNA encoding the protein according to claim 1. 8. The following DNA (c) or (d): (C) a DNA consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing (d) a base sequence in which one or several bases have been substituted, deleted, inserted or added in the base sequence shown in SEQ ID NO: 2 in the sequence listing, A DNA encoding a protein having glycerol dehydrogenase activity. 9. A vector having the DNA according to claim 7 or 8. 10. The vector according to claim 9, wherein the vector is pNTSG. 11. The vector according to claim 9, further comprising a DNA encoding glucose dehydrogenase. 12. The glucose dehydrogenase is Bacillus megaterium.
um ) -derived glucose dehydrogenase. 13. The vector according to claim 12, wherein the vector is pNTSGG1. 14. A transformant obtained by transforming a host cell with the vector according to any one of claims 9 to 13. 15. The transformant according to claim 14, wherein the host cell is Escherichia coli. 16. Escherichia coli
The transformant according to claim 15, which is HB101 (pNTSG). 17. Escherichia coli
The transformant according to claim 15, which is HB101 (pNTSGG1). 18. A method for producing an optically active alcohol, which comprises reacting the transformant according to any one of claims 14 to 17 and / or a treated product thereof with a carbonyl compound. 19. The transformant according to any one of claims 14 to 17 and / or a treated product thereof is allowed to act on a racemic alcohol to oxidize an alcohol having one steric structure, and A method for producing an optically active alcohol, characterized in that the alcohol contained therein is left.