JPH08242851A - Gene for new alcohol acetyltransferase - Google Patents

Abstract

PURPOSE: To obtain a new gene useful for producing a new alcohol acetyltransferase capable of producing an alcohol reinforced with an ester fragrance and having high stability by biotechnology. CONSTITUTION: This gene has a DNA sequence coding for a polypeptide having an amino acid sequence of the formula. The gene is obtained by a plaque hybridization method, etc., using a probe prepared of a part of a DNA chain, which has a function for producing a new alcohol acetyltransferase, obtained from a sake yeast KYOUKAI-7, Saccharomyces cerevisiae, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alcohol acetyltransferase derived from yeast (hereinafter, alcohol acetyltransferase is referred to as AATase, and the novel alcohol acetyltransferase according to the present invention is referred to as new AATase), and biotechnological production of new AATase. The present invention relates to a DNA chain having an ability and a yeast transformed with this DNA chain and having an increased ester-producing ability. INDUSTRIAL APPLICABILITY The present invention enables the production of alcoholic beverages with an enhanced ester flavor by a more stable transformant in which new AATase is enhanced as compared with known AATase.

[0002]

2. Description of the Related Art AATase is an important enzyme for the production of esters by yeasts, which produces acetic acid esters by condensing alcohols with acetyl CoA. AATas of yeast
It is a well known fact that the esters represented by isoamyl acetate produced by e influence the quality evaluation of sake such as sake and beer.

Complete purification of AATase by Minetoki et al.
Reported (Biosci. Biotech. Biochem.,57
(12), p2094, 1993). A completely purified by Minetoki et al.
ATase was used in the first column chromatography for purification.
Of the two large and small AATase peaks eluted, A
The main peak designated ATaseI was also purified.
Of. Another AATase II sub-peak
Is superior to AATase I in heat stability and
It has been reported that it is a highly frequent AATase.
Association Magazine,87(5), p334, 1992), compared to AATase I
AATaseI is present because it is present in a much smaller amount than
AATase II cannot be completely purified by the same method
Therefore, detailed elucidation of enzymatic properties and gene acquisition are not possible.
I didn't come. In this way, yeast has several types of A
There are other reports that ATase exists (Eur.
J. Biochem.210, p1015, 1992), the present inventor
To our knowledge, other AA derived from yeast and of different nature
There is no detailed report on Tase. Therefore, the known A
ATase is AATase completely purified by Minetoki et al.
It refers to the knowledge of. Regarding AATase gene
Minetoki et al. Obtained the fully purified AATase gene.
Analysis has been reported (Appl. Environ. Mic
robiol. ,60(8), p2786, 1994), other
There is no report on the DNA strand producing AATase.

It is known to utilize the gene obtained by AATase purified by Minetoki et al. As a method for producing alcoholic beverages with enhanced ester flavor by enhancing the AATase activity of yeast (Japanese Patent Laid-Open No. 06-62849). Issue).

[0005]

However, regarding the thermal stability, AATase completely purified by Minetoki et al.
Was relatively stable, but extremely unstable at higher temperatures, and there was a problem that the enzymatic activity was significantly inhibited by unsaturated fatty acids such as linoleic acid.
(Agric. Biol. Chem., 45 (10), p2183, 19
81, Agric. Biol. Chem. , 54 (6), p1485, 1
990). In the conventional production of alcoholic beverages, selection of raw materials, treatment, and fermentation control often lead to avoiding these problems. Taking sake production as an example, especially in the production of sake with enhanced ester flavor called ginjoshu, it is necessary to remove 40% or more by weight of brown rice, which is the raw material, as bran. This leads to the removal of contained fats and oils, etc., and as a result, the inhibition of AATase activity by unsaturated fatty acids such as linoleic acid is reduced. In addition, the fermentation temperature is lower than that of sake brewing that is usually performed, and the fermentation period is long.
This leads to suppression of inactivation of AATase due to heat and maintenance of its activity for a longer period of time. Controlling these processes often depends on the experience and intuition of a skilled person, from the processing of raw materials to the acquisition of produced sake. Therefore, known AA
If yeast that is more stable than Tase and capable of producing AATase can be used, the production process that requires skill will be simplified, and the production period will be shortened to enable the production of sake with enhanced ester flavor. It is a thing.

[0006]

Therefore, the present inventors have
In order to solve the above problems, other AA existing in yeast
As a result of various studies on Tase, a novel yeast-derived alcohol acetyltransferase, which is thermostable and is not inhibited by enzymatic activity by unsaturated fatty acids such as linoleic acid, was completely purified, and its gene was isolated and its structure was determined. The present invention has been completed by knowing that the transformed yeast having the ability to produce new AATase has been obtained successfully.

That is, the first aspect of the present invention is that S which is stable at a temperature of 30 ° C. and is not inhibited by unsaturated fatty acids.
It is a novel alcohol acetyltransferase derived from yeast, which has a molecular weight of about 63,000 as measured by DS polyacrylamide gel electrophoresis.

The second is a novel alcohol acetyltransferase, which is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The third is a novel alcohol acetyltransferase gene characterized by comprising a DNA sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing.

Fourth, the DN shown in SEQ ID NO: 1 in the sequence listing
It is a DNA chain having a nucleotide sequence substantially homologous to the A sequence or at least a part thereof and capable of producing a polypeptide having a novel alcohol acetyltransferase activity.

The fifth is a yeast which has been transformed by the introduction of a foreign gene containing a novel alcohol acetyltransferase gene or DNA chain of the above-mentioned third and fourth of the present invention and enriched in the ability to produce a new AATase.

In the present invention, "gene" and "DNA"
"Strand" and "DNA sequence" are used interchangeably.

The present invention will be described in detail below. The novel AATase according to the present invention is derived from yeast and is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, which is obtained by adding, inserting, deleting, deleting or substituting some amino acids. Genetic engineering or protein engineering reveals that the physiological activity of peptides may not change. Therefore, the amino acid sequence shown in the present invention is not limited to the case where it is exactly the same as the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and AATase
As long as the activity is preserved, it includes some amino acid modifications as described above.

The new AATase of the present invention is specifically Saccharomyces cerevisiae.
It is obtained from e). The Saccharomyces cerevisiae referred to in the present invention means The yeast, a Taxonomic s.
tudy 3rd. Edition (ed. By NJW Kreger-
van Rij. Elsevier Science Publishers B.M.
V. , Amsterdam, p379, 1984), and Saccharomyces cerevisiae and its synonyms or mutants.

Among the properties of the new AATase of the present invention, some typical ones different from known AATases are optimum temperature, thermostability, inhibition by unsaturated fatty acid and molecular weight. The optimum temperature for known AATase is 25
The optimum temperature of the new AATase is as high as 40 ° C.
The known AATase is easily inactivated at a temperature higher than 4 ° C, and according to the studies conducted by the present inventors,
The activity decreases to about 30% at 0 minutes, but the new AATas
Under the same conditions, e maintains an enzyme activity of 90% or more.
In addition, the known AATase, whose enzymatic activity is greatly inhibited by unsaturated fatty acids such as linoleic acid,
Tase maintains an activity of 90% or more even when 2 mM of linoleic acid is added. The known AATase has an enzyme activity reduced to 4.3% by the addition of 2 mM linoleic acid. The molecular weight of the new AATase was about 63,000 as a result of SDS polyacrylamide gel electrophoresis, and AATase (molecular weight of about 5600) which was completely purified by Minetoki et al.
0 (Journal of Japan Brewery, 87 (5), p334, 1992)). Therefore, the AATase according to the present invention has several important enzymatic properties and molecular weight differences.
Is a novel AATase. The molecular weight estimated from the nucleotide sequence is about 62,000.

The enzymatic properties of the new AATase according to the present invention are as follows. (1) Molecular weight by SDS polyacrylamide gel electrophoresis about 63000 (2) Optimal and stable pH Optimal pH: 7.5 Stable pH: 6.5-9.5 (3) Optimal and stable temperature Optimum temperature: Stable at 40 ℃: Stable below 30 ℃ (4) Effect of inhibitors Parachloromercuric benzoic acid and dithiobisbenzoic acid strongly inhibit it. (5) Effect on the activity of various fatty acids It is not strongly inhibited by saturated fatty acids and unsaturated fatty acids. (6) Km value for isoamyl alcohol and acetyl CoA Isoamyl alcohol: 84.6 mM Acetyl CoA: 82.3 μM

This new AATase is Saccharomyces
Cerevisiae Sake Yeast Association No. 7 can be obtained by a purification method comprising preparation of a solubilized crude enzyme from the content of cultured bacterial cells and various chromatographies. The actual acquisition of the new AATase consisting of these unit steps is as shown in the Examples below and is possible for those skilled in the art.

One of the methods for obtaining the new AATase gene of the present invention is to use, as a probe, a part of the DNA chain having the ability to produce the new AATase provided by the present invention, and perform plaque hybridization, colony hybridization, PCR. And the like, and all of these means are well known to those skilled in the art and can be easily implemented.

Also, useful as a gene source for obtaining a DNA chain capable of producing a new AATase is not limited to yeast, but bacteria, plants, etc. are considered, and particularly desirable gene sources are alcoholic beverages or fermentation. These are yeasts used in food production.

This DNA chain is a D encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing.
It may just correspond to the NA sequence, or may have a structure in which the DNA sequence is bound to the upstream side or the downstream side and both of them. An example of the latter is
It is in the form of a plasmid incorporated into a suitable vector.

A specific DNA sequence is shown in SEQ ID NO: 1 of the sequence listing as corresponding to the amino acid of SEQ ID NO: 2 in the sequence listing, which is analyzed for the new AATase gene obtained from Sake Yeast Association No. 7. The present invention has been carried out and is a preferred specific example of the DNA sequence according to the present invention.

The DNA chain or DNA sequence having the ability to produce a new AATase according to the present invention means a new AATas.
It refers to a DNA chain or a DNA sequence encoding a polypeptide having AATase activity exhibiting the characteristic of e, which varies according to the variation of the polypeptide as described above. It shall be understood in its broadest sense. Thus, due to degeneracy (or degeneracy), there can be multiple species of nucleic acid triplets that encode a given amino acid. The DNA chain of interest in the present invention may be derived from natural products, or may be wholly synthetic or semi-synthetic.

Methods for producing transformants are those conventionally used in the field of genetic engineering, and include, for example, the protoplast method (Proc. Natl. Acad. Sci., 7) .
5 , p. 1929, 1978) or metal treatment method (J. Ba.
cteriol. , 113 , p727, 1983). The vector used at this time may be any vector as long as it is known for yeast. For example, YEp system, YCp system, YIp system and the like are not only known in the literature but also easily prepared. You can

On the other hand, in order to express the gene of the DNA chain of the present invention in yeast, or to increase or decrease the expression, a promoter controlling transcription and translation is located in the 5'upstream region of the DNA chain of the present invention. , The terminator should be installed in the 3'downstream area. The promoter and terminator include those derived from the new AATase gene itself, as well as alcohol dehydrogenase gene (J. Bio. Chem., 257 , p3018, 198).
2), phosphoglycerate kinase gene (NucleicAci
d Res. , 10 , p7791, 1982), glycerol aldehyde-3-phosphate dehydrogenase gene (J. Bio).
l. Chem. , 254 , p9839, 1979) and the like, or any known gene-derived one or artificially improved one can be used.

In the present invention, the yeast to be transformed, that is, the yeast serving as a host, may be any taxonomically included yeast, but for the purpose of the present invention, it is for producing alcoholic beverages belonging to Saccharomyces cerevisiae. Yeast, specifically sake yeast, brewer's yeast, wine yeast, and whiskey yeast are desirable. For example, sake yeast: IFO 2347, brewer's yeast: ATCC26292, wine yeast; IFO 2
260, whiskey yeast: IFO 2112 and the like. Another preferred group of hosts is baker's yeast. Specifically, ATCC32120 can be illustrated.

The transformed yeast enriched in the ability to produce a new AATase obtained as described above can be used for each purpose. When the host yeast is a liquor-producing yeast, the ability to produce a new AATase is enriched according to the present invention, so that a liquor enriched or enriched with an ester flavor can be produced by stable AATase activity.

[0026]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. Example 1 Production of New AATase The enzyme of the present invention can be obtained from a culture of yeast which belongs to the genus Saccharomyces and produces an enzyme having the above-mentioned characteristics. An example of a preferred manufacturing method is as follows.

1- (1) Preparation of Crude Enzyme Solution AATase activity was determined by the method of Minetoki et al. (Biosci. Biotech.
Biochem. , 57 (12), p2094, 1993). Sake yeast association No. 7 was inoculated into 500 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose) and cultured at 15 ° C for 3 days. Add the culture solution to a 40-liter plastic container containing 30 liters of YPD medium.
00 ml was inoculated, and the mixture was anaerobically stirred and cultured at 25 ° C. for 12 hours. Next, the cells were collected by centrifugation (3,000 rpm / minute, 10 minutes), and the volume of the buffer solution was 10 times the buffer weight (25 mM).
Tris-HCl buffer (pH 7.5), 1.5M sorbitol)
Then, Zymori Ace 100T (obtained from Seikagaku Corporation) was added in an amount of 1/1000 of the cell weight. This was shaken at 30 ° C. for 1 hour, and the resulting protoplasts were added to 20
It was collected by centrifugation at 00 rpm for 5 minutes. Collect the collected protoplasts in 400 ml of buffer for cell disruption (25 m
Polytron PT10 type (KINE) was suspended in M imidazole hydrochloric acid buffer (pH 7.5), 0.5M sorbitol).
The cells were disrupted using a cell disruption device (made by MATICA). After crushing, the mixture was centrifuged at 4500 rpm for 10 minutes, and the supernatant was centrifuged at 14000 rpm for 20 minutes to remove the crushed residue, and ammonium sulfate was added to the supernatant to 50% saturation, After holding time, 1
A precipitate was obtained by centrifugation at 0000 rpm for 20 minutes.
The resulting precipitate was washed (25 mM imidazole hydrochloride buffer (pH 7.5), 50% saturated ammonium sulfate, 1 mM).
Dithiothreitol), and after washing, 1000
The precipitate was collected by centrifugation at 0 rpm for 20 minutes. This precipitate was added to a buffer solution (25 mM imidazole hydrochloric acid buffer solution (pH)
7.5) Dissolved in 1 mM dithiothreitol) to obtain a crude enzyme solution.

1- (2) Preparation of Microsome Fraction The crude enzyme solution obtained in 1- (1) was diluted with 100,000 × G to obtain 2
After centrifuging for an hour, the resulting precipitate (microsome fraction) was mixed with 40 ml of a buffer solution (25 mM imidazole hydrochloride buffer solution (pH 7.5),
1 mM dithiothreitol). If not used immediately, it was stored at -20 ° C.

1- (3) Preparation of solubilizing enzyme The microsome fraction obtained in 1- (2) was transferred to an Erlenmeyer flask, 1/100 volume of polyoxyethylene octylphenyl ether was added, and the mixture was bubbled at 4 ° C. Being careful not to let this happen, the mixture was stirred for 60 minutes using a magnetic stirrer.
Then, the mixture was centrifuged at 100,000 × G for 2 hours, and the supernatant was added to buffer A (25 mM imidazole hydrochloride buffer (pH 7.5),
It was dialyzed overnight against 0.1% polyoxyethylene octyl phenyl ether, 0.5% isoamyl alcohol, 1 mM dithiothreitol, 20% glycerol).

1- (4) Purification of Enzyme Purification of enzyme was carried out by repeating the procedure of 1- (1) and 1- (2) 50 times to obtain and store the microsome fraction 1 times.
-The operation of (3) was performed, and the obtained solubilized enzyme solution was used. First, the solubilized enzyme solution was divided into two portions and purified by column chromatography using Polybuffer Exchanger 94 (obtained from Pharmacia) (adsorption:
Buffer A, elution: buffer A 0.0-0.6 M sodium chloride gradient). Of the eluted active fractions, the fractions indicated by the arrow in FIG. 1 were collected and purified again by the polybuffer exchanger 94. The active fraction eluted was
Further, it was successively purified by various column chromatography described below. Ion-exchange column chromatography DEAE Toyopearl 650M (obtained from TOSOH) Adsorption: Buffer A, Elution: Buffer A 0.0-0.3M Sodium chloride concentration gradient Gel filtration column chromatography Toyopearl HW5
5 (obtained from TOSOH) Buffer B (10 mM phosphate buffer (pH 7.5), 0.1%)
Polyoxyethylene octyl phenyl ether, 0.
5% isoamyl alcohol, 1 mM dithiothreitol, 0.1 M sodium chloride, 20% glycerol) Hydroxyapatite column chromatography
(Obtained from Wako Pure Chemical Industries, Ltd.) Adsorption: Buffer B, Elution: Buffer B 10-80 mM phosphate buffer (pH 7.5) Concentration gradient Octyl Sepharose column chromatography CL
4B (obtained from Pharmacia) Adsorption: 50 mM imidazole hydrochloric acid buffer (pH 7.5),
0.5% isoamyl alcohol, 1 mM dithiothreitol, 0.1 M sodium chloride, 20% glycerol Elution: imidazole hydrochloric acid buffer (pH 7.5), 0.1
% Polyoxyethylene octyl phenyl ether, 0.0
5% isoamyl alcohol, 1 mM dithiothreitol, 0.1 M sodium chloride, 20% again like hydroxyapatite column chromatography 1-hexanol affinity column chromatography 6-amino-1-hexanol (obtained from Wako Pure Chemical Industries)
1-Hexanol Sepharose 4B was prepared using CNBr-activated Sepharose 4B (obtained from Pharmacia) according to the manual of Pharmacia as a carrier. Adsorption: 5 mM phosphate buffer (pH 7.5), 0.1% polyoxyethylene octyl phenyl ether, 20% glycerol, 1 mM dithiothreitol Elution: 5 mM
Purification was performed using a phosphate buffer (pH 7.5), 0.1% polyoxyethylene octyl phenyl ether, 20% glycerol, 1 mM dithiothreitol, and a 0.0-0.2 M sodium chloride concentration gradient. When the active fraction obtained at this stage was subjected to SDS polyacrylamide gel electrophoresis and silver staining, a small molecular weight band with no AATase activity was observed.
As shown in FIG. 2, SDS polyacrylamide gel electrophoresis and silver staining were performed as shown in FIG. 2 except for those having a small molecular weight by ultrafiltration using -50 (obtained from Kurabo Industries) according to the attached manual. A purified standard product that became a band was obtained.

Example 2 Characteristics of new AATase 2- (1) Optimal and stable pH To investigate the effect of pH on the stability of the enzyme, the pH range was 3-11 (pH 3-6: 50 mM citrate-). Phosphate buffer, pH 6-8: 50 mM Phosphate buffer, pH 8-9:
50 mM Tris-phosphate buffer, pH 9-11: 50 mM
Glycine-sodium hydroxide buffer) in the range of 4 ° C,
After standing for 22 hours, the pH was adjusted to 7.5 with 0.2 M disodium phosphate, and the enzyme activity was measured by the method of Minetoki et al. In order to investigate the effect of pH on the enzyme activity, the pH range was 3 to 11 (pH 3 to 6:50 mM citrate-phosphate buffer, pH 6 to 8:50 mM phosphate buffer, pH 8 to 9:50 mM. Tris-phosphate buffer, pH 9
~ 11:50 mM glycine-sodium hydroxide buffer)
The enzyme activity was measured by the method of Minetoki et al. Regarding pH stability, it was stable in the pH range of 6.5 to 9.5. The optimum pH was 7.5.

2- (2) Optimal and stable temperature In order to investigate the influence of temperature on the enzyme activity, the enzyme activity was measured at each temperature by the method of Minetoki et al. Further, in order to investigate the influence of temperature on the stability of the enzyme, the enzyme solution was kept at each temperature for 30 minutes, and then the enzyme activity was measured by the method of Minetoki et al. The results are shown in Fig. 3. Regarding the temperature stability, it was stable up to 30 ° C and maintained 60% activity even at 40 ° C. The optimum temperature was 40 ° C.

2- (3) Effect of Inhibitors In order to investigate the effects of various inhibitors on the enzyme activity, various inhibitors were added to the enzyme reaction solution at the concentrations shown in Table 1, and the results of Minetoki et al. The enzyme activity was measured by the method. The results are shown in Table 1. Parachloromercuric benzoic acid, dithiobis (2-
Since this enzyme was strongly inhibited by (nitro) benzoic acid, this enzyme is considered to be SH enzyme.

[0034]

[Table 1]

2- (4) Effect of fatty acid In order to investigate the effect of various fatty acids on the enzyme activity, various fatty acids shown in Table 2 were added to the enzyme reaction solution at a concentration of 2 mM, and at the peak time. The enzyme activity was measured by these methods. Table 2 shows the results. AATas purified by Minetoki et al.
The activity of e was reduced to 32% without addition of linolenic acid and to 4.3% with linoleic acid (Biosci.
Biotech. Biochem. , 57 , (12), p2094,199
3), it can be seen that this enzyme is not inhibited by any fatty acid.

[0036]

[Table 2]

Example 3 Determination of Partial Amino Acid Sequence The determination of the partial amino acid sequence was carried out according to the method (Biochemistry, 63 , p139, 1991) using Iwamatsu's polyvinylidene difluoride membrane (hereinafter abbreviated as PVDF membrane). . 1- (4)
The purified enzyme obtained in 1. was dialyzed against 3 liters of 10 mM formic acid for 1 hour and then freeze-dried. This is the migration buffer
(10% glycerol, 2.5% SDS, 2% 2-
Mercaptoethanol, 62 mM Tris-HCl buffer (p
H6.8)) and subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, the enzyme was transferred from the gel to a 10 cm × 7 cm PVDF membrane “ProBlot” (obtained from Applied Biosystems) by electroblotting. Electroblotting device is KS
"About sample pretreatment method of protein sequencer (1)" edited by Shimadzu Corporation using -8460 type (Marisol)
Electroblotting at 160 mA according to
I went on time. After the transfer, the membrane of the transferred part of the enzyme was cut off, and about 300 μl of a reducing buffer solution (6M guanidine hydrochloride-0.5M Tris hydrochloric acid buffer solution (pH 8.5), 0.3%) was added.
EDTA, 2% acetonitrile), add 1 mg of dithiothreitol, and under argon at 60 ° C, about 1
Time was reduced. To this was added 2.4 mg monoiodoacetic acid dissolved in 10 μl of 0.5N sodium hydroxide solution, and the mixture was stirred for 20 minutes in the dark. Next, PVDF
The membrane was taken out, thoroughly washed with a 2% acetonitrile aqueous solution, and then stirred in a 0.1% SDS solution for 5 minutes. Next, the PVDF membrane was lightly washed with water, immersed in 0.5% polyvinylpyrrolidone 40-100 mM acetic acid, and allowed to stand for 30 minutes. After this, wash the PVDF membrane thoroughly with water to
mm was cut into squares. This was immersed in a digestion buffer (8% acetonitrile, 90 mM Tris-HCl buffer (pH 9.0), 1 pmol of Achromobacter Protease I (obtained from Wako Pure Chemical Industries) was added, and digested at room temperature for 15 hours. Column (obtained from Wako Pure Chemical Industries, Wakosil-II
Reversed-phase high performance liquid chromatography (Hitachi, model L6200) using 5C18AR, 2.0 × 150 mm) was used for separation to obtain several kinds of peptide fragments. Furthermore, Ach
PVDF membrane after digestion with romobacter Protease I
% Aqueous solution of acetonitrile, dip it in digestion buffer (8% acetonitrile, 100 mM ammonium bicarbonate buffer (pH 7.8)), and use Endoproteinase Asp.
1 pmol of -N (obtained from Boehringer Mannheim) was added and digested at 40 ° C for 15 hours. From this digest, several kinds of peptide fragments were obtained by the reverse phase high performance liquid chromatography as described above. Peptide elution solvent is solvent A (0.05% trifluoroacetic acid), solvent B
(2-propanol / acetonitrile (7: 3) containing 0.02% trifluoroacetic acid) was used and the solvent B was 2 to
Elution was performed with a linear gradient of 50% 2-propanol / acetonitrile (7: 3) at a flow rate of 0.25 ml / min for 40 minutes. An amino acid sequence determination test was performed on the peptide fragments obtained from both protease digests by an automatic Edman degradation method using a gas phase protein sequencer PSQ-2 type manufactured by Shimadzu Corporation according to the manual. The results are shown in Table 3. The determined amino acid sequence and the amino acid sequence translated from the AATase gene obtained based on the AATase purified by Minetoki et al. (Appl. Environ. Microbio
l. , 60 (8), p2786, 1994), no identical sequence was found and it is a novel AATase.

[0038]

[Table 3]

Example 4 DNA that produces new AATase
Cloning of Chain from Sake Yeast Society No. 7 4- (1) Preparation of Chromosomal DNA of Sake Yeast Society No. 7 Sake Yeast Society No. 7 was added to 1,000 ml of YPD medium. D. 6
After culturing up to 00 = 10, the cells were collected and washed with autoclaved water. 2 ml of SCE solution (1
M sorbitol, 0.125M EDTA, 0.1M
Trisodium citrate (pH 7), 0.75% 2-mercaptoethanol, 0.01% Zymoriace 100T
(Obtained from Seikagaku Corporation)) and gently shaken at 37 ° C. for about 2 hours to completely transform the yeast into protoplasts. In addition to this, 3.5 ml of lysate per cell (0.5
M Tris-HCl buffer (pH 9), 0.2M EDTA, 3
% SDS) was added and gently stirred. This is 65
The cells were kept warm at 15 ° C for 15 minutes to completely lyse them. After lysis, the mixture was cooled to room temperature, and 23.5 ml of 10% -40% sucrose density gradient solution (0.8M sodium chloride, 0.02M) which had been prepared in Hitachi ultracentrifuge tube 40PA in advance.
Tris-HCl buffer (pH8), 0.01M EDTA, 1
10 ml each was gently placed on 0% -40% sucrose). Using Hitachi Ultracentrifuge SCP85H, 4
The mixture was centrifuged at 26000 rpm for 3 hours. After centrifuging, about 5 ml of the liquid was collected from a place as close as possible to the bottom of the tube using a Komagome pipette. 100 collected samples
0 ml TE solution (10 mM Tris-HCl buffer (pH 8), 1 m
It was dialyzed overnight against M EDTA) to obtain a chromosomal DNA.

4- (2) Preparation of genomic library Next, the chromosomal DNA thus obtained was subjected to the method of Frischau et al. (Methods in Enzymology, 152 , p183, Aca).
demic press, 1987) according to the restriction enzyme Sau3AI.
Partially digested with (obtained from Takara Shuzo) and again 10% -40%
Place on a sucrose density gradient solution, 2500 at 20 ° C
Centrifugation was performed at 0 rpm for 22 hours. After centrifugation, a hole was made in the bottom of the ultracentrifuge tube with an injection needle, and 0.5 ml each was collected in a sampling tube. A part of each of the collected fractions was subjected to agarose gel electrophoresis to confirm the molecular weight of the chromosomal DNA contained in the fractionated liquid, and then 15 to 20 kb of chromosomal DNA was collected and recovered by ethanol precipitation. 1 μg of this chromosomal DNA and 1 μg of λEMBL3 vector DNA of λEMBL3 BamHI vector kit (obtained from Stratagene) were ligated with 1 unit of T4 DNA ligase (obtained from Takara Shuzo) at 16 ° C. overnight. This was packaged using GIGAPACK GOLD (obtained from Stratagene) according to the attached manual, and infected with E. coli P2392 attached to the λEMBL3 BamHI vector kit to obtain about 20
000 plaques were obtained. The obtained plaque was blotted on a nylon membrane filter Hybond-N (obtained from Amersham) according to the protocol of Amersham.

4- (3) Synthesis and labeling of probe Among the partial amino acid sequences obtained in Example 3, AP-3
And DNA synthesizer based on AP-7 sequence
(Applied Biosystems, Model 391), the following synthetic probe (A: adenine, C:
Cytosine, G: guanine, T: thymine) were prepared.

Probe 1 AP-3 Lys Ile Ser Glu Gln 5′-AAA ATT TCT GAA CAA TT-3 ′ G C C G G A A G

Probe 2 AP-7 Lys Met Tyr Ser Asn 5′-AAA ATG TAT TCT AAT GC-3 ′ G C C C A G

All synthetic reagents (FOD phosphoramidite, etc.) used were manufactured by the same company and used according to the attached operator's manual. The obtained synthetic DNA was treated with 2 ml of 27% ammonia water at 55 ° C for 1 hour, and this was subjected to OPC.
Purified with a cartridge (obtained from Applied Biosystems). ECL3 'oligo labeling and detection system using 1 μg of these two kinds of synthetic DNA
It was labeled according to the attached protocol (obtained from Amersham) and used as a probe for plaque hybridization.

4- (4) Isolation of New AATase Gene by Plaque Hybridization Hybridization and signal detection methods are as follows:
The procedure was carried out according to the protocol attached to the ECL3 'oligo labeling and detection system. First, as the first screening, the membrane 2 to which the phage DNA of the genomic library prepared in 4- (2) was transferred
Using 5 probes, which were synthesized and labeled with 4- (3),
After performing hybridization and signal detection at 32 ° C. overnight, the probe was removed from the same membrane, and then the same operation was performed using probe 2, and both probe 1 and probe 2 showed a positive signal in common. Twenty-five plaques were used for the second screen. In addition, the removal of the probe from the membrane was performed by using a 0.4M sodium hydroxide solution at 45 ° C for 3 minutes after the detection.
After gently shaking for 0 minutes, the neutralization solution (0.2 M Tris-HCl buffer (pH 7.5), 0.1% SDS, 0.1 × SS) was added.
The membrane was transferred to C (15 mM sodium chloride, 1.5 mM sodium citrate)) and removed by gentle shaking at 45 ° C. for 30 minutes. The second screen showed a common signal for probe 1 and probe 2 2
Five plaques were punched out from the original agar medium with sterile agar and 100 μl of SM solution (0.1M each).
Sodium chloride, 10 mM magnesium sulfate, 0.0
1% gelatin, 50 mM Tris-HCl buffer (pH 7.
5)). This solution was appropriately diluted with SM solution so that the number of plaques to appear was about 50 per plate, and Escherichia coli P2392 was infected. The 25 plaques that appeared were blotted onto nylon membranes in the same manner as the library preparation. Hybridization was carried out at 35 ° C. overnight using Probe 1, and 12 positive clones were obtained. Next, the phage DNA of the isolated positive clone was analyzed by a conventional method (Molecular Cloning, p77, Cold Spring Har).
bor, USA, 1982). The obtained phage DNA was digested with various restriction enzymes and compared by agarose gel electrophoresis. As a result, 7 out of 12 positive clones were obtained.
The clone was a clone of the same site on the yeast chromosome.

4- (5) Analysis of new AATase gene Southern hybridization using probe 1 and probe 2 was carried out on a common DNA fragment contained in 7 clones, and a new DNA fragment was found on the 5.9 kb HindIII fragment. Since it was found that the AATase gene was contained, this fragment was subjected to agarose gel electrophoresis, purified with a centrifugal filtration tube Ultra Free C3HV (obtained from Millipore), and inserted into pUC18. The resulting recombinant plasmid was used to determine the nucleotide sequence with a Bst DNA sequencing kit (obtained from Bio-Rad). 5.9 kb HindI containing the full length of the DNA chain
A restriction map of the II fragment is shown in FIG. The nucleotide sequence of the portion encoding the new AATase is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the polypeptide translated from the DNA is shown in SEQ ID NO: 2 in the sequence listing. 160 new AATase genes
It had a coding region of 5 bases and encoded 535 amino acids.

4- (6) New AATase in other yeast
Existence of Genes Chromosome DNA was extracted from several yeasts and the new AATas
Its presence was examined by genomic Southern analysis using the e gene as a probe. Extraction of chromosomal DNA from yeast
Davis, R .; Method of W et al. (Method in Enzymology, 6
5 , p. 404, 1980). Extracted chromosomal DNA
After digesting 6 μg with a restriction enzyme PstI (obtained from Takara Shuzo Co., Ltd.), a conventional method (Molecular Cloning, p382, Cold Spri) was used.
ng Harbor Laboratory, USA, 1982). The probe used was the 1.5 kb PstI fragment (range indicated by the arrow) shown in Fig. 4, labeled with the ECL random prime oligo labeling detection system (obtained from Amersham), and hybridization and signal detection were carried out using the attached protocol. The hybridization and membrane washing were carried out at 50 ° C. Results are shown in FIG. All strains including laboratory strains (Beer yeast AJL2155 is Saccha
romyces uvarum , all other Saccharomyces cer
( classified as evi siae ), the same signal as the 1.5 kb signal of Sake Yeast Society No. 7 was observed, and these yeasts have the same gene as the new AATase. In addition, hybridization is usually performed (about 60 ° C).
Despite being performed under milder conditions, there is no other signal, so that the known AATase gene (Appl. Environ. Microbiol., 60 ) present in these yeasts is used.
(8), p2786, 1994), indicating that there is no other DNA fragment that does not hybridize and has homology with the new AATase gene. Further, even if hybridization is similarly carried out using a known AATase gene as a probe, a signal corresponding to the new AATase gene is not observed at all. Therefore, the AATase gene according to the present invention is a novel gene.

Example 5 Construction of vector containing new AATase gene and AATase activity of yeast transformed with this vector pUC into which the new AATase gene obtained in 4- (5) was inserted
18 to 5.4 kb Bam containing the full length of the DNA strand
The HI fragment was obtained. The obtained DNA fragment was added to yeast 2 μ
Yeast vectors YEp13 with an origin of replication and LEU2 gene mDNA marker (Gene, 8, p121,1979)
Was cleaved with the restriction enzyme BamHI (obtained from Takara Shuzo) to prepare an expression vector YAT24 (FIG. 6).
Lithium acetate method (J. Bacteriol., 153 , p163, 1
983), YAT24 was transformed into Saccharomyces cerevisiae TD4 (a, his, leu, ura, trp) to obtain a transformant TD4-AT2-4 strain. In addition, the strain has a consignment number FERMP
Deposited as-14590. Transformant TD4
-AATase activity of AT2-4 strain and control strain was measured as follows. That is, an SD liquid medium (0.67% containing an amino acid mixture except leucine) was added.
Yeast nitrogen base (removed amino acid, obtained from Difco), 2% glucose) was cultivated with shaking in 5 ml at 30 ° C. for about 16 hours, and 1 ml was added to 50 ml of the same medium.
Static culture was carried out at 30 ° C. for 24 hours. The culture solution was centrifuged at 3000 rpm for 10 minutes to collect the bacterial cells, and the solubilized solution (25 m
M imidazole-hydrochloric acid buffer (pH 7.5), 0.1% polyoxyethylene octyl phenyl ether, 0.5%
Isoamyl alcohol, 0.1M sodium chloride, 1m
M dithiothreitol, 20% glycerol) 5ml
After being washed once with, it was suspended in 0.4 ml of a solubilizing solution. 0.8g of glass beads (MK-2GX (diameter 0.25-0.
Cells (obtained from Shinmaru Enterprises Co., Ltd.), and the cells were disrupted by vigorously stirring three times for 30 seconds. 0.8 ml of solubilizing solution was added to this, and after lightly stirring,
Collect the liquid using a Pasteur pipette and
The supernatant obtained by centrifuging for 5 minutes at rotation / minute was used as AATase.
The sample was used for activity measurement. The control strains were Saccharomyces cerevisiae TD4 and the vector YEp13.
The transformed strain was used. The activity was measured according to the method of Minetoki et al., And 1 mM of linoleic acid, an unsaturated fatty acid, was added to a sample obtained from a recombinant of the new AATase gene.
Of which the activity was measured by adding
The AATase activity after standing for 1 hour was also measured. In addition, protein quantification was performed using a Bio-Rad protein assay kit (obtained from Bio-Rad) according to the attached manual. The results are shown in Table 4. The AATase activity of the recombinant was 2.8 times higher than that of the control strain, less inhibition of unsaturated fatty acid was observed, and moreover, 80% or more of the activity was retained even when left at 30 ° C for 1 hour. Admitted.

[0049]

[Table 4]

Example 6 Transformed yeast TD4-AT2-
Fermentation test with 4 strains Saccharomyces cerevisiae TD4-AT2 obtained in Example 5 and transformed with the new AATase gene
Fermentation test was carried out using 4 strains to examine the amount of acetate produced. SD liquid medium (0.67) containing TD4-AT2-4 strain added with an amino acid mixture except leucine.
%, Yeast nitrodien base (removed amino acids, obtained from Difco), 2% glucose (5 ml) at 30 ° C. for about 16 hours with shaking, the resulting culture solution was subjected to YM modified medium (0.3%).
Add 2% to yeast extract, 0.5% peptone, 0.3% malt extract, 15% glucose, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate, pH 6.0) at 30 ° C. Fermented for 48 hours. The culture solution was centrifuged (8,000 rpm, 10 minutes), and the aroma component of the supernatant was analyzed. The results are shown in Table 5. When the yeast of the present invention was used, the production amount increased 1.4 times in ethyl acetate and 4.8 times in isoamyl acetate compared with the control strain containing only vector, and the production amount of acetate ester also increased in the fermentation test. I understand that

[0051]

[Table 5]

[0052]

INDUSTRIAL APPLICABILITY By using the novel AATase gene according to the present invention, it is possible to breed a strain having a more stable AATase activity, such as isoamyl acetate, which has an increased ability to produce an acetate ester.

[0053]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1605 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Saccharomyces cerevisiae (Saccharo
myces cerevisiae) Share name: ATCC 26421 Sequence: ATG GAA GAT ATA GAA GGA TAC GAA CCA CAT ATC ACT CAA GAG TTG ATA GAC CGT GGC CAT GCA AGA CGT ATG GGC CAC TTG GAA AAC TAC TTT GCT GTT TTG AGT AGG CAG TAC TCG AAT TTT ACT GTT TAC GCG GAA TTG AAT AAA GGT GTT AAT AAG AGA CAA CTA ATG CTT GTC TTG AAA CTA TTA CTT CAA AAA TAC TCA ACT CTT GCG CAT ACA ATC ATT CCT AAG CAT TAT CCT CAT CAT GAA GCG TAC TAC TCT AGC GAA GAG TAC CTT AGT AAA CCT TTT CCA CAG CAT GAT TTC ATA AAG GTG ATT TCT CAT CTT GAA TTC GAT GAC TTG ATT ATG AAT AAT CAA CCA GAA TAC AGA GAA GTC ATG GAG AAA ATC TCA GAA CAG TTC AAA AAG GAT GAT TTC TAT AAA GTC ACC AAT AGG TTA ATC GAA TTG ATT AGC CCT GTA ATC ATA CCT GTG GGT AAT CCG AAG AGG CCT AAT TGG AGA TTG ATT TGT TTA CCA GGT AAG GAT ACT GAT GGG TTT GAA ACG TGG AAA AAC TTC GTT TAT GTC ACT AAC CAAC TGC GGC TCC GAC GGT GTC AGT GGA TCG AAT TTT TTC AAA GAT TTA GCT CTA CTC TTT TGT AAA ATC GAA GAA AAA GGG TTT GAT TAT GAT GAA GAG TTC ATC GAA GAT CAG GTC ATC ATT GAC TAT GAT CGA GAC TAC ACT GAA ATT TCT AAA TTG CCA AAA CCG ATT ACG GAT CGT ATT GAC TAC AAG CCA GCA TTG ACT TCA TTA CCC AAA TTC TTT TTA ACA ACC TTC ATT TAT GAA CAT TGT AAT TTT AAA ACC TCC AGC GAA TCT ACA CTT ACA GCT AGA TAT AGC CCC TCT ACT AAT GCT AAT GCT AGT TAC AAT TC TTG TTG CAT TTC AGT ACT AAG GAA GTA GAA CAA ATC AGA GCC CAG ATC AAG AAA AAT GTT CAC GAT GGG TGC ACC CTA ACA CCC TTC ATT CAA GCG TGC TTT CTT GTA GCC CTG TAT AGA CTA GAT AAG TTG TTC ACA AAA TCT CTT CTC GAG TAT GGG TTC GAT GTG GCT ATT CCA AGC AAC GCA AGA AGG TTT TTA CCA AAC GAT GAA GAG TTA AGA GAT TCT TAT AAA TAC GGC TCC AAC GTT GGA GGT TGT CAT TAC GCC TAT CTA ATC TCC TCA TTC GAC ATT CCC GAA GGT GAC AAT GAC AAG TTT TGG AGT CTT GTC GAA TAC TAC TAT GAC CGC TTT TTA GAA TCG TAC GAC AAC GGT GAC CAC TTG ATT GGT CTG GGG GTC CTA CAA CTT GAT ATC GTT CAA AAC AAG AAT ATA GAC AGC CTT CTT GCC AAC TCT TAT TTG CAC CAG CAA AGA GGC GGT GCA ATC ATC AGT AAT ACT GGA CTT GTC TCG CAA GAT ACG ACC AAG CCG TAC TAC GTT CGG GAT TTA ATC TTC TCG CAGCT G GC GCC TTG AGA TTT GCG TTC GGC CTA AAC GTT TGC TCC ACA AAC GTG AAT GGT ATG AAC ATG GAC ATG AGC GTG GTT CAG GGC ACT CTA CGG GAT CGT GGC GAA TGG GAA TCG TTC TGC AAG CTC TTC TAC CAA ACC ATC GGC GAA GATC GCG TCG CTT

SEQ ID NO: 2 Sequence length: 535 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence: Met Glu Asp Ile Glu Gly Tyr Glu Pro His Ile Thr Gln Glu Leu Ile Asp Arg Gly His Ala Arg Arg Met Gly His Leu Glu Asn Tyr Phe Ala Val Leu Ser Arg Gln Lys Met Tyr Ser Asn Phe Thr Val Tyr Ala Glu Leu Asn Lys Gly Val Asn Lys Arg Gln Leu Met Leu Val Leu Lys Leu Leu Leu Gln Lys Tyr Ser Thr Leu Ala His Thr Ile Ile Pro Lys His Tyr Pro His His Glu Ala Tyr Tyr Ser Ser Glu Glu Tyr Leu Ser Lys Pro Phe Pro Gln His Asp Phe Ile Lys Val Ile Ser His Leu Glu Phe Asp Asp Leu Ile Met Asn Asn Asn Gln Pro Glu Tyr Arg Glu Val Met Glu Lys Ile Ser Glu Gln Phe Lys Lys Asp Asp Phe Lys Val Thr Asn Arg Leu Ile Glu Leu Ile Ser Pro Val Ile Ile Pro Val Gly Asn Pro Lys Arg Pro Asn Trp Arg Leu Ile Cys Leu Pro Gly Lys Asp Thr Asp Gly Phe Glu Thr Trp Lys Asn Phe Val Tyr Val Thr Asn His Cys Gly Ser Asp Gly Val Ser Gly Ser Asn Phe Phe Lys Asp Leu Ala Leu Leu Phe Cys Ly s Ile Glu Glu Lys Gly Phe Asp Tyr Asp Glu Glu Phe Ile Glu Asp Gln Val Ile Ile Asp Tyr Asp Arg Asp Tyr Thr Glu Ile Ser Lys Leu Pro Lys Pro Ile Thr Asp Arg Ile Asp Tyr Lys Pro Ala Leu Thr Ser Leu Pro Lys Phe Phe Leu Thr Thr Phe Ile Tyr Glu His Cys Asn Phe Lys Thr Ser Ser Glu Ser Thr Leu Thr Ala Arg Tyr Ser Pro Ser Thr Asn Ala Asn Ala Ser Tyr Asn Tyr Leu Leu His Phe Ser Thr Lys Glu Val Glu Gln Ile Arg Ala Gln Ile Lys Lys Asn Val His Asp Gly Cys Thr Leu Thr Pro Phe Ile Gln Ala Cys Phe Leu Val Ala Leu Tyr Arg Leu Asp Lys Leu Phe Thr Lys Ser Leu Leu Glu Tyr Gly Phe Asp Val Ala Ile Pro Ser Asn Ala Arg Arg Phe Leu Pro Asn Asp Glu Glu Leu Arg Asp Ser Tyr Lys Tyr Gly Ser Asn Val Gly Gly Ser His Tyr Ala Tyr Leu Ile Ser Ser Phe Asp Ile Pro Glu Gly Asp Asn Asp Lys Phe Trp Ser Leu Val Glu Tyr Tyr Tyr Asp Arg Phe Leu Glu Ser Tyr Asp Asn Gly Asp His Leu Ile Gly Leu Gly Val Leu Gln Leu Asp Phe Ile Val Gln Asn Lys Asn Ile Asp Ser Leu Leu Ala Asn Ser Tyr Leu His Gln Gln Arg Gly Gly Ala Ile Ile Ser Asn Th r Gly Leu Val Ser Gln Asp Thr Thr Lys Pro Tyr Tyr Val Arg Asp Leu Ile Phe Ser Gln Ser Ala Gly Ala Leu Arg Phe Ala Phe Gly Leu Asn Val Cys Ser Thr Asn Val Asn Gly Met Asn Met Asp Met Ser Val Val Gln Gly Thr Leu Arg Asp Arg Gly Glu Trp Glu Ser Phe Cys Lys Leu Phe Tyr Gln Thr Ile Gly Glu Phe Ala Ser Leu

[Brief description of drawings]

FIG. 1 is a diagram showing an elution pattern of an active fraction by the polybuffer exchanger 94 of the new AATase according to the present invention.

FIG. 2 is a photograph of SDS polyacrylamide gel electrophoresis before and after ultrafiltration by Centricut, and a silver stain, of the active fraction eluted with the affinity column of the new AATase according to the present invention.

FIG. 3 is a drawing showing the influence of temperature on the enzymatic activity of the new AATase according to the present invention.

FIG. 4 is a restriction map of the new AATase gene according to the present invention.

FIG. 5 is a photograph of electrophoresis for genomic Southern analysis using the novel AATase gene according to the present invention.

FIG. 6 is a drawing showing the steps for constructing a yeast expression vector YAT24 for a new AATase gene according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1: 865) (72) Inventor Akihiko Iwamatsu 6-26-1, Jingumae, Shibuya-ku, Tokyo Kirin Beer Within the corporation

Claims (5)

[Claims] 1. A molecular weight as measured by SDS polyacrylamide gel electrophoresis, which is stable at a temperature of 30 ° C. and is not inhibited by unsaturated fatty acids, is approximately 63,000.
A novel alcohol acetyltransferase derived from yeast, characterized in that 2. A novel alcohol acetyltransferase, which is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 3. A novel alcohol acetyl transferase gene, which comprises a DNA sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing. 4. A DNA chain having a DNA sequence shown in SEQ ID NO: 1 of the sequence listing, or a nucleotide sequence substantially homologous to at least a part thereof and capable of producing a novel polypeptide having alcohol acetyltransferase activity. . 5. A novel alcohol acetyltransferase gene or DN according to claim 3 or 4, wherein the foreign gene is a yeast obtained by introducing a trait of the foreign gene by introducing the foreign gene.
A transformed and enriched yeast capable of producing a novel alcohol acetyltransferase, which is characterized by being an A chain.