JPH0799979A - New gene, transformant using the same and use thereof - Google Patents

Abstract

PURPOSE:To obtain a new gene encoding alpha-amylase derived from Aspergillus kawachii, providing a large amount of an acid-resistant, thermostable and raw starch hydrolyzing alpha-amylase by gene manipulation, capable of directly producing an alcohol by transducing it into a yeast. CONSTITUTION:A conidium of Aspergillus kawachii IFO 4308 is inoculated into a medium and subjected to rotary shaking culture at 30 deg.C for 120 hours to give a mold. The prepared cell is collected by a filter, frozen with liquid nitrogen and ground and whole RNA is extracted from the ground cell by guanidine thiocyanate method and mRNA is isolated from the whole RNA by using an oligo alphaT column. Then the mRNA is used as a template to synthesize cDNA, a cDNA library is prepared by a conventional method and screened by using a DNA fragment containing part of a gene encoding an acid-resistant alpha-amylase as a probe to select a positive clone and to recover DNA, which is treated with a restriction enzyme to give the objective new gene encoding alpha-amylase having acid resistance, thermostability and raw starch hydrolyzing proprety.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel α-amylase gene obtained from Aspergillus kawachi, a vector containing the same, a transformant obtained by transferring the vector into Aspergillus or Saccharomyces cerevisiae, and its use. Is.

The acid-resistant α-amylase of Aspergillus kawachi is an α-amylase which is not inactivated under acidic conditions, and if Aspergillus sp. Or Saccharomyces cerevisiae transformed with a vector containing a gene producing this is used. It is now possible to produce alcoholic beverages, alcohols, etc., which greatly contributes to the fermentation industry.

Acid resistance α of Aspergillus kawachi
-Because amylase can break down raw starch,
By using Aspergillus or Saccharomyces cerevisiae transformed with a vector containing a gene producing this, it becomes possible to produce alcoholic beverages, alcohols and the like from raw starch, which greatly contributes to the fermentation industry.

[0004]

2. Description of the Related Art The α-amylase currently used for sake brewing is an enzyme produced by Aspergillus oryzae, has low acid resistance, and has a stable pH in the range of 5-9. When the pH is 3 or less, 100% is deactivated. Also, it has no decomposing power for raw starch.

On the other hand, α-amylase secreted and produced by Aspergillus kawachi, which is widely used in shochu brewing and the like, has higher heat resistance and acid resistance than that of Aspergillus oryzae α-amylase (taka amylase A), and has a pH of 3
It also reacts in the following acidic range.

[0006] As a salient feature that is not found in the α-amylases of the genus Aspergillus of other species, the α-amylase of Aspergillus kawachi has the ability to decompose raw starch. It is expected to be used.

[0007] Separately, Saccharomyces cerevisiae is a microorganism widely used in alcoholic fermentation such as brewing alcohol, but since it does not have the ability to produce α-amylase, it is directly used as a raw material for alcohol production. Fermentation is not possible. However, in recent years, Saccharomyces cerevisiae capable of producing an enzyme capable of directly saccharifying starch for the purpose of simplifying the brewing process has been sought, but a strain satisfying the purpose has not been isolated.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to efficiently produce α-amylase having acid resistance, heat resistance and raw starch degrading property and to efficiently brew alcohol or liquor. Is achieved by.

[0009]

[Means for Solving the Problems] As a result of earnest research for achieving the above-mentioned object, the present inventors have focused on the acid-resistant α-amylase gene of Aspergillus kawachi and prepared total mRNA of Aspergillus kawachi, CD based on this
The NA library was prepared, and a clone encoding the acid-resistant α-amylase of Aspergillus kawachi was successfully isolated from it. Then, a transformant that succeeded in determining the nucleotide sequence of the acid-resistant α-amylase gene, ligated this cDNA to an expression vector, and then transferred into Saccharomyces cerevisiae to produce an α-amylase equivalent to Aspergillus kawachi. The present invention has been completed, and the present invention has been completed.

That is, the present invention (1) has the sequence represented by SEQ ID NO: 1 in the sequence listing as one of the basic technical ideas, and further includes the following aspects.

(2) cDNA sequence of α-amylase isolated from Aspergillus kawachi. (3) A vector for secreting α-amylase of Saccharomyces cerevisiae, which comprises the cDNA of (2). (4) A transformant which secretes α-amylase having acid resistance, heat resistance and raw starch degrading power, which is obtained by transferring the vector of (3) into Saccharomyces cerevisiae. (5) Production of acid-resistant α-amylase or efficient alcoholic fermentation by culturing the transformant of (4).

The present invention will be described below. The donor of mRNA used in the present invention is Aspergillus kawachi for shochu, and specifically, for example, Asper.
gillus kawachii (IFO 4308)
Is.

In order to carry out the present invention, Aspergillus kawachi I is produced under conditions that produce a high amount of acid-resistant α-amylase.
The FO 4308 strain is cultured, total RNA is extracted, and then cDNA is synthesized by a conventional method to prepare a cDNA gene library.

On the other hand, a primer was synthesized from the peptide sequence of the acid-resistant α-amylase purified enzyme based on the amino acid sequence near the N-terminus and the active center, a probe was prepared by the PCR method, and screening was carried out from a cDNA library to obtain a positive clone. To get

CDNA is recovered from the positive clone, and the cDNA is introduced into yeast (S. cerevisiae) with a plasmid vector to obtain a transformant. And this transformant is utilized for various purposes.

The present invention will be described in more detail below.

First, all mR from Aspergillus Kawachi
NA is extracted and a cDNA library is prepared based on the extracted NA, which is performed by the method described below.

That is, by using starch as a sole carbon source and tryptone as a sole nitrogen source, a medium for inducing α-amylase production was adjusted to pH 3.0, and cultured from Aspergillus kawachi cells. Total RNA is extracted by the method described below in the examples. From the obtained total RNA, Proc. Natl. Acad. Sci. USA , V
ol. 69, 1408-1412 (1972), poly A-containing RNA is purified using oligo (dT) -sepharose to obtain total RNA. This total mRNA
CDNA from Gene , Vol. 222,263-2
69 (1983) and synthesized by the method described in Science.
e , Vol. Was introduced into phage DNAλgt10 by the method described in 222,778~782 (1983), in
After packaging in vitro , a gene library of cDNA is prepared.

From the gene library obtained above,
Cloning of the α-amylase gene cDNA
It is carried out by plaque hybridization using a base sequence of about 0.9 Kbps as a probe by the PCR method using the base sequence deduced from the partial amino acid sequence of the enzyme α-amylase produced by Aspergillus kawachi as a primer. The isolated α-amylase gene cDNA
The coding region contained in A had a partial amino acid sequence of the enzyme α-amylase produced by Aspergillus kawachi.

Α of Aspergillus kawachi obtained next
-Inserting the cDNA for the amylase gene into a Saccharomyces cerevisiae expression vector, for example the yeast expression vector pYCDE1 with the yeast alcohol dehydrogenase gene (ADH1) promoter,
A transformant is created by transferring into S. cerevisiae. As transformation methods, known methods such as J. Bacteriol , Vol. 153,163 ~
The method described in 168 (1983) or a method similar thereto is adopted.

Examples of the present invention will be described below.

[0022]

Example 1 Construction of Aspergillus kawachi cDNA Library:

Aspergillus Kawachi IFO 4308
PH of conidiospores of 1 platinum loop with citrate buffer
It was adjusted to 3.0. Starch-peptone medium (2.0%
Starch, 0.3% NaNO ₃ , 0.1% K ₂ HPO ₄ ,
_{0.05% KCl, 0.05% MgSO 4} · 7H 2 O,
0.001% FeSO ₄ .7H ₂ O, 1.0% tryptone, pH 3.0) was inoculated and cultivated by rotary shaking at 30 ° C. for 120 hours, and the obtained cells were collected with a filter and washed with water. Next, the bacterial cells were added to a pre-cooled mortar together with several g of glass beads (diameter: about 0.5 mm), and 20 to 50 ml of liquid nitrogen was added thereto, frozen, and ground. Further, liquid nitrogen was added in the same manner, and the grinding operation was repeated twice more. Then, 10 ml of a 6M guanidine thiocyanate aqueous solution was added to the obtained crushed cells, and an equal amount of phenol was added thereto, followed by shaking for 5 minutes. . 1200 this mixture
The mixture was subjected to cooling centrifugation at 0 rpm for 10 minutes to separate the aqueous layer. This aqueous layer was treated twice with an equal amount of phenol and then twice with chloroform-phenol (1: 1) to remove contaminating proteins, and then 1/10 volume of 3M sodium acetate was added. Add 2 volumes of cold ethanol,
Total RNA was lyophilized and then dissolved in 2 ml of water. By this series of operations, 3.5 mg of total RNA was obtained.

Next, extraction of mRNA from total RNA was performed by the following method. Generally, eukaryotic mRNA has poly A at the 3'end. M of Aspergillus Kawachi
In the case of RNA as well, it is considered to have poly A.
Cloning "pp197-198, Cold Sp
It was performed by the method described in the Ring Laboratory (1982). As the oligo dT column, 0.5 g of type 7 manufactured by Pharmacia was used. 2 mg by this method
10 μg of mRNA was obtained from the total RNA.

Next, cDNA was prepared from this mRNA by the following method. The principle is Mol. Celi. Bio
l . , Vol. 2 , 161-170 (1982) and G
ene , Vol. 25, 263-269 (1983), the RNase H method was used, and cDNA was used for the actual operation.
A synthesis system plus (manufactured by Amersham) was used. By this series of operations, 1.5 μg from 5 μg mRNA
CDNA was obtained.

Next, using this cDNA, a library was prepared by the following method. First, the above-mentioned c
1 μg of DNA was treated with EcoRI methylase and c
All restriction enzyme EcoRI cleavage sites present in the DNA strand were masked. Then, the Eco
After ligating the RI linker with T4 DNA ligase, it was digested with the restriction enzyme EcoRI to obtain cDNA having EcoRI cohesive ends at both ends. These cDNA tie into the EcoRI site of the phage vector λgt10, in
After performing the vitro packaging, E. coli
NW514 was transfected to prepare a cDNA library (about 200,000 plaques). The procedure for cloning this cDNA is the cDNA cloning system λgt10 kit (manufactured by Amersham).
Was performed using.

[0027]

Example 2 Cloning of Aspergillus kawachi acid-resistant α-amylase (AAA) cDNA from a cDNA library:

After forming plaques of the phages stored in the cDNA library described in Example 1, p-hybridization was performed to detect acid-resistant α-amylase c.
Selection of clones containing DNA was performed. This series of operations is described in "Molecular Cloning" pp. 63
-67, Cold Spring HarborLab
According to the usual method described in oratory (1982). The probe used for plaque hybridization was prepared by PCR using the cDNA as a template by synthesizing a base sequence deduced from the amino acid sequence shown below as a primer.

5 ′ 3 ′ Leu-Ser-Ala-Ala-Glu-Trp-Arg-Thr-Gln 5 ′ 3 ′ Ile-Glu-Asn-His-Asp-Asn-Pro-Arg

By this method, three positive clones which hybridize with the above-mentioned probe were selected from about 4,500 plaques, and these were cloned into E. coli. coli
Ligated into the plasmid vector pUC118 of
118-AAA cDNA was obtained.

[0031]

Example 3 Acid-resistant α-amylase (AA of Aspergillus kawachi)
A) Determination of the DNA sequence of the coding region contained in the cDNA:

PUC118-AAAc described in Example 2
For DNA DN of coding region derived from cDNA
In determining the A sequence, pUC118-AAAcD
NA was treated with various restriction enzymes to prepare subclones.
By sequencing this subclone, the DNA sequence shown in SEQ ID NO: 1 of the sequence listing shown in Tables 1 and 2 below was obtained. The total DNA sequence is 2142 bp
s, which encodes a peptide consisting of 671 amino acids beginning with Met.

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

Example 4 Active Aspergillus kawachi α-amylase (AA)
Production of Saccharomyces cerevisiae that produces:

PU described in Example 2 containing the coding region for α-amylase shown in SEQ ID NO: 1 of the Sequence Listing
The cDNA of the C118-AAA cDNA clone was ligated to the expression vector of yeast Saccharomyces cerevisiae,
Secretion production of AAA becomes possible by transferring it into yeast and expressing it.

CDNA cleaved from the pUC118-AAA cDNA clone with the restriction enzyme EcoRI (2.1 k
bps) by 1% agarose gel electrophoresis,
It was ligated to an expression vector as shown in FIG. The vector used was Method in Enzymology, 1
01, pp. PYC described in 192-201 (1983)
It is DE1. This vector is 2μDN capable of replicating in yeast
A, E. E. coli pBR322, yeast TRP1 as a marker gene, alcohol dehydrogenase gene (ADH1) as a yeast promoter, and cytochrome CI gene (CYC1) as a terminator. The heterologous DNA insertion site is the EcoRI site. After treating this vector with EcoRI, the phosphate at the 5'end was removed using bacterial alkaline phosphatase, and the cDNA fragment of AAA was added thereto and ligated with T4 DNA ligase to obtain pYEAA.

The following pYEAA was transformed into E. coli HB
101, and was purified in a large amount by a conventional method. Subsequently, the purified pYEAA was added to J. Bacteriol. ，
Vol. 153, 163-168 (1983) according to the method described in yeast host DBY746 (a trpl-28).
9 his 3Δ1 leu 2-3-112 ura
3-52) and complemented with the tryptophan requirement, a transformant (A) capable of growing in a medium containing no tryptophan was obtained. This transformant was inoculated into 100 ml of synthetic medium having the following composition,
Shaking culture was carried out for 120 hours.

Synthetic medium: 2% glucose, 0.67% yeast nitrogen pace (manufactured by Difco), 24 mg /
Uracil, 24 mg / 1 L-histidine, 36 mg /
1 L-leucine, pH 5.7.

After culturing, the bacterial cells collected by centrifugation at 3000 rpm for 10 minutes were added to a pre-cooled mortar together with several g of glass beads (diameter of about 0.5 mm),
Liquid nitrogen (20 to 50 ml) was added to this, and the mixture was frozen and ground. Similarly, liquid nitrogen was added and the milling operation was repeated twice more, and then 10 ml of 0.2 M acetate buffer was added to the resulting milled cells. The obtained liquid is 15000 rpm, 1
The solution was cooled and centrifuged for 0 minutes, and the supernatant was collected. Α- in the supernatant
Amylase activity was measured and is shown in Table 3 below.

[0041]

[Table 3]

[0042]

Example 5 Acid Resistance of α-Amylase Obtained from Transformant (A): pH stability of α-amylase obtained in Example 4 after holding at 30 ° C. for 3 hours at each pH is shown. 2 shows. p
It was stable in the range of H2-7.

[0043]

Example 6 Thermostability of α-amylase obtained from transformant (A): The thermostability of α-amylase obtained from Example 4 at each temperature is shown in FIG. After 60 minutes at 60 ° C, it had a residual activity of at least 80%.

[0044]

Example 7 Raw starch degrading activity of α-amylase obtained by transformant (A):

The amount of reducing sugars released by reacting 500 ml of α-amylase (70 units / ml) obtained in Example 4 with 50 mg of raw starch at 30 ° C. was measured by the phenol-sulfuric acid method. The time course of production of reducing sugar is shown in FIG. It was confirmed that raw starch was decomposed and free reducing sugar was produced.

[0046]

Industrial Applicability According to the present invention, the sequence encoding the acid-resistant α-amylase gene was clarified for the first time, and it was successfully expressed in yeast.

The acid-resistant α-amylase has not only acid resistance but also heat resistance and raw starch degrading property, and can be widely used for various purposes. For example, when Saccharomyces cerevisiae transformed with this gene is used, saccharification and alcohol fermentation can be carried out directly using starch as a raw material under acidic conditions, and labor saving in alcohol and liquor production can be achieved.

[Brief description of drawings]

FIG. 1 is a construction diagram of a plasmid for expressing α-amylase, pYEAA.

[Fig. 2] p of α-amylase obtained by the transformant
H stability is shown.

FIG. 3 shows the thermostability of α-amylase obtained from the transformant.

FIG. 4 shows the raw starch degrading activity of α-amylase obtained by the transformant.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // (C12N 15/09 ZNA C12R 1:66) (C12N 1/19 C12R 1: 865) (C12N 9/28 C12R 1: 865) C12R 1:66) (72) Inventor Akihiro Kaneko 1-54-18 Takinogawa, Kita-ku, Tokyo Inside Institute for Brewing Resources, Inc. (72) Inventor, Gakuzo Tamura, Kita-ku, Tokyo Takinogawa 1-54-18 Stock Company Brewing Resources Institute

Claims (7)

[Claims] 1. A base sequence represented by SEQ ID NO: 1 in the sequence listing. 2. The base sequence according to claim 1, wherein the base sequence is a cDNA sequence synthesized from α-amylase mRNA of Aspergillus kawachi. 3. A vector of Saccharomyces cerevisiae, which comprises the sequence according to claim 1 or 2. 4. A transformant having the ability to produce an acid-resistant α-amylase, which is obtained by transferring the vector according to claim 3 into Saccharomyces cerevisiae. 5. A method for producing an acid-resistant α-amylase, which comprises culturing the transformant according to claim 4. 6. The method according to claim 5, wherein the α-amylase is an enzyme having acid resistance, thermostability, and raw starch decomposing ability. 7. The acid resistance of α-amylase is pH 2-7.
7. The method according to claim 6, wherein the method is stable in the range, and has a heat resistance of 60% at 60 ° C. and a residual activity of at least 80%.