JP4372986B2 - Alkaline pullulanase - Google Patents

Description

【００３１】

【００３２】

【００３３】

【００３４】

【００３５】

【００３６】

【００３７】

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【００４１】

【図面の簡単な説明】
【図１】図１は、アルカリプルラナーゼ遺伝子(Bacillus sp. KSM-AP1876株由来)のを含有したプラスミド(ｐＡＰ１００)の制限酵素地図を示す図である。
【図２】図２は、アルカリプルラナーゼのｐＨプロファイルを示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an alkaline pullulanase useful as a detergent enzyme and a gene encoding the same.
[0002]
[Prior art and problems to be solved by the invention]
Pullulanase is a kind of amylolytic enzyme that cleaves only α-1,6 glucoside bond existing in starch, glycogen, amylopectin or pullulan molecule, and finally generates maltotriose. Endo-amylase and exo-type By using in combination with amylase, malto-oligosaccharides such as glucose, maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose can be produced from starch. is there.
[0003]
On the other hand, pullulanase can also be used as a detergent enzyme because it can decompose and remove starch adhering to tableware and fibers, and the present inventors have incorporated starch puranase together with amylase into dishwashing or clothing detergents to provide starch. It has been found that the detergency against dirt is greatly improved (JP-A-2-132193).
[0004]
However, most of the pullulanases found in nature exhibit the maximum enzyme activity in the neutral to acidic region, and have the maximum enzyme activity in the alkaline region, which is a necessary condition for the detergent composition. Alkaline pullulanase has been previously reported by Nakamura and Horikoshi (Biochim. Biophys. Acta, 397, 188 (1975), Japanese Patent Publication No. 53-27786) and Bacillus sp ( Bacillus sp. .) An enzyme derived from the KSM-AP1876 strain (JP-A-3-87176) is only known.
Accordingly, there is a demand for an alkaline pullulanase that exhibits sufficient enzyme activity even in the presence of a surfactant and is highly useful as a detergent enzyme.
[0005]
An object of the present invention is to efficiently produce a large amount of an alkaline pullulanase that operates in an alkaline region and exhibits sufficient enzyme activity even in the presence of a surfactant, a gene encoding the same, and a genetic engineering technique. It is to provide a method for doing this.
[0006]
[Means for Solving the Problems]
The present inventors have found an alkaline pullulanase that acts in an alkaline region from enzymes produced by microorganisms in soil, and have succeeded in cloning a gene encoding the alkaline pullulanase and establishing a production technique by gene recombination. .
[0007]
That is, the present invention encodes an alkaline pullulanase having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added, and the alkaline pullulanase. A gene is provided.
[0008]
Furthermore, the present invention provides a recombinant vector containing the above alkaline pullulanase gene, a transformant containing the recombinant vector, and a method for producing alkaline pullulanase using these.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The alkaline pullulanase of the present invention has an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added, and has an alkaline pullulanase activity. Unless deleted, amino acid deletion, substitution or addition (hereinafter sometimes referred to as mutation) in the amino acid sequence is not particularly limited, but it is 80% or more homologous with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. It is preferable to have. Moreover, 1 to several amino acids may be added or deleted at the N-terminus in the amino acid sequence of SEQ ID NO: 1.
Here, the amino acid sequence represented by SEQ ID NO: 2 corresponds to the 209th to 1028th amino acid sequences represented by SEQ ID NO: 1, and a DNA fragment (SEQ ID NO: 4) encoding such amino acid is expressed. When ligated to the plasmid vector pHSP64 (Sumitomo, N. et al., Biosci, Biotech, Biochem. 59, 2172-1995, 1995) and expressed by Bacillus subtilis , an alkali having the amino acid shown in SEQ ID NO: 1 An alkaline pullulanase having an enzyme activity similar to that of pullulanase is obtained.
[0010]
The alkaline pullulanase gene of the present invention may be any gene as long as it encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the variant thereof as long as the alkali pullulanase activity is not impaired, and SEQ ID NO: 3 or SEQ ID NO: 4 respectively. Or a base sequence in which one or several bases of the base sequence are deleted, substituted, or added.
[0011]
The amino acid sequence of the alkaline pullulanase of the present invention and the base sequence of the gene have a unique amino acid sequence and base sequence different from those of the pullulanase known so far, and as described later, its enzymatic properties are also as follows: It is a novel enzyme different from the conventional pullulanase, including the enzyme reported by Arara (JP-A-3-87176).
[0012]
The alkaline pullulanase of the present invention is a microorganism isolated from nature, such as Bacillus sp. KSM-AP1876 strain, which has been isolated from soil in Yokohama, Kanagawa Prefecture and deposited as MICRO Kenjo No. 3049 (FERM BP-3049). These mutants can be obtained by culturing and collecting from the obtained culture, but a gene encoding the alkaline pullulanase is obtained, and a recombinant vector is prepared using the gene, and the recombinant vector is used. According to the method of transforming host cells, culturing the obtained transformant, and collecting alkaline pullulanase from the culture, mass production can be achieved.
[0013]
The alkaline pullulanase gene of the present invention can be obtained by using, for example, a DNA fragment obtained from an alkaline pullulanase-producing bacterium such as Bacillus sp. KSM-AP1876 described above by, for example, total or partial degradation of chromosomal DNA with an appropriate restriction enzyme. It can be cloned by using a shotgun method that is incorporated into Escherichia coli, introduced into E. coli, Bacillus subtilis, or the like and expressed, or by synthesizing appropriate primers and using the PCR method.
Here, the chromosomal DNA is obtained from the donor strain by, for example, the method of Marmur (J., J. Mol. Biol., 3, 208 (1961)), the method of Saito and Miura (Biochem. Biophys. Acta, 72, 619 (1963)) or What is necessary is just to acquire using another similar method, and what kind of restriction enzyme can be used if it does not cut | disconnect the said gene.
When using the PCR method, for example, based on the amino acid sequence shown in SEQ ID NO: 1, a primer having a sequence corresponding to the upstream position of the 5 ′ end and the downstream position of the 3 ′ end of the active expression essential region is synthesized, An alkaline pullulanase gene can be obtained by performing a PCR reaction using the chromosomal DNA of an alkaline pullulanase-producing bacterium as a template.
[0014]
As a host / vector system used for the production of a recombinant vector containing a gene fragment, the host strain can express the alkaline pullulanase gene of the present invention, and the recombinant DNA can be replicated and integrated in the host fungus. Any gene can be used as long as it can stably retain the gene. Examples include the EK system using Escherichia coli K-12 strain as the host, and the BM system using the Marburg strain of Bacillus subtilis as the host. Here, host strains include, for example, the HB101 strain, the C600 strain, the JM109 strain, and the BM strain in the EK strain, for example, the BD170 strain, the MI112 strain, and the ISW1214 strain, and the vectors include, for example, pBR322, Examples of pHY300PLK and pUC18, and examples of BM systems include pUB110 and pHY300PLK, and vectors constructed using these can also be used.
A preferred example of the recombinant DNA containing the gene of the present invention is plasmid pAP100 (FIG. 1). This plasmid is a 10.1 kbp recombinant plasmid comprising a fragment containing the gene of the present invention of 5.0 kbp and a part of pHY300PLK. A preferred example of the recombinant microorganism containing the recombinant DNA is E. coli HB101 (pAP100) strain.
[0015]
For example, in the case of an EK host strain, the method of transformation of a host strain with recombinant DNA is the calcium chloride method (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), etc. In the case of a BM host strain, a protoplast method (Chang, C. and Cohen. SN, Mol. Gen. Genet., 168, 111 (1978)) or the like can be used.
[0016]
For selection of recombinant microorganisms, the transformed cells are transplanted and cultured on agar plates containing pullulan or red pullulan by the replica method, etc., then colonies appear, and halos are formed on the pullulan agar plates. This can be done by detecting colonies that can do. Recombinant DNA retained by the obtained recombinant microorganism is obtained by using a normal plasmid preparation method or phage DNA preparation method (Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, New York, (1982)). Furthermore, it can be confirmed that the recombinant DNA is a combination of the vector DNA and the DNA fragment containing the alkaline pullulanase gene by analyzing the cleavage pattern of various restriction enzymes by electrophoresis or the like.
[0017]
The transformant can be cultured by inoculating a medium containing an assimilable carbon source, nitrogen source and other essential nutrients according to a conventional method. From the obtained culture broth, a generally known enzyme can be cultured. Alkaline pullulanase can be obtained by a method according to the collection and purification method.
[0018]
The alkaline pullulanase of the present invention thus obtained has the amino acid sequence represented by SEQ ID NO: 1 or 2, and its enzymatic property is that the optimum reaction pH is 8.5 to 9.5 as shown below. Has characteristics.
[0019]
<Enzymological properties>
(1) Hydrolysis of α-1,6 glucoside bond of action pullulan to produce maltotriose. It also hydrolyzes the α-1,6 glucoside bond of starch, amylopectin and glycogen.
(2) Optimum reaction pH
The pH is 8.5 to 9.5.
(3) Stable pH
It is extremely stable in the pH range of 7-10.
(4) It operates at an optimum reaction temperature of 50 to 55 ° C.
(5) Heat resistance is stable up to 50 ° C. (retains 85% or more activity in a 30-minute treatment at 50 ° C. in a buffer solution of 10 mM Tris / HCl (pH 8.5)).
(6) Molecular weight The estimated molecular weight of the wild type by SDS-polyacrylamide gel electrophoresis is 120,000 to 130,000.
[0020]
Therefore, the alkaline pullulanase of the present invention exhibits excellent enzyme activity even in the presence of a surfactant, and is used in the form of a liquid, powder, granule or the like as an enzyme for detergents, for example, detergents for clothes, dishwashing detergents, etc. can do.
[0021]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples. In addition, all the density | concentrations in a present Example show weight%.
[0022]
Example 1
Bacillus sp. KSM-AP1876, which produces alkaline pullulanase, was added to 5 mL of A medium (2.8% bactopeptone, 0.05% yeast extract, 0.5% fish extract, KH ₂ PO ₄ 0.1%, MgSO _4. 7H ₂ O 0.02%, sodium carbonate 1.0%), and after 24 hours of shaking culture at 30 ° C., 1 mL of this was inoculated into 100 mL of the same medium and further shaken at 30 ° C. for 12 hours. did. Thereafter, the cells were collected by centrifugation, and about 1 mg of chromosomal DNA was obtained according to the method of Saito and Miura.
[0023]
Example 2
An attempt was made to obtain a part of the alkaline pullulanase gene by PCR using the chromosomal DNA obtained in Example 1 as a template. The primer DNA used for amplification was designed from amino acid consensus sequences of known acidic or neutral pullulanases. The sequence of primer 1 is 5′-TATAATTGGGGT (A) TATGATCCT (A) C-3 ′ (SEQ ID NO: 5), which is designed based on the amino acid sequence consisting of YNWGYDPH. The sequence of primer 2 is 5′-ACCCATCATATCAAAT (A) CT (G) AAAA (T) CC-3 ′ (SEQ ID NO: 6), which is designed based on the amino acid sequence consisting of GFRFDMMG. As a result of PCR (94 ° C. for 1 minute, 45 ° C. for 1 minute, 60 ° C. for 1 minute for 30 cycles) using Pwo polymerase, a PCR amplified fragment consisting of about 370 bp was obtained. The base sequence of an about 370 bp-PCR amplified fragment is determined, a primer is designed based on the determined sequence, and the gene extension to the 5 'side and 3' side of the about 370 bp-PCR amplified fragment is amplified. Tried. Primer 3 is 5'-TGCCCGTTAGAGCGAAATAACTTTG-3 '(SEQ ID NO: 7), and primer 4 is 5'-CTGATGGTACGCCAAGAACAAGCTT-3' (SEQ ID NO: 8). Using a circular DNA group in which chromosomal DNA of KSM-AP1876 strain cleaved with PstI or SacI was intramolecularly bound as a template, a reverse PCR method (Triglia, T. et al., Nucleic Acids) was performed using a PCR kit (Applied Biosystems). Res., 16, 81 (1988)) (94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 5 minutes for one cycle and repeating this for 30 cycles) was performed for DNA amplification. When a template prepared using PstI was used, a DNA fragment of about 6 kbp was amplified. When a template prepared using SacI was used, a DNA fragment of about 3.5 kbp was amplified. When the base sequences of these amplified DNA fragments were determined by the direct sequencing method, a sequence of about 5 kb encoding the alkaline pullulanase gene and its upstream and downstream regions was detected. Subsequently, using the primer 5 (5′-CATTTTTAGCAGGTTAGTAAGTC-3 ′ (SEQ ID NO: 9)) and the primer 6 (5′-CGGTACCCTTGTTGAACGGACAGC-3 ′ (SEQ ID NO: 10)), the chromosomal DNA obtained in Example 1 was further obtained. As a template, an about 5 kbp DNA fragment containing the alkaline pullulanase gene was amplified again by PCR, and then the full-length base sequence of the 5 kbp DNA fragment was reconfirmed by the direct sequencing method, resulting in the determination of the sequence.
[0024]
Example 3
Based on the sequence obtained in Example 2, using the chromosomal DNA of Bacillus sp. KSM-AP1876 as a template, primer 5 (5′-CATTTTTAGCAGGTTAGTAAGTC-3 ′ (SEQ ID NO: 11)), primer 6 (5′-CGGTACCCTTGTTGAACGGACAGC − 3 ′ (SEQ ID NO: 12)) was used to amplify about 5 kb of a gene encoding alkaline pullulanase by PCR. A vector plasmid pHY300PLK cleaved with the restriction enzyme SmaI and a PCR amplified fragment were mixed, and a binding reaction with T4 DNA ligase was performed to prepare a recombinant plasmid. E. coli transformed with this recombinant plasmid mixture. The LB agar plate medium (1.0% tryptone (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 1.0% NaCl, 1.5% containing 7.5 μg / mL tetracycline) % Agar (manufactured by Wako Pure Chemical Industries, Ltd.)] and cultured for 24 hours at 37 ° C. Further, 0.2% pullulan and 0.8% red pullulan (Kanno, M. and Tomiura) were formed on the appeared transformed colonies. , E., Agric. Biol. Chem., 49, 1529 (1985)), 0.8% agar containing 1 mg / mL lysozyme, glycine-NaCl-sodium hydroxide buffer (pH 9.0) was overlaid. The reaction was allowed to proceed for 5 hours at 0 ° C. As a result, a strain having a transparent halo formed around the colony by decomposition of red pullulan was obtained, which was isolated as an alkaline pullulanase-producing recombinant microorganism.
[0025]
Example 4
The recombinant microorganism obtained in Example 3 was added to 5 mL of LB medium (1.0% tryptone (Difco), 0.5% yeast extract (Difco), 1.0% containing 15 μg / mL tetracycline. NaCl) was inoculated and cultured overnight at 37 ° C., then transferred to 500 mL of LB medium, and further cultured with shaking for 24 hours. Bacteria were collected from this culture by centrifugation, and about 500 μg of a recombinant plasmid was prepared according to a conventional method (Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982)). When a restriction enzyme cleavage map of the obtained recombinant plasmid was prepared, it was revealed that the approximately 5 kb fragment shown in FIG. 1 was contained, and this was designated as plasmid pAP100. In addition, E. coli HB101 strain transformed with pAP100 was named HB101 (pAP100) strain.
[0026]
Example 5
1 mL of a culture solution of HB101 (pAP100) strain cultured overnight in 45 mL of LB medium (including tetracycline) was inoculated into 100 mL of LB medium (including tetracycline), and cultured with shaking at 37 ° C. for 24 hours. After culturing, the cells collected by centrifugation were suspended in Tris-HCl buffer (pH 8.0) and disrupted by ultrasonic waves. After crushing, insoluble matters were removed by centrifugation, and the resulting supernatant was used as a cell-free extract. As a control, cell-free extracts were similarly prepared for the HB101 (pBR322) strain, and the pullulanase activity in these extracts was measured. Note that the pullulanase activity was reduced after the reaction was carried out at 40 ° C. for 30 minutes in a reaction solution containing 40 mM glycine-NaCl-NaOH buffer (pH 10) and pullulan (final concentration 0.25%). Was measured by 3,5-dinitrosalicylic acid (DNS) method, and the amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute was defined as 1 unit.
[0027]
As a result, pullulanase activity was observed in the cell-free extract of HB101 (pAP100) strain. Furthermore, as a result of measuring the optimum pH of action of the produced pullulanase, as shown in FIG. 2, the pullulanase activity of the enzyme is an alkaline pullulanase having an optimum action pH of about pH 8.5 to 9.5. It became clear. In addition, Britton-Robinson buffer (HTSBritton, G. Welford, J. Chem. Soc., 1848 (1937)) was used for the measurement of enzyme activity.
[0028]
Example 6
In addition, B. subtilis ISW1214 strain transformed with pAP100 was added to 100 mL of B medium (2.8% bactopeptone, 0.05% yeast extract, 0.5% fish extract, KH ₂ PO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.02%, sodium carbonate 1.0%, tetracycline 7.5 μg / mL), and further cultured with shaking at 30 ° C. for 3 days, followed by centrifugation to obtain a crude enzyme solution of alkaline pullulanase. It was. The crude enzyme solution was subjected to purification steps in the order of adsorption by DEAE-cellulose, Sepharose-α-cyclodextrin affinity column treatment, and Sephacryl S-200 column treatment to obtain an enzyme sample that was electrophoretically uniform. As a result of measuring the optimum pH of the pullulanase activity of this enzyme in the same manner as in Example 5, the optimum pH was 8.5 to 9.5.
[0029]
【The invention's effect】
The alkaline pullulanase of the present invention has an optimum reaction pH of 8.5 to 9.5 and has the maximum activity in the alkaline region, and thus is useful as a detergent for clothes or tableware. In addition, by using the alkaline pullulanase gene of the present invention, it becomes possible to produce the alkaline pullulanase in a single and a large amount.
[0030]
[Sequence Listing]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[Brief description of the drawings]
FIG. 1 is a view showing a restriction enzyme map of a plasmid (pAP100) containing an alkaline pullulanase gene (derived from Bacillus sp. KSM-AP1876 strain).
FIG. 2 is a diagram showing a pH profile of alkaline pullulanase.

Claims (6)

An alkaline pullulanase having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. A gene encoding the alkaline pullulanase according to claim 1. The alkaline pullulanase gene according to claim 2, which has the base sequence shown in SEQ ID NO: 3 or 4 or a base sequence in which one or several bases of the base sequence are deleted, substituted or added. A recombinant vector containing the gene according to claim 2 or 3. A transformant comprising the recombinant vector according to claim 4. The method for producing an alkaline pullulanase according to claim 1, wherein the transformant according to claim 5 is cultured.

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