JP2002112776A - Alkaline pullulanase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an alkaline pullulanase that acts at an alkaline region and shows and adequate enzymatic activity even in the presence of a surfactant and a gene encoding the enzyme as well as a method for efficiently mass-producing the alkaline pullulanase by the genetic engineering technique. SOLUTION: The objective alkaline pullulanase is found in enzymes produced by microorganisms in the soil, and a gene encoding the enzyme is cloned. The enzyme has a specific amino acid sequence derived from a bacterium belonging to the genus Bacillus, or has an amino acid sequence modified by deleting, substituting or adding one amino acid or more from, in or to the amino acid sequence. A gene encoding the enzyme, a recombinant vector containing the gene, a transformant containing the recombinant vector, and a method for producing the enzyme using these are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an alkaline pullulanase useful as an enzyme for detergents and a gene encoding the same.

[0002]

2. Description of the Related Art Pullulanase cleaves only the α-1,6 glucosidic bond present in starch, glycogen, amylopectin or pullulan molecules, and finally produces maltotriose. It is a kind of degrading enzyme and can produce maltooligosaccharides such as glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose from starch by using in combination with endo-amylase and exo-amylase. It is an enzyme that has attracted attention in the starch manufacturing industry.

[0003] On the other hand, pullulanase has been attempted to be used as a detergent enzyme because it can decompose and remove starch attached to tableware and fibers. The present inventors have proposed that pulnarase be combined with amylase in dishwashing or clothing detergents. As a result, it has been found that the detergency of starch stains is dramatically improved (JP-A-2-132193).

[0004] However, most of the pullulanases found in nature exhibit the maximum enzymatic activity in a neutral or acidic region, and exhibit the maximum enzymatic activity in an alkaline region which is a necessary condition for a detergent composition. So-called alkaline pullulanase is an enzyme previously reported by Nakamura and Horikoshi (Biochim. Biophys. Acta, 39
7,188 (1975), JP-B-53-27786) and Bacillus sp .
s sp.) Enzyme derived from KSM-AP1876 strain (JP-A-3-8717)
No. 6) is only known. Therefore, there is a demand for an alkaline pullulanase which exhibits sufficient enzyme activity even in the presence of a surfactant and is highly useful as a detergent enzyme.

It is an object of the present invention to operate in the alkaline region,
An object of the present invention is to provide an alkaline pullulanase exhibiting a sufficient enzyme activity even in the presence of a surfactant, a gene encoding the same, and a method for efficiently mass-producing the alkaline pullulanase by a genetic engineering technique.

[0006]

Means for Solving the Problems The present inventors have found an alkaline pullulanase that acts in an alkaline region from among enzymes produced by microorganisms in soil, cloned a gene encoding the alkaline pullulanase, Successful establishment of production technology by replacement.

That is, the present invention relates to an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2,
Another object of the present invention is to provide an alkaline pullulanase having an amino acid sequence in which several amino acids have been deleted, substituted or added, and a gene encoding the alkaline pullulanase.

Further, the present invention provides a recombinant vector containing the above-mentioned alkaline pullulanase gene, a transformant containing the recombinant vector, and a method for producing an alkaline pullulanase using the same.

[0009]

BEST MODE FOR CARRYING OUT 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. The deletion, substitution or addition (hereinafter, sometimes referred to as mutation) of an amino acid in the amino acid sequence is not particularly limited as long as the alkaline pullulanase activity is not lost, but the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 And preferably 80% or more homology. Further, at the N-terminal in the amino acid sequence of SEQ ID NO: 1, one to several amino acids may be added or deleted. Here, the amino acid sequence represented by SEQ ID NO: 2 corresponds to positions 209 to 1028 of the amino acid sequence represented by SEQ ID NO: 1, and a DNA fragment (SEQ ID NO: 4) encoding such an amino acid is expressed. Plasmid vector pHSP64 (Sumito
mo, N. et al., Biosci, Biotech, Biochem. 59, 2172-
1995, 1995) and expressed by Bacillus subtilis , an alkaline pullulanase having the same enzymatic activity as the alkaline pullulanase having the amino acid shown in SEQ ID NO: 1 is obtained.

The alkaline pullulanase gene of the present invention may be any one which encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a mutant thereof as long as the alkaline pullulanase activity is not impaired. It is preferable to have a base sequence represented by SEQ ID NO: 4 or a base sequence in which one or several bases of the base sequence are deleted, substituted or added.

The amino acid sequence and the nucleotide sequence of the gene of the alkaline pullulanase of the present invention have a unique amino acid sequence and nucleotide sequence different from those of the pullulanase known so far. In terms of properties, the enzyme is a novel enzyme different from conventional pullulanase, including the enzyme reported by Ara (Japanese Unexamined Patent Publication No. 3-87176).

The alkaline pullulanase of the present invention is isolated from microorganisms isolated from the natural world, for example, soil from Yokohama city, Kanagawa prefecture, and deposited as Bacillus sp. KSM- No. 3049 (FERM BP-3049). The strain can be obtained by culturing the AP1876 strain or a mutant thereof and collecting it from the obtained culture.However, a gene encoding the alkaline pullulanase is obtained, and a recombinant vector is prepared using the gene, and the recombinant is prepared. According to a method in which a host cell is transformed with a vector, the obtained transformant is cultured, and alkaline pullulanase is collected from the culture, mass production can be achieved.

[0013] The alkaline pullulanase gene of the present invention comprises:
Alkaline pullulanase-producing bacteria, such as Bacillus s
p. From the KSM-AP1876 strain or the like, for example, a DNA fragment obtained by total digestion or partial digestion of chromosomal DNA by an appropriate restriction enzyme is incorporated into an appropriate vector, and introduced into Escherichia coli, Bacillus subtilis, or the like, and expressed by the shotgun method. The appropriate primers can be synthesized and cloned by using the PCR method. Here, chromosomal DNA was obtained from the donor strain by, for example, the method of Marmur (J., J. Mol. Biol., 3,208).
(1961)) and the method of Saito and Miura (Biochem. Biophys.
2,619 (1963)) or other similar methods, and any restriction enzyme can be used as long as it does not disrupt the gene. In addition, PCR
When the method is used, for example, 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 based on the amino acid sequence of SEQ ID NO: 1 to produce alkaline pullulanase. If a PCR reaction is performed using the chromosomal DNA of the bacterium as a template, an alkaline pullulanase gene can be obtained.

[0014] As a host-vector system used for preparing a recombinant vector containing a gene fragment, a host strain can express the alkaline pullulanase gene of the present invention, and the recombinant DNA can be replicated in the host bacterium. Any gene can be used as long as it can stably retain the incorporated gene. For example, E. coli using Escherichia coli K-12 strain as a host
Examples include the K system and the BM system using the Marburg strain of Bacillus subtilis as a host. Here, the host strain is, for example, HB101 strain, C600 strain, J
For the M109 strain and the BM strain, for example, BD170 strain, MI1
12 strains, ISW1214 strain, and the like. As the vector, in the case of the EK system, for example, pBR322, pHY300
PLK, pUC18, etc., and in the BM system, for example, pUB
110, pHY300PLK, etc., and vectors constructed using these can also be used. Suitable examples of the recombinant DNA containing the gene of the present invention include plasmid pAP100 (FIG. 1). This plasmid is constructed by combining a fragment containing 5.0 kbp of the gene of the present invention with pHY300.
It is a 10.1 kbp recombinant plasmid consisting of a part of PLK. Preferred examples of recombinant microorganisms containing recombinant DNA include E. coli HB101 (pAP10
0) strains.

A method for transforming a host strain with a recombinant DNA is, for example, a calcium chloride method (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (197)
0)) and the like, and in the case of the BM host strain, the protoplast method (Chang, C. and Cohen. SN, Mol. Gen. Genet., 168, 11).
1 (1978)) and the like.

[0016] Selection of the recombinant microorganism is performed by transplanting the transformed cells onto an agar plate containing pullulan or red pullulan by a replica method or the like, culturing the colonies, allowing the colonies to appear, and pulling the colonies on a pullulan agar plate. The detection can be performed by detecting a colony capable of forming a halo. Recombinant D retained by the obtained recombinant microorganism
NA is determined by a conventional plasmid preparation method or phage DN.
A Preparation Method (Maniatis, T. et al., Molecular Cloning, Cold
Spring Harbor Laboratory, New York, (1982)), and by analyzing the cleavage patterns by various restriction enzymes by electrophoresis or the like, the recombinant DNA can be extracted.
Can be confirmed to be a combination of a vector DNA and a DNA fragment containing an alkaline pullulanase gene.

The culture of the transformant may be performed by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients which can be used by microorganisms, and conducting the culture according to a conventional method. Alkaline pullulanase can be obtained by a method according to a method for collecting and purifying an enzyme.

The thus-obtained alkaline pullulanase of the present invention has the amino acid sequence represented by SEQ ID NO: 1 or 2, and its enzymatic properties are as follows,
It has a feature that H is 8.5 to 9.5.

<Enzymatic Properties> (1) Action The α-1,6 glucosidic bond of pullulan is hydrolyzed,
Generate maltotriose. It also hydrolyzes α-1,6 glucosidic bonds between starch, amylopectin and glycogen. (2) Optimal reaction pH The pH is 8.5 to 9.5. (3) Stable pH Extremely stable in the range of pH 7 to 10. (4) Works at an optimum reaction temperature of 50 to 55 ° C. (5) Heat resistance Stable up to 50 ° C (Tris / hydrochloric acid 10 mM (pH 8.
5) Retains 85% or more activity in a buffer at 50 ° C. for 30 minutes). (6) Molecular weight The estimated molecular weight of wild type by SDS-polyacrylamide gel electrophoresis is 120,000 to 130,000.

Therefore, the alkaline pullulanase of the present invention exhibits excellent enzymatic activity even in the presence of a surfactant, and is used as an enzyme for detergents, for example, liquid detergent, powder detergent, granule detergent, etc. Can be used in form.

[0021]

The present invention will be specifically described below with reference to examples. In addition, all the concentrations in the present examples indicate% by weight.

Example 1 Bacillus sp. KSM-AP producing alkaline pullulanase
The 1876 strain was transformed into 5 mL of A medium (2.8% bactopeptone, 0.05% yeast extract, 0.5% fish meat extract, KH ₂ P
O ₄ 0.1%, MgSO ₄ 7H ₂ O 0.02%, sodium carbonate 1.0%), and after shaking culture at 30 ° C. for 24 hours, 1 mL of this was inoculated into 100 mL of the same medium. And cultured with shaking at 30 ° C. for another 12 hours. Thereafter, the cells were collected by centrifugation, and the cells were collected for about 1 hour according to the method of Saito and Miura.
mg of chromosomal DNA was obtained.

Example 2 Using the chromosomal DNA obtained in Example 1 as a template, an attempt was made to obtain a part of the alkaline pullulanase gene by PCR. The primer DNA used for the amplification was designed from the amino acid common sequence of known acidic or neutral pullulanase. The sequence of primer 1 is 5'-TATAATTGGGGT (A) TATG
ATCCT (A) C-3 '(SEQ ID NO: 5) and YNWGYDPH
This was designed based on the amino acid sequence consisting of
The sequence of primer 2 is 5'-ACCCATCATATCAAAT (A) CT (G) AAA
A (T) CC-3 '(SEQ ID NO: 6), which was designed based on the amino acid sequence consisting of GFRFDMMG. P
PCR using wo polymerase (94 ° C for 1 minute, 45 minutes)
As a result, a PCR amplified fragment consisting of about 370 bp was obtained. The base sequence of the amplified fragment of about 370 bp-PCR is determined, and primers are designed based on the determined sequence to amplify the extended portion of the gene to the 5 'side and 3' side of the amplified fragment of about 370 bp-PCR. Tried. The designed primer sequence is that primer 3 is 5'-TGCCCGTTA
GAGCGAAATAACTTTG -3 '(SEQ ID NO: 7) and primer 4
Is 5'-CTGATGGTACGCCAAGAACAAGCTT-3 '(SEQ ID NO: 8). Using a cyclic DNA group in which chromosomal DNA of the KSM-AP1876 strain cut with PstI or SacI was intramolecularly bound as a template, an inverse PCR method (Triglia, T. et al., N.
ucleic Acids Res., 16, 81 (1988)) at 94 ° C for 1 minute, 5
One cycle of 5 ° C. for 1 minute and 72 ° C. for 5 minutes constitute 30 cycles.
(Repeat cycle) DNA amplification was performed. When a template prepared using PstI was used, a DNA fragment of about 6 kbp was amplified, and 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, primer 5 (5'-CATTTTTAGCAGGTTAG
TAAGTC -3 '(SEQ ID NO: 9)), primer 6 (5'-CGGT)
Using ACCCTTGTTGAACGGACAGC-3 '(SEQ ID NO: 10)) and the chromosomal DNA obtained in Example 1 as a template, about 5 kbp DN containing an alkaline pullulanase gene.
A fragment was again amplified by PCR, and its 5 kbp DNA
The full-length nucleotide sequence of the fragment was reconfirmed by the direct sequencing method, and the sequence was determined.

Example 3 Based on the sequence obtained in Example 2, Bacillus sp. KSM-AP1876
Using the chromosomal DNA of the strain as a template, primer 5 (5'-CAT
Using TTTTAGCAGGTTAGTAAGTC-3 '(SEQ ID NO: 11) and primer 6 (5'-CGGTACCCTTGTTGAACGGACAGC-3' (SEQ ID NO: 12)), about 5 kb of the gene encoding alkaline pullulanase was amplified by PCR. Restriction enzyme Sma
The vector plasmid pHY300PLK digested with I and the PCR amplified fragment were mixed, and a ligation reaction was performed with T4 DNA ligase to prepare a recombinant plasmid. E. coli transformed with this recombinant plasmid mixture was used. c
The oli suspension was mixed with LB agar plate medium containing 7.5 μg / mL tetracycline (1.0% tryptone (Difco), 0.5% yeast extract (Difco),
1.0% NaCl, 1.5% agar (manufactured by Wako Pure Chemical Industries, Ltd.)] and cultured at 37 ° C. for 24 hours. Furthermore, 0.2% pullulan and 0.8% red pullulan (Kanno, M. and Tomiura, E., Agric. Biol. Chem.,
49,1529 (1985)), 1 mg / mL lysozyme, glycine-N
0.8% agar containing an aCl-sodium hydroxide buffer (pH 9.0) was overlaid and reacted at 37 ° C for 5 hours.
As a result, one strain in which a transparent halo was formed around the colony by decomposition of red pullulan was obtained, and this strain was isolated as an alkaline pullulanase-producing recombinant microorganism.

Example 4 The recombinant microorganism obtained in Example 3 was combined with 5 mL of LB medium containing 15 μg / mL tetracycline (1.0% tryptone (Difco), 0.5% yeast extract (Difco) ), 1.0% NaCl) and cultured overnight at 37 ° C., then transferred to 500 mL of LB medium, and
The cells were cultured with shaking for hours. The cells were collected from this culture by centrifugation, and the cells were collected by a conventional method (Maniatis, T. et al., Molecular Cloni
ng, Cold Spring Harbor Laboratory (1982))
About 500 μg of the recombinant plasmid was prepared. When a restriction map of the obtained recombinant plasmid was prepared,
It was found that the fragment contained the about 5 kb fragment shown in FIG. 1, and this was named plasmid pAP100. In addition, E. coli HB1 transformed with pAP100
The 01 strain was named HB101 (pAP100) strain.

Example 5 One mL of a culture of the HB101 (pAP100) strain cultured overnight in 45 mL of LB medium (including tetracycline)
00 mL of LB medium (containing tetracycline) was inoculated and cultured with shaking at 37 ° C. for 24 hours. After the culture, the cells collected by centrifugation are separated into Tris-HCl buffer (pH 8.0).
And crushed by ultrasonic waves. After crushing, insoluble materials were removed by centrifugation, and the obtained supernatant was used as a cell-free extract. As a control, HB101 (pBR32
2) Cell-free extracts were similarly prepared for the strains, and the pullulanase activity in these extracts was measured. The pullulanase activity was determined by performing a reaction at 40 ° C. for 30 minutes in a reaction solution containing a 40 mM glycine-NaCl-NaOH buffer (pH 10) and pullulan (final concentration: 0.25%). To 3,5-dinitrosalicylic acid (3,5-dinitr
It was measured by quantification by the osalicylic acid (DNS) method, and the amount of the enzyme that produces reducing sugar corresponding to 1 μmol of glucose per minute was defined as 1 unit.

As a result, pullulanase activity was observed in the cell-free extract of the HB101 (pAP100) strain. Further, as a result of measuring the optimal pH of the produced pullulanase, as shown in FIG. 2, the pullulanase activity of this enzyme was found to be an alkaline pullulanase having an optimal action pH of about pH 8.5 to 9.5. It became clear.
The enzyme activity was measured using Britton-Robinson buffer (HTSBritton, G. Welford, J. Chem. Soc., 1848 (193)
7)) was used.

Example 6 In addition, B. subtil transformed with pAP100
is ISW1214 strain in 100 mL of B medium (Bacto-peptone)
2.8%, yeast extract 0.05%, fish meat extract 0.5
%, KH ₂ PO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.0
2%, sodium carbonate 1.0%, tetracycline
7.5 .mu.g / mL) and cultured with shaking at 30.degree. C. for further 3 days, followed by centrifugation to obtain a crude enzyme solution of alkaline pullulanase. The crude enzyme solution was subjected to DEAE-cellulose adsorption, separation on a Sepharose-α-cyclodextrin affinity column, and separation on a Sephacryl S-200 column, in order to obtain an electrophoretically uniform enzyme preparation. Optimal pH for pullulanase activity of this enzyme
Was measured in the same manner as in Example 5, and as a result, the optimum pH was 8.5 to 8.5.
9.5.

[0029]

Industrial Applicability The alkaline pullulanase of the present invention has an optimum reaction pH of 8.5 to 9.5 and has the maximum activity in an alkaline region, and thus is useful as an enzyme for clothing or dish detergent. In addition, by using the alkaline pullulanase gene of the present invention, it becomes possible to produce the alkaline pullulanase singly and in large quantities.

[0030]

[Sequence List] SEQUENCE LISTING <110> KAO CORPORATION <120> Alkali pullularase <130> P04771210 <160> 12 <170> PatentIn Ver. 2.1 <210> 1 <211> 1142 <212> PRT <213> Bacillus sp. KSM -AP1876 <400> 1 Leu Lys Gly Lys Leu Lys Lys Phe Leu Val Leu Ser Met Val Phe Ile 1 5 10 15 Leu Thr Phe Ser Ser Phe Gly Met Met Gly Thr Thr Thr Gln Leu Val Glu 20 25 30 Ala Glu Thr Lys Ser Ser Ile Glu Glu Ser Glu Ile Pro Glu Asn Thr 35 40 45 Leu Arg Ile His Tyr Gln Asn Ser Ser Gly Asp Tyr Ser Asn Leu Gly 50 55 60 Val Trp Thr Trp Asp Asp Val Glu Ser Pro Thr Asp Asn Trp Pro Ser 65 70 75 80 Gly Gly Ile Pro Phe Thr Glu Glu Gln Val Thr Asp Tyr Gly Ala Tyr 85 90 95 Val Asp Val Pro Leu Glu Ser Gly Ala Arg Asn Val Gly Phe Leu Ile 100 105 110 Leu Asp Ile Val Thr Gly Gly Lys Asp Asp Ala Gly His Asn Gly Gly 115 120 125 Asp Lys Arg Leu Glu Leu Phe Ser Pro Asn Ile Asn Glu Val Trp Ile 130 135 140 Ser Glu Gly Ser Asp Glu Ile Ser Phe Ile Glu Pro Val Asp Leu Pro 145 150 155 160 Glu Asp Thr Val Arg Val His Tyr Lys Lys Ala Asp Gly A sn Tyr Glu 165 170 175 Asn Trp Ser Leu Trp Phe Trp Gly Asp Val Gln Ser Pro Ser Glu Thr 180 185 190 Gln Gly Asp Phe Pro Ser Gly Ala Thr Gly Phe Ser Glu Glu Gln Ile 195 200 205 Gly Lys Tyr Gly Ala Tyr Ile Asp Ile Pro Leu Asn Glu Gly Ala Glu 210 215 220 Glu Ile Gly Leu Val Val Val Asn Lys Glu Thr Asn Glu Gln Glu Ala 225 230 235 240 Asp Arg Ser Phe Thr Gln Leu Asn Gln Phe Asn Gln Ile Phe Ile Val 245 250 255 Glu Gly Asp Gly Ala Val Tyr Ser Asn Pro Tyr Gly Ala Ile Ala Thr 260 265 270 Glu Leu Leu Ser Ala Glu Gln Leu Ser Asp Glu Lys Ile Val Leu Tyr 275 280 285 Phe Ser Arg Thr Asp Glu Leu Thr Glu Glu Ala Leu Leu Glu Gln Val 290 295 300 Gly Ile Arg Asp Ile Glu Glu His Thr Val Asp Val Asn Glu Val Glu 305 310 315 320 Ile Lys Thr Asp Asn Arg Thr Val Glu Val Lys Gly Asp Phe Asn Leu 325 330 335 Asp Gln Ser Pro Tyr Thr Val Ser Leu Gly Glu Val Ser Val Pro Ala 340 345 350 Lys Ile Gly Trp Arg Leu Ile Asp Glu Met Tyr Ala Tyr Asp Gly Glu 355 360 365 Leu Gly Ala Gln Leu His Gln Asp Gly Thr Ala Thr Ile Lys L eu Trp 370 375 380 Ser Pro Lys Ala Glu His Val Gln Val Ile Leu Tyr Asp Lys Asp Asn 385 390 395 400 Pro Asp Asp Ile Val Thr Glu Val Glu Met Lys Leu Gly Asp Arg Gly 405 410 415 Val Trp Glu Val Gln Leu Thr Lys Glu Asn Thr Gly Leu Asp Ser Leu 420 425 430 Arg Gly Tyr Tyr Tyr His Tyr Glu Ile Asp His Asp Gly Asp Lys Lys 435 440 445 Ile Ala Leu Asp Pro Tyr Ala Lys Ser Leu Ser Ala Trp Asn Ser Asp 450 455 460 Glu Gln Gly Pro Tyr Ala Lys Ala Ala Ile Val Asp Pro Ser Ser Ile 465 470 475 480 Gly Pro Glu Leu Glu Phe Ala His Ile Glu Gly Phe Glu Lys Lys Glu 485 490 495 Asp Thr Ile Ile Tyr Glu Val His Val Arg Asp Phe Thr Ser Asp Pro 500 505 510 His Ile Ala Asp Glu Leu Thr Ala Gln Phe Gly Thr Phe Ala Ser Phe 515 520 525 Val Asp Lys Leu Asp Tyr Ile Glu Asp Leu Gly Val Thr His Ile Gln 530 535 540 Leu Leu Pro Val Met Ser Tyr Phe Phe Ala Asn Glu Phe Lys Asn Asp 545 550 555 560 Glu Arg Met Leu Asp Phe Ser Ser Thr Asn Thr Asn Tyr Asn Trp Gly 565 570 575 Tyr Asp Pro Gln Ser Tyr Phe Ala Leu Thr Gly Met Tyr SerGlu Asp 580 585 590 590 Pro Thr Asp Pro Glu Leu Arg Ile Lys Glu Phe Lys Lys Leu Ile Asp 595 600 605 Glu Ile His Ser Arg Gly Met Gly Val Val Leu Asp Val Val Tyr Asn 610 615 620 His Thr Ala Arg Val Gly Ile Phe Glu Asp Leu Val Pro Asn Tyr Tyr 625 630 635 640 His Phe Met Asp Ala Asp Gly Thr Pro Arg Thr Ser Phe Gly Gly Gly 645 650 655 Arg Leu Gly Thr Thr His Glu Met Ser Arg Arg Ile Leu Val Asp Ser 660 665 670 Ile Thr Tyr Trp Val Glu Glu Phe Lys Ile Asp Gly Phe Arg Phe Asp 675 680 685 Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Ile Ala Tyr Asp Lys 690 695 700 Ala Lys Glu Ile Asn Pro Asn Ile Val Met Ile Gly Glu Gly Trp Ile 705 710 715 720 Thr Tyr Ala Gly Asp Glu Asp Asp Pro Asn Val Gln Ala Ala Asp Gln 725 730 735 His Trp Met Gln Tyr Thr Glu Ser Val Gly Val Phe Ser Asp Glu Phe 740 745 750 Arg Asn Glu Leu Lys Ser Gly Phe Gly His Glu Gly Glu Pro Arg Phe 755 760 765 Leu Thr Gly Gly Ala Arg Asn Ile Asp Leu Ile Phe Asp Asn Ile Lys 770 775 780 Ala Gln Pro His Asn Phe Ile Ala Asp Asp Pro Gly Asp Val ValPro 785 790 795 800 Tyr Ile Glu Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala Met 805 810 815 Ala Ile Gln Lys Asp Pro Asp Asp His Gln Glu Glu Ile His Gln Arg 820 825 830 830 Ile Arg Leu Gly Asn Leu Met Thr Leu Thr Ser Gln Gly Ile Ala Phe 835 840 845 845 Leu His Ala Gly Gln Glu Tyr Gly Arg Thr Lys Gln Phe Arg Ala Glu 850 855 860 860 Thr Ser Glu Pro Pro Tyr Lys Ser Thr Tyr Met Thr Asp Glu Asn Gly 865 870 875 880 Glu Pro Phe Arg Tyr Pro Tyr Phe Ile His Asp Ser Tyr Asp Ser Thr 885 890 895 Asp Ile Ile Asn Arg Phe Asp Trp Glu Arg Ala Thr Asn Ala Asp Ala 900 905 910 Tyr Pro Ile Gln Asn Leu Thr Arg Glu Tyr Thr Thr Gly Leu Ile Glu 915 920 925 Leu Arg Arg Ser Ser Asp Ala Phe Arg Leu Gly Thr Lys Asp Leu Val 930 935 940 Asp Glu Lys Val Thr Gln Leu Asn Ile Pro Glu Ile Glu Glu Thr Asp 945 950 955 960 Leu Val Val Ala Tyr Arg Ile Glu Ala Thr Thr Gly Glu Ala Phe Tyr 965 970 975 Val Phe Val Asn Ala Asp Asp Glu Glu Arg Thr Leu Thr Leu Glu Glu 980 985 990 Asp Leu Thr Val Gly Glu Phe Val Val Asp Gly Lys Thr Ala Gly Val 995 1000 1005 Thr Ala Ile Ala Glu Pro Gln Gly Val Glu Leu Ser Ala Glu Gln Ile 1010 1015 1020 Lys Leu Asp Pro Leu Thr Ala Ala Ile Val Gln Val Gly Gly Ile Glu 1025 1030 1035 1040 Ala Pro Glu Lys Pro Thr Pro Pro Lys Glu Glu Pro Lys Glu Pro Lys 1045 1050 1055 Glu Pro Lys Glu Pro Lys Glu Pro Lys Glu Pro Lys Asp Pro Lys Asp 1060 1065 1070 Pro Lys Asp Pro Lys Asp Pro Leu Asp Pro Pro Gly Thr Asp Asp Gln 1075 1080 1085 Asn Gly Glu Thr Pro Lys Gly Asn Glu Glu Lys Lys Glu Glu Gln Lys 1090 1095 1100 Gly Asp Asn Leu Pro Asn Thr Ala Thr Ser Asn Tyr Gln Phe Ile Ala 1105 1110 1115 1120 Leu Gly Ile Ala Leu Val Leu Met Ala Thr Ala Ile Tyr Trp Trp Lys 1125 1130 1135 Arg Lys Arg Gln Val Ala 1140

<210> 2 <211> 821 <212> PRT <213> Bacillus sp.KSM-AP1876 <400> 2 Ile Gly Lys Tyr Gly Ala Tyr Ile Asp Ile Pro Leu Asn Glu Gly Ala 1 5 10 15 Glu Glu Ile Gly Leu Val Val Val Asn Lys Glu Thr Asn Glu Gln Glu 20 25 30 Ala Asp Arg Ser Phe Thr Gln Leu Asn Gln Phe Asn Gln Ile Phe Ile 35 40 45 Val Glu Gly Asp Gly Ala Val Tyr Ser Asn Pro Tyr Gly Ala Ile Ala 50 55 60 Thr Glu Leu Leu Ser Ala Glu Gln Leu Ser Asp Glu Lys Ile Val Leu 65 70 75 80 Tyr Phe Ser Arg Thr Asp Glu Leu Thr Glu Glu Ala Leu Leu Glu Gln 85 90 95 Val Gly Ile Arg Asp Ile Glu Glu His Thr Val Asp Val Asn Glu Val 100 105 110 Glu Ile Lys Thr Asp Asn Arg Thr Val Glu Val Lys Gly Asp Phe Asn 115 120 125 Leu Asp Gln Ser Pro Tyr Thr Val Ser Leu Gly Glu Val Ser Val Pro 130 135 140 Ala Lys Ile Gly Trp Arg Leu Ile Asp Glu Met Tyr Ala Tyr Asp Gly 145 150 155 160 Glu Leu Gly Ala Gln Leu His Gln Asp Gly Thr Ala Thr Ile Lys Leu 165 170 175 Trp Ser Pro Lys Ala Glu His Val Gln Val Ile Leu Tyr Asp Lys Asp 180 185 190 Asn Pro Asp Asp Ile Val Thr Glu Val Glu Met Lys Leu Gly Asp Arg 195 200 205 Gly Val Trp Glu Val Gln Leu Thr Lys Glu Asn Thr Gly Leu Asp Ser 210 215 220 Leu Arg Gly Tyr Tyr Tyr His Tyr Glu Ile Asp His Asp Gly Asp Lys 225 230 235 240 Lys Ile Ala Leu Asp Pro Tyr Ala Lys Ser Leu Ser Ala Trp Asn Ser 245 250 255 Asp Glu Gln Gly Pro Tyr Ala Lys Ala Ala Ile Val Asp Pro Ser Ser 260 265 270 Ile Gly Pro Glu Leu Glu Phe Ala His Ile Glu Gly Phe Glu Lys Lys 275 280 285 Glu Asp Thr Ile Ile Tyr Glu Val His Val Arg Asp Phe Thr Ser Asp 290 295 300 Pro His Ile Ala Asp Glu Leu Thr Ala Gln Phe Gly Thr Phe Ala Ser 305 310 315 320 Phe Val Asp Lys Leu Asp Tyr Ile Glu Asp Leu Gly Val Thr His Ile 325 330 335 Gln Leu Leu Pro Val Met Ser Tyr Phe Phe Ala Asn Glu Phe Lys Asn 340 345 350 Asp Glu Arg Met Leu Asp Phe Ser Ser Thr Asn Thr Asn Tyr Asn Trp 355 360 365 Gly Tyr Asp Pro Gln Ser Tyr Phe Ala Leu Thr Gly Met Tyr Ser Glu 370 375 380 Asp Pro Thr Asp Pro Glu Leu Arg Ile Lys Glu Phe Lys Lys Leu Ile 385 390 395 400 Asp Glu IleHis Ser Arg Gly Met Gly Val Val Leu Asp Val Val Tyr 405 410 415 Asn His Thr Ala Arg Val Gly Ile Phe Glu Asp Leu Val Pro Asn Tyr 420 425 430 Tyr His Phe Met Asp Ala Asp Gly Thr Pro Arg Thr Ser Phe Gly Gly 435 440 445 Gly Arg Leu Gly Thr Thr His Glu Met Ser Arg Arg Ile Leu Val Asp 450 455 460 Ser Ile Thr Tyr Trp Val Glu Glu Phe Lys Ile Asp Gly Phe Arg Phe 465 470 475 480 480 Asp Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Ile Ala Tyr Asp 485 490 495 Lys Ala Lys Glu Ile Asn Pro Asn Ile Val Met Ile Gly Glu Gly Trp 500 505 510 Ile Thr Tyr Ala Gly Asp Glu Asp Asp Pro Asn Val Gln Ala Ala Asp 515 520 525 Gln His Trp Met Gln Tyr Thr Glu Ser Val Gly Val Phe Ser Asp Glu 530 535 540 Phe Arg Asn Glu Leu Lys Ser Gly Phe Gly His Glu Gly Glu Pro Arg 545 550 555 560 Phe Leu Thr Gly Gly Ala Arg Asn Ile Asp Leu Ile Phe Asp Asn Ile 565 570 575 Lys Ala Gln Pro His Asn Phe Ile Ala Asp Asp Pro Gly Asp Val Val 580 585 590 590 Pro Tyr Ile Glu Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala 595 600 605 Met Ala Ile Gln Lys Asp Pro Asp Asp His Gln Glu Glu Ile His Gln 610 615 620 Arg Ile Arg Leu Gly Asn Leu Met Thr Leu Thr Ser Gln Gly Ile Ala 625 630 635 635 640 Phe Leu His Ala Gly Gln Glu Tyr Gly Arg Thr Lys Gln Phe Arg Ala 645 650 655 Glu Thr Ser Glu Pro Pro Tyr Lys Ser Thr Tyr Met Thr Asp Glu Asn 660 665 670 Gly Glu Pro Phe Arg Tyr Pro Tyr Phe Ile His Asp Ser Tyr Asp Ser 675 680 685 Thr Asp Ile Ile Asn Arg Phe Asp Trp Glu Arg Ala Thr Asn Ala Asp 690 695 700 Ala Tyr Pro Ile Gln Asn Leu Thr Arg Glu Tyr Thr Thr Gly Leu Ile 705 710 715 715 720 Glu Leu Arg Arg Ser Ser Asp Ala Phe Arg Leu Gly Thr Lys Asp Leu 725 730 735 Val Asp Glu Lys Val Thr Gln Leu Asn Ile Pro Glu Ile Glu Glu Thr 740 745 750 Asp Leu Val Val Ala Tyr Arg Ile Glu Ala Thr Thr Gly Glu Ala Phe 755 760 765 Tyr Val Phe Val Asn Ala Asp Asp Glu Glu Arg Thr Leu Thr Leu Glu 770 775 780 Glu Asp Leu Thr Val Val Gly Glu Phe Val Val Asp Gly Lys Thr Ala Gly 785 790 795 800 Val Thr Ala Ile Ala Glu Pro Gln Gly Val Glu Leu Ser Ala Glu Gln 805 810 815 Ile Lys Leu Asp Pro 820

[210] 3 <211> 3429 <212> DNA <213> Bacillus sp. gtgtatggac atgggatgat gtggaatctc ctacagacaa ttggccaagt 240 gggggaattc cttttactga agaacaagta acagattatg gtgcatatgt agatgttccg 300 ttagaatcag gtgctagaaa cgtaggattc ctaattcttg atattgtgac aggtgggaaa 360 gatgatgcag gtcataacgg tggagataaa cgattagagc ttttttcccc caatattaat 420 gaagtgtgga tttcagaagg gtctgatgag atctcattca ttgagcctgt tgatttaccg 480 gaagatacag taagggttca ttataaaaaa gctgatggta attatgagaa ttggagcttg 540 tggttttggg gagatgttca aagcccatct gagactcaag gtgactttcc atcaggggct 600 acaggtttct ctgaagagca aatagggaag tatggagcat atatcgatat tcctttaaat 660 gaaggagcag aagaaattgg gctggttgtt gtgaataaag agactaacga gcaagaagct 720 gatcgctcat ttacgcaatt aaatcaattt aatcaaatat ttatagttga aggtgatggg 780 gcc gtatt caaacccgta tggtgcgatt gcaacagaat tactttctgc tgagcagtta 840 tccgatgaga aaatcgtctt atatttttct agaacggatg agttaacaga ggaagctctg 900 cttgaacaag tgggcattcg agatatcgaa gagcacacgg tagatgttaa tgaagtagaa 960 attaaaacag ataatcgtac ggtagaagtg aagggagatt ttaacctcga tcaatctcca 1020 tatacggtaa gtcttggcga agtgagtgtt cctgctaaga ttggctggag gctgattgat 1080 gagatgtatg cctatgatgg agagttaggg gcacagttac accaagatgg aacagctacg 1140 attaagctgt ggtcaccaaa agcagagcat gttcaggtga ttttgtacga taaagataac 1200 ccagatgata tcgtaacaga ggttgaaatg aagttaggtg atcgtggagt ttgggaagtg 1260 caattgacta aagaaaacac gggtctagat agtctgagag gctactacta tcattatgaa 1320 attgatcatg atggagataa aaaaattgct ttagatccat atgctaaatc tttatcggct 1380 tggaatagtg atgaacaagg tccatatgcg aaagcagcga ttgtcgatcc gagctcaatt 1440 ggacctgagt tagaatttgc tcatattgaa ggctttgaga aaaaagaaga tacaattatt 1500 tatgaagttc atgtgagaga tttcacttca gaccctcata ttgccgatga gttaaccgct 1560 caatttggta catttgcaag ctttgtagat aagcttgatt atattgaaga cttgggtgtg 1620 acacatattc agc ttttacc agttatgagt tatttctttg cgaacgagtt taagaatgat 1680 gaaagaatgc tcgacttttc ttcgacgaat acaaattata attggggata tgacccgcaa 1740 agttatttcg ctctaacggg catgtattca gaagatccaa cagaccctga attacgcatt 1800 aaagagttca agaaattaat tgatgaaatt cacagtcgtg gaatgggtgt tgttttggat 1860 gttgtgtata atcacacagc aagagttggt atctttgaag atttagttcc aaattattat 1920 cattttatgg atgctgatgg tacgccaaga acaagctttg gtggtggacg cctcggtaca 1980 acacacgaga tgtcacgtcg aattctcgtt gattccatta cctattgggt agaagagttt 2040 aagattgatg gattccgttt tgatatgatg ggcgatcatg atgcagagtc aattcaaatt 2100 gcctatgata aagctaaaga aatcaatccg aatattgtga tgattggtga ggggtggatt 2160 acgtacgctg gtgatgaaga tgacccgaat gtacaagctg ctgatcagca ttggatgcag 2220 tatacagaaa gtgtcggtgt attttctgat gagttccgta atgagttaaa gtctggtttt 2280 ggccatgaag gtgagccgcg tttcttaacg ggtggtgcaa gaaatattga cctgattttt 2340 gacaatatta aagctcaacc acataacttt atcgctgatg atccaggtga tgttgtgccg 2400 tatattgaag cacatgataa tttaacttta catgatgtca ttgcgatggc gattcaaaaa 2460 gatccagatg accatcaag a agaaatccac caacgcattc gtctaggaaa cttgatgacg 2520 ttgacttctc aaggtattgc tttcttacat gcaggtcaag agtatggacg cacgaagcaa 2580 tttagagcgg aaacatcaga gccaccgtat aaatctacct atatgactga tgaaaatggt 2640 gagcctttcc gttacccata ctttattcat gactcgtatg attctacaga tatcatcaat 2700 cgttttgatt gggaaagagc aacgaatgct gatgcttatc cgattcaaaa tttaacgaga 2760 gagtatacaa caggtttaat tgagttaaga cgttctagcg atgctttccg attaggaaca 2820 aaagatcttg tagacgaaaa agtaacgcaa ttaaacattc cagaaattga agaaacagat 2880 ttagttgtgg cttatcgcat tgaggcgaca acaggtgaag cattttatgt atttgtgaat 2940 gcggatgatg aagaaagaac attaacatta gaagaggatt taaccgtggg tgaatttgtc 3000 gttgatggaa aaacagctgg tgtgacagcg attgccgagc cacaaggggt ggagttgagt 3060 gctgagcaaa taaagctgga tccattaact gcagcgattg tacaagtagg tggtattgaa 3120 gcaccagaaa aaccaacacc tcctaaagaa gaaccgaaag aaccaaaaga gccaaaagag 3180 ccaaaagagc caaaagagcc aaaagatcca aaagatccaa aagatccaaa agatccacta 3240 gacccgcctg gaaccgatga tcaaaatgga gaaacaccaa aaggaaacga agagaaaaaa 3300 gaggagcaaa aaggtgataa ctt gccaaat acggcgacat ctaactatca attcattgcc 3360 ttaggtatcg cacttgtatt aatggcaacc gcgatttatt ggtggaaaag aaaacggcaa 3420 gtagcgtaa 3429

<210> 4 <211> 2463 <212> DNA <213> Bacillus sp. caacagaatt actttctgct gagcagttat ccgatgagaa aatcgtctta 240 tatttttcta gaacggatga gttaacagag gaagctctgc ttgaacaagt gggcattcga 300 gatatcgaag agcacacggt agatgttaat gaagtagaaa ttaaaacaga taatcgtacg 360 gtagaagtga agggagattt taacctcgat caatctccat atacggtaag tcttggcgaa 420 gtgagtgttc ctgctaagat tggctggagg ctgattgatg agatgtatgc ctatgatgga 480 gagttagggg cacagttaca ccaagatgga acagctacga ttaagctgtg gtcaccaaaa 540 gcagagcatg ttcaggtgat tttgtacgat aaagataacc cagatgatat cgtaacagag 600 gttgaaatga agttaggtga tcgtggagtt tgggaagtgc aattgactaa agaaaacacg 660 ggtctagata gtctgagagg ctactactat cattatgaaa ttgatcatga tggagataaa 720 aaaattgctt tagatccata tgctaaatct ttatcggctt ggaatagtga tgaacaaggt 780 ccata tgcga aagcagcgat tgtcgatccg agctcaattg gacctgagtt agaatttgct 840 catattgaag gctttgagaa aaaagaagat acaattattt atgaagttca tgtgagagat 900 ttcacttcag accctcatat tgccgatgag ttaaccgctc aatttggtac atttgcaagc 960 tttgtagata agcttgatta tattgaagac ttgggtgtga cacatattca gcttttacca 1020 gttatgagtt atttctttgc gaacgagttt aagaatgatg aaagaatgct cgacttttct 1080 tcgacgaata caaattataa ttggggatat gacccgcaaa gttatttcgc tctaacgggc 1140 atgtattcag aagatccaac agaccctgaa ttacgcatta aagagttcaa gaaattaatt 1200 gatgaaattc acagtcgtgg aatgggtgtt gttttggatg ttgtgtataa tcacacagca 1260 agagttggta tctttgaaga tttagttcca aattattatc attttatgga tgctgatggt 1320 acgccaagaa caagctttgg tggtggacgc ctcggtacaa cacacgagat gtcacgtcga 1380 attctcgttg attccattac ctattgggta gaagagttta agattgatgg attccgtttt 1440 gatatgatgg gcgatcatga tgcagagtca attcaaattg cctatgataa agctaaagaa 1500 atcaatccga atattgtgat gattggtgag gggtggatta cgtacgctgg tgatgaagat 1560 gacccgaatg tacaagctgc tgatcagcat tggatgcagt atacagaaag tgtcggtgta 1620 ttttctgatg agt tccgtaa tgagttaaag tctggttttg gccatgaagg tgagccgcgt 1680 ttcttaacgg gtggtgcaag aaatattgac ctgatttttg acaatattaa agctcaacca 1740 cataacttta tcgctgatga tccaggtgat gttgtgccgt atattgaagc acatgataat 1800 ttaactttac atgatgtcat tgcgatggcg attcaaaaag atccagatga ccatcaagaa 1860 gaaatccacc aacgcattcg tctaggaaac ttgatgacgt tgacttctca aggtattgct 1920 ttcttacatg caggtcaaga gtatggacgc acgaagcaat ttagagcgga aacatcagag 1980 ccaccgtata aatctaccta tatgactgat gaaaatggtg agcctttccg ttacccatac 2040 tttattcatg actcgtatga ttctacagat atcatcaatc gttttgattg ggaaagagca 2100 acgaatgctg atgcttatcc gattcaaaat ttaacgagag agtatacaac aggtttaatt 2160 gagttaagac gttctagcga tgctttccga ttaggaacaa aagatcttgt agacgaaaaa 2220 gtaacgcaat taaacattcc agaaattgaa gaaacagatt tagttgtggc ttatcgcatt 2280 gaggcgacaa caggtgaagc attttatgta tttgtgaatg cggatgatga agaaagaaca 2340 ttaacattag aagaggattt aaccgtgggt gaatttgtcg ttgatggaaa aacagctggt 2400 gtgacagcga ttgccgagcc acaaggggtg gagttgagtg ctgagcaaat aaagctggat 2460 cca 2463

<210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 tataattggg gwtatgatcc wc 22

<210> 6 <211> 24 <212> DNA <213> Artificial Sequence <400> 6 acccatcata tcaaawckaa awcc 24

<210> 7 <211> 25 <212> DNA <213> Artificial Sequence <400> 7 tgcccgttag agcgaaataa ctttg 25

<210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 ctgatggtac gccaagaaca agctt 25

<210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 catttttagc aggttagtaa gtc 23

<210> 10 <211> 24 <212> DNA <213> Artificial Sequence <400> 10 cggtaccctt gttgaacgga cagc 24

<210> 11 <211> 23 <212> DNA <213> Artificial Sequence <400> 11 catttttagc aggttagtaa gtc 23

<210> 12 <211> 24 <212> DNA <213> Artificial Sequence <400> 12 cggtaccctt gttgaacgga cagc 24

[Brief description of the drawings]

FIG. 1 shows the alkaline pullulanase gene ( Bacillu
s sp. KSM-AP1876 strain) (pA
It is a figure which shows the restriction enzyme map of (P100).

FIG. 2 is a diagram showing a pH profile of alkaline pullulanase.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12N 9/44 (C12N 9/44 // (C12N 15/09 ZNA C12R 1:07) C12R 1:07) C12N 15/00 ZNAA (C12N 9/44 5/00 A C12R 1:07) C12R 1:07) (72) Inventor Katsuhisa Saeki 2606 Kabanecho, Kaigamachi, Haga-gun, Tochigi Pref. Kao Corporation Research Institute (72) Inventor Shuji Kawai 2606 Akabane, Kakaicho, Haga-gun, Tochigi Prefecture F-term in the Kao Corporation Research Laboratories (reference) 4B024 AA03 BA12 CA02 CA04 DA06 DA07 EA04 GA11 HA01 4B050 CC01 DD02 FF11E FF12E FF14E LL04 4B065 AA15X AA15CA0257

Claims (6)

[Claims] 1. 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 have been deleted, substituted or added. 2. A gene encoding the alkaline pullulanase according to claim 1. 3. The nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or one or several nucleotides of the nucleotide sequence are deleted,
3. The alkaline pullulanase gene according to claim 2, which has a substituted or added base sequence. 4. A recombinant vector containing the gene according to claim 2 or 3. A transformant comprising the recombinant vector according to claim 4. 6. The method for producing alkaline pullulanase according to claim 1, wherein the transformant according to claim 5 is cultured.