JPH09271385A - New dna fragments, plasmid and recombined microorganism holding these fragments and production of debranching enzyme therewith - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA fragment containing a debranching enzyme gene produced by a microorganism of the genus Bacillus, etc., having a specific restriction enzyme map, and useful for the genetic engineering production of an enzyme expressing a heat-resistant and acidic α-1,6-glucosidase activity, etc. SOLUTION: This DNA codes a debranching enzyme containing an amino acid sequence of formula I or its part and an amino acid sequence homologous thereto, and gives a restriction enzyme map of formula II. The DNA is useful for producing a heat-resistant enzyme having an acidic α-1,6-glucosidase (debranching enzyme) activity and used for the saccharification of starch, etc., in a high yield by a genetic engineering method. The DNA fragment is obtained by culturing Bacillus SP APC-9603 strain (FERM BP-4204), collecting the cells by a centrifugal method, isolating the chromosomal DNA from the cells by a conventional method, treating the DNA with a restriction enzyme, binding the obtained fragments to a vector, transducing the vector into Escherichia coli, culturing the transformant, selecting a strain decomposing a colored pullulan derivative to form a harrow, and subsequently recovering the DNA of the strain.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is heat-resistant and acidic α-1,6-
A DNA fragment having the genetic information of a protein having glucosidase activity (hereinafter, also referred to as debranching enzyme), an expression vector containing the DNA fragment, a transformant transformed with the expression vector, and the transformant are cultured. The present invention relates to a method for efficiently producing heat-resistant and acidic α-1,6-glucosidase.

More specifically, the heat resistance produced by Bacillus sp. APC-9603 (FERM BP-4204),
The gene for acid α-1,6-glucosidase is produced by Bacillus subtilis
The present invention relates to a method for efficiently producing the enzyme by expressing the enzyme.

[0003]

2. Description of the Related Art What is α-1,6-glucosidase?
It is an enzyme that cleaves 1,6-glucoside bonds to produce linear amylose, and is mainly classified into isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41) and the like based on its substrate specificity. In particular, pullulanase is widely used in the saccharification industry in combination with endo-type and exo-type amylase in the production of maltooligosaccharides such as glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and the like.

That is, a debranching enzyme that hydrolyzes an α-1,6-glucoside bond is added at the saccharification stage of liquefied starch in order to convert starch into glucose at a high level. As a result, it was found in yeast for a long time, and its production has been reported in various bacteria in recent years.

[0005] For example, as the producing bacteria, Aerobacter aerogenes, Escherichia
Escherichia intermedia, Pseudomonas amyloderamos
a), Streptococcus mites
tis), Cytophaga genus, Streptomyces genus and Flavochromogenes (Flav)
genus, etc.

On the other hand, the α-1,6-glucosidase produced by the genus Bacillus is Bacillus cereus IFO 3001, Bacillus cereus.
Bacillus fermus IFO 3330, Bacillus acidopullulyt
icus) and Bacillus sectramas (Bacillus sectorr)
amus) etc. are known.

[0007] Bacillus sp. APC-96 is a strain producing a novel debranching enzyme having heat resistance, optimum pH on the acidic side, and wide substrate specificity.
03 (FERM BP-4204) has been reported (JP-A-5-29296).
2).

That is, the debranching enzyme derived from Bacillus sp. APC-9603 is a more suitable enzyme to be used for saccharification of starch as compared with the debranching enzyme produced by conventional strains.
However, this productivity is not sufficient, and a more effective production method has been demanded.

[0009]

The present invention utilizes a Bacillus subtilis harboring a novel plasmid, which is thermostable, has an optimum pH on the acidic side, and has a wide substrate specificity.
The present invention relates to a method for producing -1,6-glucosidase.

The novel debranching enzyme used in the present invention is secreted and produced by Bacillus sp. APC-9603.
This strain was isolated from soil in Aichi prefecture.

Bacillus sp. APC-9603 has the following mycological properties. This strain has been deposited as FERM BP-4204 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

A. Morphology (1) Cell shape Straight rod shape (2) Cell size 0.5 to 0.8 × 3.0 to 4.0 μ (3) Motility positive (4) Sporangial circular swelling (1.0 to 1.5 μ) (5) ) Position of spores Endo-quasi-edge (6) Properties of colony Semi-transparent, glossy circular convex, moss is slightly yellowish (7) Gram stain variable (positive in immature cells, undefined in old culture)

B. Growth state and physiological properties (1) Growth negative in normal broth (2) Aerobic growth positive (3) Anaerobic growth negative (4) Growth temperature 13 to 45 ° C (5) Optimal growth temperature 33-38 ℃ (6) Growth pH 4.0-6.0 (7) Optimal growth pH 4.5-5.5 (8) Growth range in NaCl 0-5.0% (9) Nitrate reduction positive (10) Pigment formation negative (11 ) Use of citric acid Negative (12) Use of propionate Negative (13) Negative of catalase activity (14) Negative degradation of tyrosine (15) Negative hydrolysis of starch (16) Negative of VP reaction (17) Negative of lecithinase (18) Indole negative (19) Esculin decomposition positive (20) Litmus milk reaction unchanged (30 days) (21) Gelatin decomposition positive (weak) (22) Casein decomposition negative (23) Phenylalanine deamino negative (24) VP broth pH 5.0 (25) Utilization of sugars Glucose is acid-positive (delayed) Mannitol is acid-positive Arabinose is acid-positive Xysis Over scan acid from the acid-positive sucrose-positive (delayed) acid positive lactose (delay) Gas-negative trehalose from acid positive mannitol

In the above test, Gram stain, catalase, morphology, etc. are inspected by a designated yeast extract inorganic salt medium, growth test (pH, temperature) is Tributycase soy agar (BBL), and other physiological tests are all. The pH was adjusted to 5.0 ± 0.1 and the cells were cultured for 14 days.

The above-mentioned mycological properties are based on the Burj's Manual of Determinative Bacteriology (Be
rgey's Manual of Determinative Bacteriology) 8th
Edition and The Genus Bacillus
Ruth, B. Gordon, Agriculture Handbook No. 427, Agri
culture Research Service, USDepartment of Agricul
Strains were identified as described by ture Washington DC (1973).

That is, it is clear that this strain belongs to the genus Bacillus because it is Gram-staining positive, has spores, and grows aerobically. This strain is very similar to the mycological properties of Bacillus circulans, except that it is negative for catalase. Examples of the genus Bacillus known to be negative for catalase include Bacillus larb
vae), Bacillus popilliae or Bacillus lentimorbu
s) etc., but this strain is clearly different from these. Therefore, the present inventors named this strain Bacillus sp. APC-9603. Next, α-1,6 produced by this strain
-The enzymic chemistry of glucosidase is detailed below.

1 Action: This enzyme acts on the α-1,6-glucoside bond to produce linear amylose.

2 Substrate specificity: The relative activities of corn soluble starch, corn amylopectin, potato amylopectin and oyster glycogen with respect to the pullulan activity of this enzyme are shown in Table 1. The activity is shown relative to 1% concentration of substrate (pH 5.0, 100 mM McIlvain buffer).

[0019]

[Table 1]

3 Optimum pH: about 5.2 (100 mM McIlvain buffer) is shown. (Shown in Figure 1)

4 pH stability: Stable up to pH 4.0 to 6.0 at 40 ° C. for 30 minutes (100 mM McIlvain buffer). (Shown in Figure 2)

5 Optimum temperature: around 65 ° C (pH 5.0, 30
Minute reaction). (Shown in FIG. 3)

6 Temperature stability: 70 ° C., 30 minutes treatment
80% stable. (Shown in FIG. 4)

7 Effect of inhibitors: Slightly inhibited by p-mercuribenzoic acid (hereinafter referred to as p-CMB) and EDTA, and N
-Not inhibited by ethylmaleimide (hereinafter referred to as N-ENI) and monoiodoacetic acid (hereinafter referred to as MIA). The effect of each inhibitor is the residual activity after treatment for 30 minutes at 50 mM acetate buffer, pH 5.0, 40 mM at a final concentration of 1 mM of the inhibitor.
Shown in

[0025]

[Table 2]

8 Effect of metal salt: Mg ²⁺ , Co ²⁺ , Ca ²⁺ , Ni
Almost unaffected by ²⁺ , Ba ²⁺ , Cu ²⁺ , Zn ²⁺ and Ag ³⁺ , slightly inhibited by Mn ²⁺ , Fe ³⁺ , Sn ²⁺ and Cd ²⁺ , Hg ²⁺
Is strongly hindered by. The effect of various metal salts is that the final concentration of the metal salt is 1 mM per activity of 14 u / ml and 50 mM acetate buffer.
It shows the residual activity after treatment at pH 5.0, 40 ° C. for 30 minutes.

[0027]

[Table 3]

9 molecular weight: about 98,000 (by SDS-polyacrylamide gel electrophoresis)

10 Isoelectric point: about 2.9

11 Activity measuring method Branched dextrin DE7-9 (manufactured by Sanmatsu Industry Co., Ltd.) 0.4%
(W / v) and 0.05N acetate buffer (pH 5.0) dissolved substrate 6
Add 1.0 ml of enzyme solution to ml and react at 50 ° C for 30 minutes, then 2 ml
0.4M trichloroacetic acid-0.25N sulfuric acid solution was added and mixed,
Add 1 ml of 0.005N iodine solution, mix, and leave at 25 ° C for 15 minutes.

The absorbance (E ₃₀ ) at 610 nm of this solution is measured. As a blank, the enzyme was added to a 0.4 M trichloroacetic acid-0.25 N acetic acid solution, and after stirring, 6 ml of a substrate was added.

Next, 1.0 ml of 0.005N iodine solution was added and mixed well, and after leaving at 25 ° C. for 15 minutes, the absorbance at 610 nm (E ₀ )
Is measured.

One unit of the debranching enzyme activity is the amount of the enzyme that causes an increase in the absorbance of 1.0 under the above conditions, and is calculated from the following equation. Debranching enzyme activity (u / ml) = dilution _{× (E 30 -E 0) /1.0} E 30: reaction absorbance E _0: target absorbance

The DNA fragment encoding the debranching enzyme of the present invention has the activity of the debranching enzyme which can be produced by the above-mentioned Bacillus sp. APC-9603, and has the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. Or a part thereof and D encoding a debranching enzyme containing an amino acid sequence homologous thereto
It is characterized in that it contains an NA fragment. Here, "a part and homologous thereto" means that, as long as it has the activity of debranching enzyme, some deletions, insertions, substitutions, etc. of amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. Indicates that there may be.

For the DNA of the present invention, SEQ ID NO: in the sequence listing:
It goes without saying that the DNA consisting of 3934 bases shown in 2 is included, but the present invention is not limited to this. It should be noted that the modification of DNA so as to cause some deletions, insertions, substitutions, etc. of amino acids in the amino acid sequence encoded by DNA is carried out by a well-known method such as a site-directed mutagenesis method using synthetic oligonucleotides It can be performed appropriately.

Further, the whole gene of the debranching enzyme contains at least a promoter region sequence required for transcription, a signal sequence involved in secretion, a nucleotide sequence encoding a mature debranching enzyme, and a transcription and transfer termination region.

Further, the DNA fragment of the present invention preferably contains at least a nucleotide sequence encoding the mature protein of the debranching enzyme of Bacillus sp. APC-9603, a signal sequence enabling secretion, a transcription promoter and a transcription termination region. .

In the present invention, the modified debranching enzyme means an enzyme in which at least one amino acid differs from the wild-type enzyme in the amino acid sequence, and these modifications are carried out by a conventional method for mutagenesis of DNA, for example, ultraviolet irradiation. , Treatment with nitrous acid, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, or a genetic engineering method [Molcular cloning-alaboratory manual, Samnrook,
F. Ritsah, Maniatis-secound edition (1989)].

In order to express the gene of the present invention in a microorganism, it is first necessary to insert this gene into a plasmid vector or phage vector which is stably present in the microorganism. Further, in order to express the DNA of the present invention in a microorganism, it is necessary to transcribe / translate the genetic information contained therein. For that purpose, a promoter corresponding to a unit that controls transcription / translation should be a DN of the present invention.
The terminator may be incorporated in the upstream of 5′-side of A and in the downstream of 3′-side, respectively. As the promoter and terminator, it is necessary to use a promoter and terminator known to function in the microorganism used as the host.

For example, in the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors, and they can be directly inserted into the chromosome. Also, apr as a promoter and terminator
(Alkaline protease), npr (neutral protease), amy (α-amylase) and the like can be used.

In the present invention, Bacillus sp.
More preferably, the transcription promoter and secretory signal sequence in the entire gene encoding the cellulase of N-2 (FERM P-8809) are used.

That is, it is preferable to connect the structural gene encoding the mature debranching enzyme downstream of the transcription promoter and secretion signal sequence.

Further, the present invention also provides a recombinant strain which has been introduced by the genetic engineering method encoding a debranching enzyme. This gene is ligated to a plasmid and introduced into a host bacterium.

The microorganism to be transformed in the present invention may be any microorganism as long as it is transformed with a DNA encoding a polypeptide having a debranching enzyme activity and can express the debranching enzyme activity. ,
Specifically, the genus Escherichia, Bacillus
(Bacillus) genus, Pseudomonas genus, Penicillium genus, Serratia
Genus, Brevibacterium genus, Corynebacterium genus, Streptococcu genus, Lactobacillus
Kleberomyces (Saccharomyces), a bacterium for which a host-vector system such as a genus has been developed,
genus luyveromyces, Schizosacch
aromyces), Zygosaccharomyces
ces), Yarrowia spp., Trichosporon (Tric
hosporon) genus, Rhodosporidium genus,
The yeasts of the genera Hansenula, Pichia, Candida, etc., Neurospora (Neurospora)
ora), Aspergillus, Cephalosporium, Trichoderma
ma) Includes molds that belong to the genus.

As a preferred host, a bacterium of the genus Bacillus,
Alternatively, it is selected from the group including filamentous fungi of the genus Aspergillus and the genus Penicillium. More preferably, it is preferably selected from Bacillus bacteria.

The host is particularly preferably selected from Bacillus subtilis, Bacillus licheniformis, Bacillus pumilas, Bacillus amyloliquefaciens, Bacillus lentus and the like. Good results have been obtained when the host for expression of the debranching enzyme of the present invention is Bacillus subtilis strain IA289.

The procedure or method for producing a transformant can be carried out according to the techniques commonly used in the fields of ordinary molecular biology, biotechnology and genetic engineering.

The present invention also provides a method for preparing a debranching enzyme by culturing the recombinant bacterium, which comprises isolating a DNA fragment encoding the debranching enzyme, inserting the DNA fragment into an appropriate vector, and Also provided is a method comprising culturing a transformant obtained by introducing this vector into an appropriate host, expressing a debranching enzyme gene, and recovering the debranching enzyme produced in the culture solution.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Hereinafter, unless otherwise stated,% is expressed in W / V.

[0050]

【Example】

Example 1 Preparation and purification of debranching enzyme Bacillus sp. APC-9603 (FREM BP-4204) was prepared using MB medium Mist P1G (Asahi Breweries) 1.5%, branched dextrin 2.5%, KH ₂ PO ₄ 0.1%, MgSO ₄・ 7H ₂ O 0.03%, FeS
_{O 4 · 7H 2 O 0.01%} , 37 ℃ at _{(NH 4) 2 SO 4 0.025} % (pH5.3)
Culture with aeration and stirring.

After culturing for 65 hours, centrifugation (KUBOTA RA
(8, 5000 rpm, 30 minutes) and collect the supernatant as crude debranching enzyme solution.

Then, the solution is concentrated by ultrafiltration to obtain a debranching enzyme concentrate. The resulting concentrated debranching enzyme solution is α-CD
(Cyclodextrin) Sepharose to adsorb the enzyme, and then 0.2% γ-CD, 50 mM acetate buffer (pH 5.
Elution was carried out in 5) and the active fraction was collected.

The obtained eluent was mixed with 10 mM phosphoric acid (KH ₂ PO ₄ -K ₂
MonoQ after dialysis against HPO ₄ ) buffer (pH 6.0) overnight
Adsorbed on a column (Pharmacia), NaCl 60-200
Elution was performed with a linear gradient of mM.

The main peak was collected, and the obtained eluate was dialyzed against 10 mM acetate buffer (pH 5.5) to obtain a purified debranching enzyme. When the obtained debranching enzyme was subjected to SDS-polyacrylamide gel electrophoresis, it was confirmed to be a single band.

Example 2 Bacillus sp. APC-9603
Cloning of Debranching Enzyme Gene Produced by Bacillus sp. APC-9603 (FREMBP-4204), a α-1,6-Glucosidase-Producing Bacterium
(Manufactured by Asahi Breweries), 1.5% branched dextrin (manufactured by Sanmatsu Kogyo Co., Ltd.), 0.1% potassium phosphate, 0.05% magnesium sulfate heptahydrate, 0.01% ferrous sulfate heptahydrate, 0.0
100 ml of a medium consisting of 25% ammonium sulfate (pH 5.2) was used and cultured with shaking at 37 ° C. for 24 hours.

After the completion of the culture, the cells obtained by collecting the cells by centrifugation are subjected to the method of Saito et al. [Biochim. Biophys. Acta, 72 ,
619-629 (1963)] to obtain chromosomal DNA.

The obtained chromosomal DNA was digested with the restriction enzyme Sau3AI
DNA partially digested with (Takara Shuzo) and restriction enzyme
PUC11 obtained by dephosphorylation after digestion with BamHI (Takara Shuzo)
8 (Takara Shuzo) was ligated with a DNA ligation kit (Takara Shuzo).

Using this ligation product, E. coli DH5
Was transformed to obtain an ampicillin-resistant transformant. The obtained ampicillin-resistant transformed colonies were transferred to sterilized filter paper (manufactured by Advantech Toyo Co., Ltd.), and then Tsukagoshi
And the like [Mol. Gen. Genet., 193 , 58-63 (1984)], followed by Rinder Knecht et al. [Experimenti
a, 23 , 805 (1967)], and layered on an agar plate medium containing 1% of a colored derivative of pullulan (pullulan red), and allowed to stand overnight at 37 ° C to decompose pullulan red to form halo. A transformant strain was obtained. The plasmid contained in this transformant was designated as pBC24.

Example 3 Determination of amino acid sequence Determination of N-terminal amino acid sequence The purified debranching enzyme obtained in Example 1 was determined for its amino acid sequence by a protein sequencer by the solid phase method. The determined sequence is as shown in SEQ ID NO: 3 from left to right in the amino-carboxyl orientation.

Determination of Amino Acid Sequence of Debranching Enzyme The nucleotide sequence of a DNA fragment of about 4.0 Kb which was excised with Sau3AI from the plasmid pBC24 containing the gene encoding the debranching enzyme obtained in Example 2 was prepared by Applied Biosystems. Determined using the DNA Sequence Kit and DNA Sequencer 373A.

Analysis of this sequence begins with ATG 2853bas
The existence of an open reading frame consisting of es was shown. It was confirmed that a sequence corresponding to the N-terminal amino acid sequence of the debranching enzyme was present in the amino acid sequence obtained by translating this open reading frame. From this result, it was revealed that the debranching enzyme was synthesized as a precursor having a pre-sequence consisting of 29 amino acids.

This pre-sequence is characteristic of the secretory signal sequence involved in the extracellular secretion of the protein. (Freu
dl, Journal of Biotechnology, 23 , 231-240 (199
2)]. The 29 amino acid sequence is shown in SEQ ID NO: 4.

Example 4 Amino Acid Distribution Table 4 summarizes the amino acid distribution of the mature debranching enzyme determined from the amino acid sequence of the debranching enzyme (of Example 3).

[0064]

[Table 4]

Example 5 Construction of plasmid and production of debranching enzyme by transformant pB24 obtained by cleaving pBC24 with restriction enzyme EcoRI and dephosphorylating
A DNA fragment obtained by cleaving C24-RI-BAP and pUB110 (vector for Bacillus subtilis) with EcoRI was ligated with a DNA ligation kit (Takara Shuzo). Using the ligation reaction solution,
Escherichia coli DH5 was transformed, and a plasmid pUBC24 (FIG. 6) which was capable of replicating in Escherichia coli and Bacillus subtilis and which contained a debranching enzyme gene derived from Bacillus sp. APC-9603 was obtained from the obtained transformant resistant to ampicillin.

The Bacillus subtilis IA289 strain was subjected to the protoplast method using pUBC24 [S. Chang, SNChoen, Mol. Gen. Genet., 16
8 , 111-115 (1978)] and transformed into recombinant Bacillus subtilis IA
289 (pUBC24) strain was acquired. Obtained recombinant Bacillus subtilis IA289
(pUBC24) in TS medium [1.0% tryptone, 0.75% SMS peptide (manufactured by Fuji Oil Co., Ltd.), 0.5% NaCl, 0.25% K ₂ HPO ₄ ,
0.02% MgSO ₄ , 0.75% glucose (pH 7.0)] at 37 ° C
Shaking culture for 24 hours produced 6.8 units of debranching enzyme per ml of medium.

Example 6 Cloning of Cellulase Gene Bacillus sp. N-2 (FERM P-8809) was supplemented with 1% tryptone (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 0.05M Sucrose, 1% Na ₂ 200 m of CO ₃ medium
The cells were cultivated with shaking at 37 ° C. for 16 hours.

After culturing, centrifugation (KUBOTA RA6, 8000
rpm, 10 min) and collected from the bacterial cells in the same manner as in Example 2.
Chromosomal DNA was obtained by the method described in o.

5 μg of the obtained chromosomal DNA was digested with the restriction enzyme Hi.
Partially decomposed with ndIII (Takara Shuzo). Then this is Hin
The mixture was mixed with 1 μg of plasmid pBR322 that had been dephosphorylated after digestion with dIII and ligated using a DNA ligation kit (Takara Shuzo).

The ligation product was used to transform E. coli HB101 strain into H
anahan et al. [Douglas Hanahan, DNA Cloning, 1 , 1
09-135]. Bacterial fluid after treatment
LB agar medium containing 100 μg / ml ampicillin (manufactured by Sigma) [1% tryptone (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 1.0% NaCl, 1.5% agar (manufactured by Wako Pure Chemical Industries)] And was cultured overnight at 37 ° C.

A colony of the ampicillin-resistant transformant that appeared was LB / CMC / agar medium [LB agar, 1% carboxymethyl cellulose (CMC) (manufactured by Tokyo Kasei), 100 μg / ml.
Ampicillin] and cultured at 37 ° C for 16 hours to decompose CMC around the colony using the Congo red method [RM Teath
er, PJWood., Appl. Environ. Microbiol., 43 , 777-
780 (1982)], and an Escherichia coli transformant in which the cellulase gene was cloned was obtained.

Example 7 The recombinant plasmid harbored by the recombinant Escherichia coli obtained in Example 6 was prepared by a conventional method [eg, T. Maniatis et al., Molecular Cloning,
Spring Cold Harbar Laboratry (1982)] and analyzed the recognition sites of various restriction enzymes by agarose gel electrophoresis [JA Meyer et al., J. Bacteriol., 127 , 1529-153.
7 (1976)] and subcloning, it was revealed that the cellulase gene exists on a DNA fragment of about 1.7 Kb cut out with HindIII-ScaI.

The plasmid containing this DNA fragment was designated as pCN21.
2. The E. coli HB101 strain retaining pCN212 was designated as HB101 (pCN212) strain.

Inserted DNA fragment of pCN212 About 1.7 Kb HindIII-
The ScaI region was modified by the Sanger method [F. Sanger et al., Proc. Natl. Aca
d. Sci. USA, 74 , 5463-5467 (1977)] and found a structural gene of cellulase consisting of 1227 bp.

At the N-terminal of the amino acid sequence deduced from this structural gene, a signal peptide-like sequence characteristic of a secretory protein was observed. A ribosome binding site (SD sequence) was found upstream of the translation initiation point (ATG).

Example 8 Expression of Cellulase Gene in Bacillus subtilis About 1.7 excised with ScaI-HindIII shown in Example 6
A DNA fragment containing the Kb cellulase gene was cloned into pUBH1 [F.Kaw
amura, RHDoi, J. Bacteriol., 160 (1), 422-444 (198
4)] was inserted and ligated into the HindIII-EcoRV site. Transformation of the obtained Bacillus subtilis RM125 strain (hsdR, hsdM, arg15, leuB8) was carried out by the protoplast method [S. Chang, SNChoen., Mol. Gen.
Gent., 168 , 111-115 (1978)], 300 μg / ml
DM3, a protoplast regeneration medium containing kanamycin
Medium [0.5% sodium succinate (pH 7.3), 0.5% casamino acid (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 0.35% K ₂ HPO ₄ , 0.15% KH ₂ PO ₄ , 0.5% glucose] , 20 mM MgCl ₂ , 0.01% bovine serum albumin (manufactured by Sigma)]. As a result, a recombinant Bacillus subtilis RM125 (pCNU212) strain containing the recombinant plasmid was obtained.

The obtained RM125 (pCNU212) strain and RM125 (pUBH1) strain carrying the plasmid vector pUBH1 were added at 5 μg / m 2.
Medium containing l kanamycin [5.0% soluble starch, 2.0
% Polypeptone, 2.0% Proten F (manufactured by Ajinomoto Co.), 0.
5% meat extract, 0.2% yeast extract, 0.3% KH ₂ PO ₄ , 1%
K ₂ HPO ₄ , 0.2% NaCl, 0.1% MgSO ₄ (pH 7.0)] 37
When the cellulase activity in the culture solution was measured by shaking culture at 48 ° C. for 48 hours, the RM125 (pCNU212) strain showed 270 times more cellulase productivity than the RM125 (pUBH1) strain.

[0078]

[Table 5]

From these results, Bacillus sp. N-2
It was revealed that the cellulase gene of Bacillus subtilis was efficiently expressed in Bacillus subtilis by its promoter and secreted the gene product.

Example 9 For the purpose of linking the region encoding the mature protein of the debranching enzyme gene derived from Bacillus sp. APC-9603 strain, to the downstream of the cellulase gene promoter and signal peptide derived from Bacillus sp. N-2 strain, A new restriction enzyme Aor51HI recognition site is constructed inside the cellulase gene and debranching enzyme gene.

Oligonucleotides (for cellulase gene: nucleotide 1, for debranching enzyme gene: nucleotide 2) for mutating each gene were prepared as follows.

Nucleotide 1: 5'-ATAGGAAACACGAGCGCTG
CTGATGATTATTC-3 'nucleotide 2: 5'-TTTCAAAGGCCAGCGCTCATGCGGACGGAAC
G-3 '

Mutations were introduced into each gene by the KunKel method using these two kinds of oligonucleotides [Kunkel, TA et al., Methods in Enzymology, 154 , 367 (1).
987)]. The KunKel method was carried out using a Mutan-K kit (manufactured by Takara Shuzo Co., Ltd.), and the operation followed the protocol.

That is, pBC24 obtained in Example 2 was replaced with BamHI-HindI.
After cutting with II, a 937 bp DNA fragment containing the promoter of the debranching enzyme gene and the N-terminal region was cloned into M13mp19 phage DNA, and single-stranded DNA was prepared according to a conventional method. Was used to introduce mutations.

42 obtained from pCN212 obtained in Example 7
A 6 bp EcoRI-BamHI digested fragment was cloned into M13 phage mp19, and single-stranded DNA was obtained by a conventional method. A mutation was introduced into the cellulase gene using the obtained single-stranded DNA and nucleotide 1.

Each of the DNA fragments containing the mutation was ligated to pUC118 to obtain pMCP1 (mutant cellulase) and pMBCP1 (mutant debranching enzyme).

A fragment of about 3440 bp obtained by double-cutting pMCP1 with restriction enzymes Aor51HI and HindIII, and pMBCP1 with Aor51HI and Hin.
A 375 bp DNA fragment obtained by double-cutting with dIII was ligated into pCB
I got N1.

A DNA fragment of about 650 bp obtained by double-cutting pCBN1 with EcoRI-HindIII, a DNA fragment of about 3.0 Kbp obtained by double-digesting pBC24 with HindIII-ClaI, and pCNU212.
Using a ligation solution obtained by ligating a DNA fragment of about 4.0 Kb obtained by double digestion with coRI-ClaI with a DNA ligation kit (Takara Shuzo), in the same manner as in Example 5, Bacillus subtilis IA
The 289 strain was transformed by the protoplast method and transformed into IA289 (pUBC
32) A strain was obtained. The structure of pUBC32 is shown in FIG.

Example 10 IA289 (pUBC24) obtained in Example 5 and IA289 (pUBC32) obtained in Example 9 were added to a medium containing kanamycin (5 μg / ml) [6% Paindex # 3 (Matsuya Chemical Co., Ltd. Made),
2.0% SMS peptide (manufactured by Fuji Oil Co., Ltd.), 0.2% Meast p
1G (manufactured by Asahi Breweries, Ltd.), (manufactured by Ajinomoto Co.) 1.0% sodium _{glutamate, 0.5% K 2 HPO 4,} 0.05% MgSO 4 · 7H 2 O,
0.01% FeSO ₄ .7H ₂ O (pH 7.2)] was shake-cultured at 37 ° C. for 48 hours. The debranching enzyme activity in each culture is
The IA289 (pUBC32) strain showed about 9.6 times the debranching enzyme productivity of the IA289 (pUBC24) strain.

[0090]

[Table 6]

Example 11 Bacillus subtilis IA289 (pUBC32) in a medium containing kanamycin (5 μg / ml) [6% pa index # 3, 2.0% SMS peptide, 0.2% meest p1G, 1.0% sodium glutamate,
_{0.5% K 2 HPO 4, 0.05} % MgSO 4 · 7H 2 O, 0.01% FeSO 4 · 7H 2 O
(PH7.2)], the cells were cultured by shaking at 37 ° C. for 48 hours, and then the cells and solids were removed by centrifugation (KUBOTA RA6, 8000 rpm, 10 min) to obtain a centrifugation supernatant. After the centrifugal supernatant was concentrated by ultrafiltration, the debranching enzyme was purified by α-CD sepharose column chromatography and MonoQ column chromatography in the same manner as in Example 1.

The N-terminal amino acid sequence of the obtained recombinant purified enzyme was determined by a protein sequencer by the solid phase method. This sequence is based on the Bacillus
The debranching enzyme derived from SPC APC-9603 and the N-terminal amino acid sequence determined from the debranching enzyme gene were identical.

[0093]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a large-scale and efficient method for producing a debranching enzyme that is thermostable, works in an acidic region, and has broad substrate specificity by recombinant DNA technology. It opens up. Moreover, the enzyme produced from the transformant according to the present invention is an enzyme whose entire amino acid sequence has been clarified, and induces a enzyme with higher utility value in terms of substrate specificity or stability against heat and pH. It is also possible.

[0094]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 922 Sequence type: Amino acid sequence Asp Gly Thr Thr Thr Asn Val Ile Val His Tyr Phe Arg Pro Gly 15 Gly Asp Tyr Gln Ser Trp Ser Leu Trp Met Trp Pro Glu Gly Gly 30 Asp Gly Asn Asn Tyr Asn Phe Asn Gly Thr Asp Ser Tyr Gly Glu 45 Ile Ala Asn Val Ser Ile Pro Gly Ser Pro Ser Lys Val Gly Ile 60 Ile Val Arg Thr Gln Asp Trp Ala Lys Asp Val Ser Gln Asp Arg 75 Tyr Ile Asp Leu Ser Lys Gly His Glu Val Trp Leu Val Gln Gly 90 Asn Ser Gln Ile Phe Tyr Asn Glu Lys Asp Ala Glu Asp Ala Ala 105 Glu Pro Ala Val Ser Asn Ala Tyr Leu Asp Ala Pro Asn Lys Val 120 Leu Val Lys Leu Ser Gln Pro Phe Thr Leu Gly Glu Gly Ala Ser 135 Gly Phe Thr Val His Asp Asp Thr Ala Asn Gly Asp Ile Pro Val 150 Thr Lys Val Lys Asn Ala Asn Lys Val Glu Asp Val Thr Ala Ile 165 Leu Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser Asp Trp Ala 180 Pro Asp Asn His Ser Thr Gln Leu Lys Lys Val Asn Asp Asn Leu 195 Tyr Gln Phe Ser Gly Glu Leu Pro Gly Gly Ser Tyr Gln Tyr Lys 210 Val Ala Leu Asn Asp Ser Trp Asn Ser Ser Tyr Pro Ser Asp Asn 225 Ile Asn Leu Thr Val Pro Asp Gly Gly Ala His Val Thr Phe Ser 240 Tyr Val Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro 255 Gly Ala Asn Leu Pro Leu Asp Gly Ser Gly Ile Lys Thr Asp Leu 270 Val Thr Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu 285 Ser Ile Gln Thr Asp Gly Phe Lys Thr Gly Arg Val Ile Pro Arg 300 Asn Val Leu Asp Phe Ser Gln Tyr Tyr Tyr Ser Gly Glu Asp Leu 315 Gly Asn Thr Tyr Thr Lys Lys Ala Thr Thr Phe Lys Val Trp Ala 330 Pro Thr Ser Thr Lys Val Asn Val Leu Leu Tyr Asn Lys Ala Ala 345 Gly Ala Leu Thr Lys Thr Val Pro Met Lys Ala Ser Gly His Gly 360 Val Trp Ser Val Thr Val Pro Gln Asn Leu Glu Asn Trp Tyr Tyr 375 Leu Tyr Glu Val Thr Gly Gln Gly Ser Thr Arg Thr Ala Val Asp 390 Pro Tyr Ala Thr Ala Ile Ala Pro Asn Gly Thr Arg Gly Met Val 405 Val Asp Leu Ala Lys Thr Asn Pro Thr Gly Trp Lys Ser Asp Lys 420 His Met Thr Pro Lys Asn Ile Glu Asp Glu Val Ile Tyr Glu Met 435 His Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met Thr Asn 450 LysGly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly Pro 465 Glu Asn Val Lys Thr Gly Val Asp Ser Leu Lys Gln Leu Gly Ile 480 Thr His Val Gln Leu Gln Pro Val Phe Ala Phe Asn Ser Val Asp 495 Glu Thr Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn 510 Tyr Asn Val Pro Glu Gly Gln Tyr Ala Thr Asp Ala Asn Gly Thr 525 Thr Arg Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Arg 540 Asn His Ile Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe 555 Ala Thr Gln Ile Ser Asp Phe Asp Lys Ile Val Pro Gln Tyr Tyr 570 Tyr Arg Thr Asp Asp Ala Gly Asn Tyr Thr Asn Gly Ser Gly Thr 585 Gly Asn Glu Val Ala Ala Glu Arg Pro Met Val Gln Lys Phe Ile 600 Ile Asp Ser Leu Lys Tyr Trp Val Asn Glu Tyr His Ile Asp Gly 615 Phe Arg Phe Asp Leu Met Ala Leu Leu Gly Lys Asp Thr Met Ala 630 Lys Ala Ala Gln Glu Leu His Ala Ile Asp Pro Gly Ile Ala Leu 645 Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala Leu Pro Thr Asp 660 Gln Leu Leu Thr Lys Gly Val Gln Lys Gly Met Gly Val Ala Val 675 Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Asn Val Phe Asp 690 Ala Ser Ser Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr Asp 705 Val Ile Lys Lys Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ser 720 Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr 735 Thr Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu 750 Ala Asp Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Val Val Val 765 Thr Ser Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu 780 Arg Thr Lys Gly Gly Asn Ser Asn Ser Tyr Asn Ala Gly Asp Ala 795 Val Asn Glu Phe Asp Trp Ser Arg Lys Ala Gln Tyr Ser Asp Val 810 Phe Asn Tyr Tyr Ser Gly Leu Ile His Leu Arg Leu Ala His Pro 825 Ala Phe Arg Met Thr Thr Ala Asn Gln Ile Lys Glu His Leu Gln 840 Phe Ile Asp Ser Pro Asp Asn Thr Val Ala Tyr Glu Leu Thr Asn 855 His Ala Asn Lys Asp Lys Trp Gly Asn Ile Val Val Ile Tyr Asn 870 Pro Asn Lys Thr Ala Glu Thr Val Asn Leu Pro Ser Gly Lys Trp 885 Ala Ile Asn Ala Thr Asn Gly Lys Ile Gly Glu Ser Thr Leu Ser 900 His Ala Glu Gly His Val Gln Val Pro Gly Ile Ser Met Met Ile 915 Leu His Gln Glu Thr Asn Lys 922

[0095] SEQ ID NO: length of the two sequences: 3934 Type of sequence: nucleic acid sequence GATCCTTTTT TGACTACAAA ATTTCAAATT GGAGTGAAGG TCAATCTTGA AACTCTCAGG 60 GGGGAAAAAC AGCGAATGGC AATAGCATGG ATGTTCCTTA AAAAACCACC GTTCGTTGAT 120 AAATAAAAGA AGGAGCACTC ACTCACACTG GTGCCTTCCT TTTGTATATG AGCTGTGCTT 180 AATAGTTTTT GTTCGAGATA TTTATAAAAA GTAGGTATGA CTTTAGAACT ATGGTACGGA 240 GTTATGAGGT GGACTAAAGT AGGTTTTTTG AAAAAATGGG ACTCTGTCCT TTCATTCGAC 300 AGTTTTTTTA AAAGAATGAC GGTATAATTG AAATCGGTGT AAGCGCTTTA TAATCAAATG 360 GAGGGGGGAG CCGTTTGAAA TATAAAAGTG TTTTTTGAGA AAGATTTGCT TTCTTTAATC 420 ATTAAGTGCA ACCGGTTGCC TTTTGTGCTA TTTAATGCAA CCGATTGCAT ATAGGAGGAC 480 AC ATG ATG AAA AAA GTA ATT TAC GTG CTT TTA AGT TTA TGT TTA GTG 527 Met Met Lys Lys Val Ile Tyr Val Leu Leu Ser Leu Cys Leu Val -15 TTG TCA TGG GCT TTT AAT TTC AAA GGC CAG TCT GCT CAT GCG GAC 572 Leu Ser Trp Ala Phe Asn Phe Lys Gly Gln Ser Ala His Ala Asp 1 GGA ACG ACA ACA AAC GTC ATT GTT CAT TAT TTT CGG CCA GGT GGT 617 Gly Thr Thr Thr Asn Val Ile Val His Tyr Phe Arg Pro Gly Gly 16 GAT TAC CAA TCA TGG AGT CTT TGG ATG TGG CCA GAA GGT GGT GAT 662 Asp Tyr Gln Ser Trp Ser Leu Trp Met Trp Pro Glu Gly Gly Asp 31 GGG AAC AAT TAT AAT TTT AAT GGA ACA GAT TCG TAT GGG GAA ATT 707 Gly Asn Asn Tyr Asn Phe Asn Gly Thr Asp Ser Tyr Gly Glu Ile 46 GCC AAT GTT TCG ATT CCA GGG AGT CCA AGT AAG GTT GGA ATT ATT 752 Ala Asn Val Ser Ile Pro Gly Ser Pro Ser Lys Val Gly Ile Ile 61 GTT CGC ACC CAG GAT TGG GCA AAA GAT GTG AGT CAA GAC CGC TAC 797 Val Arg Thr Gln Asp Trp Ala Lys Asp Val Ser Gln Asp Arg Tyr 76 ATA GAT CTT AGC AAA GGT CAT GAG GTT TGG CTT GTC CAG GGA AAT 842 Ile Asp Leu Ser Lys Gly His Glu Val Trp Leu Val Gln Gly Asn 91 AGC CAA ATT TTT TAT AAT GAA AAG GAT GCT GAA GAT GCC GCC GAG 887 Ser Gln Ile Phe Tyr Asn Glu Lys Asp Ala Glu Asp Ala Ala Glu 106 CCC GCT GTG AGC AAT GCT TAT TTA GAT GCG CCA AAT AAG GTA TTG 932 Pro Ala Val Ser Asn Ala Tyr Leu Asp Ala Pro Asn Lys Val Leu 121 GTT AAG CTT AGT CAG CCG TTT ACT CTT GGT GAA GGG GCT AGC GGT 997 Val L ys Leu Ser Gln Pro Phe Thr Leu Gly Glu Gly Ala Ser Gly 136 TTT ACG GTT CAC GAT GAC ACT GCA AAT GGG GAT ATC CCG GTT ACT 1022 Phe Thr Val His Asp Asp Thr Ala Asn Gly Asp Ile Pro Val Thr 151 AAG GTG AAG AAT GCC AAT AAG GTC GAG GAT GTT ACC GCC ATT CTT 1067 Lys Val Lys Asn Ala Asn Lys Val Glu Asp Val Thr Ala Ile Leu 166 GCA GGG ACC TTC CAA CAT ATT TTT GGT GGT TCC GAT TGG GCA CCT 1112 Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser Asp Trp Ala Pro 181 GAT AAT CAT AGT ACG CAG CTT AAA AAA GTA AAT GAT AAC CTT TAC 1157 Asp Asn His Ser Thr Gln Leu Lys Lys Val Asn Asp Asn Leu Tyr 196 CAA TTT TCC GGT GAG TTG CCA GGG GGG AGT TAC CAA TAT AAA GTG 1202 Gln Phe Ser Gly Glu Leu Pro Gly Gly Ser Tyr Gln Tyr Lys Val 211 GCA TTA AAT GAT AGC TGG AAT AGT AGC TAC CCA TCT GAC AAC ATT 1247 Ala Leu Asn Asp Ser Trp Asn Ser Ser Tyr Pro Ser Asp Asn Ile 226 AAT TTA ACT GTA CCG GAT GGA GGC GCA CAT GTG ACG TTC TCT TAT 1292 Asn Leu Thr Val Pro Asp Gly Gly Ala His Val Thr Phe Ser Tyr 241 GTC CCG TCC ACC CAT GCT GTA TAT GAC AC G ATT AAT AAT CCA GGA 1337 Val Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro Gly 256 GCT AAT TTA CCT TTA GAT GGG AGC GGT ATC AAA ACA GAT CTA GTA 1382 Ala Asn Leu Pro Leu Asp Gly Ser Gly Ile Lys Thr Asp Leu Val 271 ACG GTG ACT TTG GGA GAA AAC CCA GAT GTC TCG CAT ACC ACC CTT TCT 1427 Thr Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu Ser 286 ATC CAA ACA GAC GGT TTT AAG ACA GGA AGG GTC ATC CCT CGA AAT 1472 Ile Gln Thr Asp Gly Phe Lys Thr Gly Arg Val Ile Pro Arg Asn 301 GTG CTG GAC TTC TCT CAA TAT TAT TAT TCA GGA GAG GAT CTT GGG 1517 Val Leu Asp Phe Ser Gln Tyr Tyr Tyr Ser Gly Glu Asp Leu Gly 316 AAC ACC TAT ACA AAG AAA GCA ACA ACC TTT AAA GTG TGG GCT CCA 1562 Asn Thr Tyr Thr Lys Lys Ala Thr Thr Phe Lys Val Trp Ala Pro 331 ACT TCT ACT AAG GTG AAT GTT CTG CTT TAT AAC AAA GCA GCG GGT 1607 Thr Ser Thr Lys Val Asn Val Leu Leu Tyr Asn Lys Ala Ala Gly 346 GCT CTT ACA AAG ACT GTT CCT ATG AAG GCA TCA GGA CAT GGC GTG 1652 Ala Leu Thr Lys Thr Val Pro Met Lys Ala Ser Gly His Gly Val 361 TGG TC A GTG ACC GTT CCA CAA AAC CTA GAA AAT TGG TAT TAC TTG 1697 Trp Ser Val Thr Val Pro Gln Asn Leu Glu Asn Trp Tyr Tyr Leu 376 TAT GAG GTA ACT GGT CAA GGC TCT ACC CGA ACA GCG GTT GAC CCT 1742 Tyr Glu Val Thr Gly Gln Gly Ser Thr Arg Thr Ala Val Asp Pro 391 TAT GCC ACC GCA ATC GCA CCA AAT GGA ACG AGA GGC ATG GTT GTT 1787 Tyr Ala Thr Ala Ile Ala Pro Asn Gly Thr Arg Gly Met Val Val 406 GAC CTA GCC AAA ACA AAC CCG ACT GGT TGG AAG AGC GAC AAA CAT 1832 Asp Leu Ala Lys Thr Asn Pro Thr Gly Trp Lys Ser Asp Lys His 421 ATG ACA CCG AAG AAC ATA GAG GAT GAA GTT ATT TAT GAA ATG CAT 1877 Met Thr Pro Lys Asn Ile Glu Asp Glu Val Ile Tyr Glu Met His 436 GTC CGA GAC TTC TCC ATT GAT TCC AAT TCA GGT ATG ACA AAT AAA 1922 Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met Thr Asn Lys 451 GGG AAG TAC TTA GCC CTT ACT GAA AAA GGG ACC AAA GGC CCT GAA 1967 Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly Pro Glu 466 AAT GTC AAA ACA GGT GTG GAT TCA TTG AAG CAG CTT GGC ATT ACC 2012 Asn Val Lys Thr Gly Val Asp Ser Leu Ly s Gln Leu Gly Ile Thr 481 CAT GTT CAG CTT CAG CCT GTC TTC GCC TTT AAC AGC GTT GAT GAA 2057 His Val Gln Leu Gln Pro Val Phe Ala Phe Asn Ser Val Asp Glu 496 ACG GAT CCC ACC CAA TAT AAC TGG GGC TAT GAT CCT CGT AAC TAT 2102 Thr Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr 511 AAT GTT CCG GAA GGG CAG TAT GCA ACG GAT GCA AAC GGC ACA ACT 2147 Asn Val Pro Glu Gly Gln Tyr Ala Thr Asp Ala Asn Gly Thr Thr 526 CGG ATT AAA GAG TTT AAA GAA ATG GTT CTT TCC CTC CAT CGA AAC 2192 Arg Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Arg Asn 541 CAC ATT GGA GTC AAC ATG GAT GTG GTG TAT AAT CAT ACC TTT GCC 2237 His Ile Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe Ala 556 ACA CAA ATA TCT GAC TTT GAT AAG ATT GTC CCG CAA TAT TAT TAC 2282 Thr Gln Ile Ser Asp Phe Asp Lys Ile Val Pro Gln Tyr Tyr Tyr 571 CGG ACA GAT GAT GCG GGG AAC TAC ACC AAT GGG TCA GGT ACA GGA 2327 Arg Thr Asp Asp Ala Gly Asn Tyr Thr Asn Gly Ser Gly Thr Gly 586 AAC GAA GTG GCA GCT GAA CGG CCA ATG GTT CAA AAA TTT ATT ATT 2372 Asn Gl u Val Ala Ala Glu Arg Pro Met Val Gln Lys Phe Ile Ile 601 GAT TCA CTT AAG TAT TGG GTG AAT GAG TAC CAT ATT GAC GGC TTC 2417 Asp Ser Leu Lys Tyr Trp Val Asn Glu Tyr His Ile Asp Gly Phe 616 CGG TTT GAC TTA ATG GCA TTA CTT GGA AAA GAT ACA ATG GCA AAA 2462 Arg Phe Asp Leu Met Ala Leu Leu Gly Lys Asp Thr Met Ala Lys 631 GCG GCA CAA GAG CTT CAT GCG ATA GAT CCA GGG ATT GCC CTT TAT 2507 Ala Ala Gln Glu Leu His Ala Ile Asp Pro Gly Ile Ala Leu Tyr 646 GGT GAG CCT TGG ACG GGA GGC ACA TCA GCG CTA CCA ACC GAT CAG 2552 Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala Leu Pro Thr Asp Gln 661 CTT TTA ACA AAA GGC GTT CAG AAA GGC ATG GGT GTG GCT GTG TTT 2597 Leu Leu Thr Lys Gly Val Gln Lys Gly Met Gly Val Ala Val Phe 676 AAT GAC AAT CTG CGA AAC GGG CTG GAT GGC AAC GTT TTT GAT GCC 2642 Asn Asp Asn Leu Arg Asn Gly Leu Asp Asn Gly Asn Val Phe Asp Ala 691 TCC TCT CAG GGC TTT GCC ACA GGG GCA ACA GGC TTA ACA GAT GTT 2687 Ser Ser Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr Asp Val 706 ATT AAA AAG GGT GTT GAA GGG AGT ATC AAT GAC TTC ACC TCG TCA 2732 Ile Lys Lys Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ser Ser 721 CCA GGT GAG ACA ATC AAC TAT GTC ACA AGT CAT GAT AAC TAT ACG 2777 Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr Thr 736 CTC TGG GAT AAG ATT GCT CAA AGT AAT CCT AAC GAT TCT GAA GCG 2822 Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu Ala 751 GAT CGA ATA AAA ATG GAT GAA CTG GCT CAA GCT GTC GTG GTG ACG 2867 Asp Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Val Val Val Thr 766 TCA CAA GGG GTT CCG TTC ATG CAG GGG GGG GAA GAG ATG CTT CGC 2912 Ser Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg 781 ACG AAA GGT GGA AAC AGT AAT AGC TAT AAT GCA GGT GAT GCG GTC 2957 Thr Lys Gly Gly Asn Ser Asn Ser Tyr Asn Ala Gly Asp Ala Val 796 AAT GAA TTT GAT TGG AGC CGA AAA GCC CAA TAC TCA GAT GTT TTC 3002 Asn Glu Phe Asp Trp Ser Arg Lys Ala Gln Tyr Ser Asp Val Phe 811 AAC TAT TAT AGC GGA CTC ATC CAC CTT CGT CTT GCT CAC CCC GCC 3047 Asn Tyr Tyr Ser Gly Leu Ile His Leu Arg Leu Ala His Pro Ala 826 TTC CGT ATG ACG ACA GCG AAT CAA ATA AAA GAG CAT CTC CAA TTC 3092 Phe Arg Met Thr Thr Ala Asn Gln Ile Lys Glu His Leu Gln Phe 841 ATA GAT AGC CCG GAC AAT ACC GTT GCT TAT GAG TTA ACG AAT CAT 3137 Ile Asp Ser Pro Asp Asn Thr Val Ala Tyr Glu Leu Thr Asn His 856 GCA AAC AAA GAC AAA TGG GGA AAT ATT GTG GTT ATA TAT AAT CCC 3182 Ala Asn Lys Asp Lys Trp Gly Asn Ile Val Val Ile Tyr Asn Pro 871 AAT AAA ACA GCA GAA ACG GTG AAT TTA CCG AGT GGA AAA TGG GCC 3227 Asn Lys Thr Ala Glu Thr Val Asn Leu Pro Ser Gly Lys Trp Ala 886 ATT AAT GCT ACA AAT GGA AAA ATT GGA GAA TCC ACT CTA AGT CAT 3272 Ile Asn Ala Thr Asn Gly Lys Ile Gly Glu Ser Thr Leu Ser His 901 GCA GAG GGG CAC GTT CAA GTC CCA GGC ATT TCT ATG ATG ATT CTT 3317 Ala Glu Gly His Val Gln Val Pro Gly Ile Ser Met Met Ile Leu 916 CAT CAA GAA ACG AAT AAA TGAAT AAAAAGTAAA AGAGACTTAA TC 3370 His Gln Glu Thr Asn Lys 922 ACCTCTTATG GAATAATCCA TAAGAGGTTT TTGGGGTGAC TTTAGTTATA AAAAAACGTA 3430 GAGTTTTTCT GTGTAATTCT CTATTCAGTA GAAGGGGTGG CAAAAAGTTC TGTTAGAAGA 3490 AGGGAGACTG AACCTATGAC GGTGGCCTTC TCACCTAATT CTGAAATCAG AATCTGAGTT 3550 TGTTTGGCTT TTTCCGTTAA GACATGTTGG GCAATAGTCG CTTTTAAGCT ATTCATAATA 3610 TAGGGACCAG ATTTAGCGAC GCCTCCTCCA ATAATGATCC GCTCTGGATT CAACGTGTGA 3670 ATGAGGTTCG TGATTCCAAT ACCAAGATAA ATTCCCGTTT GACTTAAAAC CTTACATGCA 3730 AGCGGATCTC CCAGTTTAGC CGCTTCATAA ACGATTTCAC CTGAAATTTG CGACAGATCC 3790 CCATGAACGA GTTCTTTTAT TAAGCTCTCT GTGCCTGACT CCAATTCTTT AATCGCTTTA 3850 CTAGCAATTG AGGGTCCAGA CGCAAGAGCT TGGAGGCACC CATGATTCCC ACAACTACAC 3910 CTAGGTCCAT CGATATCAAT GATC 3934

SEQ ID NO: 3 Sequence length: 21 Sequence type: Amino acid sequence Asp Gly Thr X X Asn Val Ile Val His Tyr Phe Arg Pro Gly 15 Gly Asp Tyr Gln X Trp 21 (X is unidentified)

SEQ ID NO: 4 Sequence length: 29 Sequence type: Amino acid sequence ATG ATG AAA AAA GTA ATT TAC GTG CTT TTA AGT TTA TGT TTA GTG 45 Met Met Lys Lys Val Ile Tyr Val Leu Leu Ser Leu Cys Leu Val 15 TTG TCA TGG GCT TTT AAT TTC AAA GGC CAG TCT GCT CAT GCG 87 Leu Ser Trp Ala Phe Asn Phe Lys Gly Gln Ser Ala His Ala 29

SEQ ID NO: 5 Sequence length: 10 Sequence type: Amino acid sequence Asp Gly Thr Thr Asn Val Ile Val His 10

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH of a debranching enzyme obtained in the present invention.

FIG. 2 is a diagram showing pH stability of the debranching enzyme obtained in the present invention.

FIG. 3 is a diagram showing the optimum temperature of the debranching enzyme obtained in the present invention.

FIG. 4 is a diagram showing the temperature stability of the debranching enzyme obtained in the present invention.

FIG. 5 is a diagram showing a restriction enzyme map of a DNA fragment containing the debranching enzyme gene of the present invention.

FIG. 6: Plasmid pU containing the debranching enzyme gene of the present invention
It is a figure showing BC24.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:07) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:07) (C12N 9 / 44 C12R 1:07) (C12N 9/44 C12R 1:19)

Claims (9)

[Claims] 1. A DNA fragment containing a debranching enzyme gene and giving the restriction map shown in FIG. 2. A DNA fragment encoding a debranching enzyme containing the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing or a part thereof and an amino acid sequence homologous thereto. 3. A DNA fragment containing the nucleotide sequence set forth in SEQ ID NO: 2 of the Sequence Listing or a modification enzyme derived therefrom. 4. The DNA fragment is Bacillus sp. APC-96.
D containing a nucleotide sequence encoding a debranching enzyme produced by strain 03 (FREM BP-4204) or a modification enzyme derived therefrom
NA fragment. 5. A vector comprising the DNA fragment according to any one of claims 1 to 4. 6. The vector of claim 5 is Bacillus sp.
DNA encoding the promoter region and signal peptide region of the cellulase gene of N-2 (FERM P-8809)
A vector comprising a DNA fragment obtained by ligating a DNA fragment encoding a mature debranching enzyme downstream of the fragment. 7. A recombinant microorganism transformed with the DNA according to claim 5 or 6. 8. The transformed recombinant microorganism according to claim 7, wherein the microorganism is Bacillus subtilis. 9. A method for producing a debranching enzyme, which comprises culturing the microorganism according to claim 7 or 8 and obtaining a debranching enzyme from the culture.