JP2000262292A - Gene coding for protopectinase, transformant containing the gene, and refinement of fiber using the transformant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene coding for a protein which has a specific amino acid sequence, has a protopectinase activity, releases pectin from a plant tissue, and is useful for producing the enzyme which allows simple and inexpensive refinement of a large amount of fiber. SOLUTION: This is a new gene coding for a protein which has the specific amino acid sequence shown by the formula or an amino acid sequence obtained by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into the amino acid sequence, has a protopectinase activity, has an activity of releasing pectin from a plant tissue, and allows simple and inexpensive refinement of a large amount of fiber using the gene and transformants. This gene is obtained by completely digesting genomic DNA in Bacillus subtilis strain BS with restriction enzym(s), electrophoresing, linking a DNA fragment to a plasmid, transforming Escherichia coli, screening by the colony hybridization method using its partial sequence as a probe, followed by collecting DNA from positive strains.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel gene encoding a protopectinase, and more particularly, to a recombinant vector containing the gene, a transformant, a method for producing the transformant, and the transformant. And a fiber treated by the method.

[0002]

2. Description of the Related Art Cellulose fibers represented by cotton fibers are:
It is composed of a secondary film made of cellulose and a primary film covering this film via a winding layer. The primary membrane is
It consists of pectin, wax and protein, with pectin being the main component. In order to improve the water absorption, dyeability, decolorization and feel of the fiber, it is necessary to remove the primary film, and this treatment is generally called scouring.
Conventionally, for scouring, a method of treating fibers at a high temperature using a mixed solution of sodium hydroxide and a scouring aid has been adopted. However, this method has a problem that it requires a lot of energy and requires a great deal of cost to treat the strongly alkaline waste liquid. To solve this problem, a scouring method using protopectinase has been proposed (JP-A-6-220772).

[0003] Protopectinase is a general term for enzymes that release pectin from plant tissues, and is an A type that releases pectin from plant tissues and limits the decomposition of free pectin (fermentation and industry: 37, 928-938, 1978). B type (Agric. Biol. Chem., 52,109) which releases pectin from plant tissue but has no activity of degrading free pectin.
1-1093, 1988; Agric. Biol. Chem., 53, 1213-1223, 198
9; Agric. Biol. Chem., 54, 879-889, 1990; Eur. J. Bioche.
m., 226, 285-291, 1994; Biosci. Biotech. Biochem., 5
8, 353-358, 1994). Particularly with regard to type B, the present inventors have found a protopectinase that acts on arabinan present between pectin and cellulose and releases pectin from plant tissues, and has shown the use of this enzyme in scouring (PCT / JP / 02794 and PCT / JP / 02795
issue).

[0004]

However, in scouring, the required properties vary depending on the type or use of the fiber, so that not only those acting on arabinan but also pectin from the fiber by another mechanism of action. There is a need to make available a protopectinase that can be released. Protopectinase is used to produce pectin, which is used as a raw material for foods, pharmaceuticals and cosmetics
No. 8322), it is desired to develop a method that can produce and use such enzymes at low cost because they are useful in various fields including treatment of plant tissues.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies on a protein derived from a bacterium belonging to the genus Bacillus obtained by chromatography under various salt concentration conditions. And found a novel gene encoding a protopectinase that releases this from the fiber, and production of a transformant containing the gene;
In addition, a method for scouring fibers using the transformant was established, and the present invention was completed. Therefore, according to the present invention, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in this sequence and having protopectinase activity Are provided. Also provided are a recombinant vector containing the gene, a transformant, a method for producing the transformant, a method for scouring a fiber using the transformant, and a fiber treated by the method.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the gene encoding protopectinase is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid in which one or several amino acids have been deleted, substituted or added. It is a gene consisting of a sequence and encoding a protein having protopectinase activity. Amino acids deleted, substituted or added can be obtained by subjecting a nucleotide sequence encoding the amino acid to a method well known to those skilled in the art such as site-directed mutagenesis or point mutation. The number, type and position of amino acids
The protein produced is protopectinase (hereinafter referred to as PP
It is not particularly limited as long as it has an activity.

[0007] Here, the PPase activity refers to an action of releasing pectin from plant tissues, that is, an action of releasing water-soluble pectin by limited hydrolysis of water-insoluble protopectin in plant tissues. More specifically, a protein having PPase activity can be measured using protopectin derived from lemon peel as a substrate and has the following enzymatic properties: 1) has protopectinase activity; 2) SDS-
The molecular weight on a polyacrylamide gel electrophoresis gel is about
43,000; 3) optimum pH is 8.0; 4) optimum temperature is 60
° C; and 5) inhibited by Cu, Hg, Mn and Zn. Such a protein will be referred to hereinafter as PPase-BS.

[0008] The gene of the present invention may be added with any signal peptide for the purpose of secreting the finally produced protein, ie, PPase-BS, out of the host cell. In this case, the gene to which the signal peptide has been added is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in this sequence, and has a protopectinase An active protein can be encoded.

The gene of the present invention can have the nucleotide sequence represented by nucleotide numbers 358 to 1557 of the sequence shown in SEQ ID NO: 3, and when a signal peptide is added, the sequence of SEQ ID NO: 3 Has a base sequence represented by base numbers 295 to 1557.

[0010] These genes are primers designed based on a partial amino acid sequence of a protein having PPase activity when protopectin derived from lemon peel is used as a substrate, obtained by subjecting a culture of a Bacillus bacterium to chromatography. Was isolated and determined by using the polymerase chain reaction (PCR) method. Therefore, the gene of the present invention is a bacterium belonging to the genus Bacillus or a mutant thereof, preferably Bacillus subtilis (Bacillus subtilis) BS
From the strain (Accession No. FERM BP-6031, Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry)
Can be obtained by amplification. Bacillus subtilis
The BS strain is a gram-positive bacillus, which has a spore-forming ability, a low protease-producing ability, and a feature that production of protopectinase is suppressed by glucose. This microorganism is located at 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture (postal code 3
05-0046) and the accession number FERM BP described above as of July 30, 1997
Deposited under -6031.

[0011] The gene of the present invention obtained as described above can be used to produce a recombinant vector by introducing it into a vector using genetic engineering techniques. Any type of vector can be used as long as it is a vector suitable for expressing a gene in a desired host. In general, a multicopy vector having a strong promoter is preferably used. For example, pBlue
Script II SK + (Stratagene) or pUB110 (TJG
ryczan, S. Contente and D. Dubuan: J. Bacteriol., 34,
318-329 (1978)).

The recombinant vector containing the gene of the present invention can be transformed into any host by a conventional method such as a protoplast method, a calcium chloride method, an electroporation method or a particle gun to produce a transformant. can do. The host may be a microorganism, a plant or an animal, but is preferably a microorganism. In addition to the genus Bacillus, the host may be Escherichia coli.
li) Gram-negative bacteria such as Saccharomyces (Sac
charomyces yeast, Aspergillus
Fungi of s) and the like can be used. Of these, the use of Bacillus subtilis or Escherichia coli is particularly preferred. When the vector is pBlueScriptII SK +, Escherichia coli can be used, and pUB110 can be Bacillus subtilis. The selection of the transformant is not particularly limited, but it can be easily performed depending on the properties of the vector such as antibiotic resistance.

The transformant containing the gene of the present invention comprises:
Under aerobic or anaerobic conditions depending on the host used, PPase-BS is inoculated into an appropriate medium, either liquid medium or solid medium, and subjected to aeration stirring or shaking culture, etc.
Can be produced. The medium components are not particularly limited, but it is preferable to use a medium containing a carbon source, a nitrogen source, and other essential nutrients that can be assimilated by the host under appropriate conditions. Examples of the carbon source include galactose, glucose, sucrose, and acetic acid. Examples of the nitrogen source include inorganic ammonium salts and nitrates, yeast extract, and soybean powder. The pH of the medium can be adjusted to any pH using sodium carbonate or potassium carbonate. Culture conditions are determined depending on the host and the like, but it is generally preferable to culture at 37 ° C. for about 16 to 48 hours.

The culture of the transformant obtained as described above may be subjected to a reaction after immersing unsculpted cellulose fibers or cloth such as cotton or hemp in a heated buffer, keeping the temperature warm, and then reacting. Thereby, the fiber can be scoured under mild conditions. The culture is a culture supernatant or a culture purified product, that is,
Any form of PPase-BS itself may be used.For example, the culture supernatant may be obtained by removing host cells from the culture solution by centrifugation or filtration, or by recovering the host cells from the culture solution, followed by enzyme treatment or ultrafiltration. It can be obtained by subjecting to sonication and then centrifuging. In addition, PPase-BS is produced from these solutions by ordinary means, for example, concentration by drying or ultrafiltration, precipitation by salting out or solvent precipitation, dialysis, gel filtration chromatography, or ion exchange chromatography. It can be obtained by combining protein purification means. The treatment conditions are appropriately determined depending on the form of the culture, the type of the fiber to be treated, the properties or the use of the fiber desired after the treatment, for example, when using a culture supernatant for the culture, the range of 50 to 60 ° C. The culture supernatant can be added to the heated buffer so that the concentration of PPase-BS is about 25 units per 1 g of fiber, and the mixture can be treated for several hours. When the fibers are pasted, the fibers may be preliminarily treated with a desizing agent, or the desizing agent may be put into a treatment liquid and processed simultaneously.

[0015] According to a quantitative test of free pectin eluted in the treatment solution, it has been confirmed that a large amount of pectin is released from the fiber scoured using the transformant of the present invention or a culture thereof. In addition, these fibers have no difference in strength and the like from those treated by a usual method, but rather can be obtained as fibers having good dyeability or texture.

Further, by using the gene or the transformant of the present invention, an organism which does not originally produce PPase-BS or produces only a small amount of the enzyme can be used as a host.
High-purity PPase-BS can be produced in large quantities and easily, or its amount can be increased.

[0017]

The present invention will be described below in detail with reference to examples. Note that the present invention is not limited by these examples. Example 1 Purification of Enzyme Bacillus subtilis BS strain was transformed into SP medium (1.5% soybean powder (Fuji Oil), 1.2% KH ₂ PO ₄ , 2.8% K ₂ HPO ₄ , pH 7.0)
And cultured with shaking at 37 ° C. for 22 hours. The culture was centrifuged to obtain a supernatant. The supernatant was concentrated by a rotary evaporator, dialyzed overnight at 4 ° C. against 20 mM acetate buffer (pH 6.0) containing 50% saturated ammonium sulfate, and Butyl-Toyopearl 650M (manufactured by Tosoh) equilibrated with the same buffer. ) Applied to the column. After washing well with the same buffer, 50-30%
The fraction having PPase activity was collected by elution with a continuous linear gradient of saturated ammonium sulfate.

The PPase activity was determined by the method of Sakai et al. (Method) using protopectin derived from lemon peel as a substrate as follows.
s in Enzymology, 161, 335-350, 1988, WA Wood and T. Kellogg, eds., Academic Press); 990 μl of acetate buffer containing 100 μg of substrate (100
mM, pH 6.0), add 10 μl of the enzyme solution, incubate at 37 ° C for 1 hour, and filter the reaction solution: filtrate 125
μl, 125 μl of water, 250 μl of ethanol containing 0.2% carbazole, and 3 ml of 31.5 N sulfuric acid.
Incubate at 75 ° C for 20 minutes. After cooling the reaction solution on ice, the absorbance at 525 nm is measured. The PPase activity was calculated and displayed from the relational expression between the amount of galacturonic acid of 1 μmol per minute and the degree of color development.

The fraction having PPase activity collected as above was dialyzed overnight at 4 ° C. against 20 mM acetate buffer (pH 6.0), and equilibrated with 20 mM acetate buffer (pH 6.0). CM-To
It was applied to a yopearl 650M (Tosoh) column. After thorough washing with the same buffer, elution was carried out with a continuous linear gradient of 0-0.5 M salt, and fractions having the same activity were further collected and collected. Next, this fraction was dialyzed overnight at 4 ° C. against 20 mM acetate buffer (pH 6.0) containing 50% saturated ammonium sulfate, and Butyl-Toyopearl 650M equilibrated with the same buffer.
(Tosoh) column. After thorough washing with the same buffer, elution was carried out with a continuous linear gradient of ammonium sulfate 50-30% saturation, and fractions having enzyme activity were collected by fractionation. This fraction was applied to a Superdex 75 (Pharmacia Biotech) column equilibrated with 20 mM acetate buffer (pH 6.0) containing 100 mM NaCl. 20 containing 100 mM NaCl
After elution with a mM acetate buffer (pH 6.0), fractions having PPase activity were collected and collected. Since the obtained enzyme solution showed a single band on SDS-polyacrylamide gel electrophoresis,
This was used as a purified enzyme sample in the following operation.
Table 1 shows the results of the purification.

[Table 1]

Example 2 Characterization of Enzyme 2.1 Molecular Weight The molecular weight of PPase-BS was about 43,000 as estimated from the mobility on a 10% SDS-PAGE gel. Also, this enzyme
The molecular weight was estimated to be about 32,000 by using a Superdex 75 gel filtration column equilibrated with 20 mM acetate buffer (pH 6.0) containing 100 mM NaCl. 2.2 pH and temperature stability After PPase-BS was incubated at 37 ° C. for 16 hours at various pHs, the enzyme activity was measured by the method described in Example 1. As a result, 80% or more remained at pH 3 to 10. Also PPase-BS
Was incubated in 20 mM acetate buffer (pH 6.0) at various temperatures for 30 minutes, and the enzyme activity was measured. As a result, 80% or more remained at a temperature of 60 ° C. or lower. 2.3 Optimum pH and temperature The PPase activity was measured by the method described in Example 1 except that the reaction pH or the reaction temperature was changed.
It showed the highest activity at 60 ° C. 2.4 Influence of various ions PPase activity was measured at pH 8.0 and 50 ° C in a reaction solution containing 1 mM of various substances shown in Table 2 below. The activity was CuSO ₄ , HgCl ₂ , MnCl ₂ And almost completely inhibited in the presence of ZnCl ₂ .

[Table 2]

Example 3 Determination of Partial Amino Acid Sequence 3.1 Determination of N-Terminal Amino Acid Sequence 22.5 μg of purified PPase-BS was reduced with 5% mercaptoethanol and then separated on 10% PAGE. Separated proteins in blotting solution (10% methanol / 1
0mM CAPS / NaOH (pH11)) with PVDF membrane (Immobilon Ps
q) The sample was electrically (100 V, 1.5 hr) blotted. this
After washing the PVDF membrane with distilled water, 0.1% Coomassie Blue R-
Stained with 250/50% methanol. Decolorization was performed with 50% methanol, the stained band was cut out, washed with distilled water, and the N-terminal amino acid sequence was identified by Applied Biosy.
This was performed using stems model 492. Of the identified PPase-BS
The result of the N-terminal amino acid sequence is shown in SEQ ID NO: 5. 3.2 Identification of partial amino acid sequence 22.5 μg of purified PPase-BS 70% containing 1% bromocyan
100 μl of formic acid was added to dissolve and react. After the reaction,
The residue was dried under a stream of nitrogen, redissolved in about 100 μl of 70% formic acid, and then dried. The resulting peptide fragments were fractionated by reverse phase HPLC, and the partial amino acid sequence was identified. The result is shown in SEQ ID NO: 6.

Example 4 : Amplification of gene fragment by PCR method 4.1 Preparation of genomic DNA The genomic DNA of Bacillus subtilis BS was obtained by using Molecular
Prepared using CTAB according to Cloning 3rd Edition (1993). 4.2 Preparation of primers In order to amplify a partial fragment of the PPase-BS gene from genomic DNA by the PCR method, two types of degenerat
e oligonucleotide primer was synthesized with an automatic DNA synthesizer. Primer N-1 (SEQ ID NO: 7) was designed as a base sequence corresponding to the partial sequence of amino acids 1 to 7 of the sequence shown in SEQ ID NO: 5. Primer N-2 (SEQ ID NO: 8) was designed as a complementary strand of the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 6.

4.3 PCR Using the LA-PCR kit (Takara Shuzo), PCR was performed from genomic DNA of Bacillus subtilis BS under the following reaction conditions. Reaction solution: 10x buffer 5.0 μl dNTP mix 8.0 μl Primer N-1 2.5 μl (final concentration 10 μM) Primer N-2 2.5 μl (final concentration 10 μM) LA Taq polymerase 0.5 μl Genomic DNA 1.5 μl (500 ng) H ₂ O 30 μl total 50 μl Amplification conditions: 94 ° C., 1 minute / 1 cycle 94 ° C., 1 minute; 50 ° C., 1 minute; 72 ° C., 2 minutes / 30 cycles After completion of the reaction, the reaction solution was subjected to agarose gel electrophoresis.
The amplified DNA fragment of about 380 bp was recovered by a glass adsorption method. This fragment was digested with T4 ligase in the usual way using EcoRV of plasmid pBlueScript II SK + (Stratagene).
After binding to the cleavage site, Escherichia coli JM109 was transformed. The resulting transformant was cultured in an LB medium, and the recovered cells were subjected to an alkaline SDS method [Birnboim (Birn
boim) et al., Nucleic Acids Res., 7, 1513. 1979], extracted the plasmid, deproteinized by RNaseA treatment, PEG (polyethylene glycol) precipitation and phenol extraction, and prepared plasmid DNA by ethanol precipitation. . Determination of the nucleotide sequence of the thus prepared plasmid DNA
This was performed using an LF DNA sequencer (Pharmacia). The nucleotide sequence of the obtained DNA fragment is shown in SEQ ID NO: 4.

Example 5 : Cloning of PPase-BS gene 5.1 Southern blotting After completely digesting 250 ng of Bacillus subtilis BS genomic DNA with the restriction enzymes EcoRI or HindIII, 1%
Agarose gel electrophoresis was performed. Denature gel after electrophoresis
(1.5 M NaCl / 0.5 M NaOH, 45 minutes), neutralization (1.5 M NaCl /
1M Tris-HCl (pH 8.0), 30 minutes), and the separated DNA was transferred to a nylon membrane (HybondN +, manufactured by Amersham) and fixed by ultraviolet irradiation. The plasmid DNA containing the PCR fragment obtained in Example 4 was digested with restriction enzymes EcoRI and HindIII, electrophoresed on a 1% agarose gel, and a DNA fragment of about 380 kb recovered by a glass adsorption method was used to obtain a full-length PPase-BS gene. It was used as a probe DNA for cloning.
Probe DNA is available from Fluoresein Gene Images labeling
Fluorescent labeling was performed using it (manufactured by Amersham). Hybridization, washing and signal detection were performed using Fluo
Using a resein Gene Images detection kit (manufactured by Amersham), the procedure was performed according to the attached protocol. As a result, a band of 1.5 kb was detected by EcoRI and a band of about 1.3 kb by HindIII. Gene cloning was performed using these bands.

5.2 Cloning of Gene After completely digesting 10 μg of Bacillus subtilis BS genomic DNA with the restriction enzymes EcoRI or HindIII, 1%
Agarose gel electrophoresis, around 1.5 kb and 1.3 k
The fractions near b were collected by the glass adsorption method.
These DNA fragments were separately converted into plasmid pBlueScript I
EcoRI or HindIII from ISK + (Stratagene)
After binding to the cleavage site, Escherichia coli JM109 was transformed. The DNA of the obtained transformant was immobilized on a nylon membrane, and a positive clone was screened by a colony hybridization method using the PCR fragment of about 380 kb obtained in Example 4 as a probe. As a result, E
CoRI pPPBSE1 having an insert of about 1.5 kb from the eluted DNA around 1.5 kb, and eluted DNA around 1.3 kb HindIII
Gave pPPBSH1 having an insert of about 1.3 kb. 5.3 Analysis of clones The DNA base sequences of the obtained two fragments (inserted fragments of pPPBSE1 and pPPBSH1) were determined and compared with the sequence of the PCR fragment obtained in Example 4. As a result, it was confirmed that pPPBSE1 pP
PBSH1 encodes 3 'end, 395bp common EcoRI-HindI
It was found to have the II fragment. This nucleotide sequence is shown in SEQ ID NO: 3.

Example 6 : Identification of amino acid sequence The nucleotide sequence (SEQ ID NO: 3) analyzed in Example 5 contains an open reading frame consisting of 1263 bases from base number 295 to base number 1557, and this open reading frame Was predicted to encode a protein consisting of 420 amino acids. In this amino acid sequence, the N-terminal amino acid sequence of PPase-BS shown in SEQ ID NO: 5 and the partial amino acid sequence of the Bromcian-cleaved peptide fragment shown in SEQ ID NO: 6 were found, respectively. The protein was determined to be PPase-BS. This amino acid sequence is shown in SEQ ID NO: 2.
The first to 21 amino acid residues of SEQ ID NO: 2 are considered to be secretory signal sequences because a charged amino acid Lys in the N-terminal region and a cluster of hydrophobic amino acids in the center are recognized. That is, PPas
Since the sequence of the N-terminal 20 amino acid residues of e-BS was identified as shown in SEQ ID NO: 5, PPase-BS was first synthesized after being biosynthesized as a protein having the sequence of SEQ ID NO: 2. As a mature protein having the amino acid sequence shown in SEQ ID NO: 1 (corresponding to bases 358 to 1557 in SEQ ID NO: 3) which is cleaved between the 21st amino acid Ala and the 22nd amino acid Ala from the amino acid residue It is thought that it is secreted outside the cells.

Example 7 : Production of a transformant using Escherichia coli as a host The PPase-BS gene is composed of an EcoRI 1.5 kb fragment and HindIII 1.3
Since the clone was divided into kb fragments and cloned, both fragments were ligated to construct a complete PPase-BS gene. That is,
A PPase-BS expression vector pPPBS-1 was prepared by inserting the EcoRI 1.1 kb fragment of pPPBSE1 into the EcoRI cleavage site of pPPBSH1, and the pPPBS-
Thus, Escherichia coli DH1 retaining 1 was obtained. The obtained transformant was cultured overnight at 37 ° C. in an L-broth medium (containing 50 μg / ml ampicillin), and the production of PPase-BS was examined. The culture solution is separated into a culture supernatant and a precipitate by centrifugation, and the precipitate is suspended in a buffer solution and then centrifuged to remove the supernatant (periplasm fraction) and the precipitate by ultrasonication. After the treatment, the supernatant was separated by centrifugation (intracellular fraction). When the pectate lyase activity of each of these fractions was measured, all the fractions showed activity, but the strongest activity was detected in the periplasm fraction (Table 3). Therefore, it is considered that the recombinant PPase-BS passes through the intracellular membrane and is secreted and produced.

[Table 3]

Example 8 : Preparation of a transformant using Bacillus subtilis as a host The plasmid pUB110 (described above) was used for expression of PPase-BS in Bacillus subtilis. An XbaI cleavage site was added to the PPase-BS gene by PCR to insert the PPase-BS gene into the XbaI cleavage site of pUB110.
That is, primer N-3 (SEQ ID NO: 9) corresponding to nucleotides 155 to 174 of SEQ ID NO: 3 and 1654 of SEQ ID NO: 3
Primer N-4 (SEQ ID NO: 10) corresponding to the 鎖 1666th complementary strand was synthesized by an automatic DNA synthesizer. Using these,
Using pPPBS-1 prepared in Example 7 as a template, PCR was performed under the same conditions as in Example 4.3. The amplified DNA fragment of about 1.5 kb was completely digested with the restriction enzyme XbaI, subjected to 1% agarose gel electrophoresis, and recovered by a glass adsorption method. The amplified DNA fragment was ligated to the XbaI cleavage site of plasmid pUC18 (Amersham Pharmacia Biotech), and Escherichia coli JM109 was transformed. The plasmid of the obtained transformant was extracted, its nucleotide sequence was determined, and it was confirmed that there was no mutation in the PPase-BS gene. This plasmid was designated as pUC18-BS. A 1.5 kb PPase-BS gene fragment obtained by digesting pUC18-BS with XbaI
110 XbaI cleavage site, and transformed into Bacillus subtilis MI112 by the protoplast method (5 μg / ml).
PUB110-BS).

Bacillus subtilis carrying pUB110-BS was cultured overnight at 37 ° C. in the above L-broth, and the enzyme activity was measured in the same manner as in Example 7. As a result, the culture supernatant showed about 5 to 8 times the PPase activity as compared to that obtained from Bacillus subtilis holding pUB110 alone, indicating that the PPase-BS gene was expressed in Bacillus subtilis. Turned out to be.

[Table 4]

Example 9 : Scouring of cotton cloth using a transformant using Bacillus subtilis as a host The scouring of cotton fibers was evaluated according to the following method. 0.1% Woomin TE (Tokai Oil Industry Co., Ltd.)
An unscrambled cotton cloth (about 5 mm in diameter) after desizing 40 mg was immersed in 1 ml of 100 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM calcium chloride at 50 ° C. Then, 10 μl of a 100-fold dilution of the culture supernatant prepared in Example 8 was added, and the mixture was reacted at the same temperature for 2 hours. The reaction solution was filtered through filter paper, and the amount of pectin eluted in the obtained filtrate was determined. Was measured by the carbazole sulfate method described in Example 1. When using the culture supernatant of Bacillus subtilis carrying pUB110-BS, 9
6.5 μg of pectin eluted. PUB110 compared to this
2 in the culture supernatant of Bacillus subtilis
Since only 2.1 μg of pectin was eluted, it was revealed that PPase-BS expressed and produced by Bacillus subtilis solubilized pectin on cotton cloth and could be used for scouring.

[0031]

According to the present invention, there are provided a gene encoding protopectinase, a recombinant vector containing the gene, a transformant, and the like. By using these genes and transformants, a large amount of fibers can be scoured easily and inexpensively. In addition, the genes can be applied in various fields including treatment of plant tissues.

[0032]

[Sequence List] SEQUENCE LISTING <110> Takuo Sakai <120> Gene coding for protopectinase, transformant including the gene, and method for purifying fibers using the transformant. <130> PTS-8839 <160> 10 <210> 1 <211> 399 <212> PRT <213> Bacillus subtilis <400> 1 Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn Asp Gly Trp Gly Ala 1 5 10 15 Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala Ser Ser Ser His Val 20 25 30 Tyr Thr Val Ser Asn Arg Asn Gln Leu Val Ser Ala Leu Gly Lys Asp 35 40 45 Thr Asn Thr Thr Pro Lys Ile Ile Tyr Ile Lys Gly Thr Ile Asp Met 50 55 60 Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu Asn Asp Tyr Lys Asp 65 70 75 80 Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala Tyr Asp Pro Ser Thr 85 90 95 Trp Gly Lys Lys Glu Pro Ser Gly Thr Leu Glu Glu Ala Arg Ala Arg 100 105 110 Ser Gln Lys Asn Gln Lys Ala Arg Val Met Val Asp Ile Pro Ala Asn 115 120 125 Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys Ile Val Gly Gly Asn 130 135 140 Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg Asn Ile Glu Phe Gln 145 150 155 160 Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro Thr Asp Gly Ser Ser 165 170 175 Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr Ile Asn Gly Gly Thr 180 185 190 His Ile Trp Ile Asp His Cys Thr Phe Asn Asp Gly Ser Arg Pro Asp 195 200 205 Ser Thr Ser Pro Lys Tyr Phe Gly Arg Lys Tyr Gln His His Asp Gly 210 215 220 Gln Thr Asp Ala Ser Asn Gly Ala Asn Tyr Ile Thr Met Ser Tyr Asn 225 230 235 240 Tyr Tyr His Asp His Asp Lys Ser Ser Ile Phe Gly Ser Ser Asp Ser 245 250 255 Lys Thr Ser Asp Asp Gly Lys Leu Lys Ile Thr Leu His His Asn Arg 260 265 270 Tyr Lys Asn Ile Val Gln Arg Ala Pro Arg Val Arg Phe Gly Gln Val 275 280 285 His Val Tyr Asn Asn Tyr Tyr Glu Gly Ser Thr Ser Ser Ser Asp Tyr 290 295 300 Ala Phe Ser Tyr Ala Trp Gly Ile Gly Lys Ser Ser Lys Ile Tyr Ala 305 310 315 320 Gln Asn Asn Val Ile Asp Val Pro Gly Leu Ser Ala Ala Lys Thr Ile 325 330 335 Ser Val Phe Ser Gly Gly Thr Ala Leu Tyr Asp Ser Gly Thr Leu Leu 340 345 350 Asn Gly Thr Gln Ile Asn Ala Ser AlaAla Asn Gly Leu Ser Ser Ser 355 360 365 Val Gly Trp Thr Pro Ser Leu His Gly Thr Ile Asp Ala Ser Ala His 370 375 380 Val Lys Ser Asn Val Ile Ser Gln Ala Gly Ala Gly Lys Leu Asn 385 390 395 <210> 2 <211> 420 <212> PRT <213> Bacillus subtilis <400> 2 Met Lys Lys Val Met Leu Ala Thr Ala Leu Leu Leu Gly Leu Thr Pro -20 -15 -10 Ala Gly Pro Asn Ala Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn -5 -1 1 5 10 Asp Gly Trp Gly Ala Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala 15 20 25 Ser Ser Ser His Val Tyr Thr Val Ser Asn Arg Asn Gln Leu Val Ser 30 35 40 Ala Leu Gly Lys Asp Thr Asn Thr Thr Pro Lys Ile Ile Tyr Ile Lys 45 50 55 Gly Thr Ile Asp Met Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu 60 65 70 75 Asn Asp Tyr Lys Asp Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala 80 85 90 Tyr Asp Pro Ser Thr Trp Gly Lys Lys Glu Pro Ser Gly Thr Leu Glu 95 100 105 Glu Ala Arg Ala Arg Ser Gln Lys Asn Gln Lys Ala Arg Val Met Val 110 115 120 Asp Ile Pro Ala Asn Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys 125 130 135 Ile Val Gly Gly Asn Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg 140 145 150 155 Asn Ile Glu Phe Gln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro 160 165 170 Thr Asp Gly Ser Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr 175 180 185 Ile Asn Gly Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp 190 195 200 Gly Ser Arg Pro Asp Ser Thr Ser Pro Lys Tyr Phe Gly Arg Lys Tyr 205 210 215 Gln His His Asp Gly Gln Thr Asp Ala Ser Asn Gly Ala Asn Tyr Ile 220 225 230 235 Thr Met Ser Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser Ile Phe 240 245 250 Gly Ser Ser Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys Ile Thr 255 260 265 Leu His His Asn Arg Tyr Lys Asn Ile Val Gln Arg Ala Pro Arg Val 270 275 280 Arg Phe Gly Gln Val His Val Tyr Asn Asn Tyr Tyr Glu Gly Ser Thr 285 290 295 295 Ser Ser Ser Asp Tyr Ala Phe Ser Tyr Ala Trp Gly Ile Gly Lys Ser 300 305 310 315 Ser Lys Ile Tyr Ala Gln Asn Asn Val Ile Asp Val Pro Gly Leu Ser 320 325 330 Ala Ala Lys Thr Ile Ser Val Phe Ser Gly Gly Thr Ala Leu Tyr Asp 350 340 345 Ser Gly Thr Leu Leu Asn Gly Thr Gln Ile Asn Ala Ser Ala Ala Asn 350 355 360 Gly Leu Ser Ser Ser Val Gly Trp Thr Pro Ser Leu His Gly Thr Ile 365 370 375 Asp Ala Ser Ala His Val Lys Ser Asn Val Ile Ser Gln Ala Gly Ala 380 385 390 395 Gly Lys Leu Asn <210> 3 <211> 1729 <212> DNA <213> Bacillus subtilis <400> 3 tttttctgag gatgaagccg ctcaattcga aaaacgttta gatgaaggga aagtg gatga tg tca tg ga tga tg tga tg tga tg tga tg tg ggtgtatgtg ttgcagaatg 180 ccgcaataag atagcggaac attttcggtt ctggatgtcc ctcaatttgc tatcatatct 240 ttgtgagaaa ttggaatata atctcacaaa atagaaaatg ggggttcata gtga atg 297 aaa aaa gtg atg tta gcc acg gct ctg ctt tta ggg ttg act cca gct 345 ggg ccg aac gcg gcg gat tta ggc cat caa aca tta gga tca aat gat 393 ggc tgg ggc gcg tac tcg acc ggc aca aca ggc gga tca aaa gct tcg 441 tca tcc cac gtg tat acc gtc agc aac aga aac cag ctt gtc tcg gca 489 tta ggc aag gac acc aca ata atc atc aag gga 537 acg att gac atg aac gtc gat gac aat ctg aag ccg ctt ggt cta aat 585 gat tat aaa gat cca gag tac gat ttg gac aaa tat ttg aaa gcc tat 633 gac cct agc aca tgg ggc aaa aag gag ccg cc gg gc g cg gc g cg gc g cg gc g cg gc g cg gc g gc aga gca cga tct cag aaa aat caa aaa gca cga gtc atg gtg gat 729 att ccg gca aac acg acg atc gtc ggt tca ggg aca aat gcc aaa atc 777 gtg ggc gga aat ttc cag atc aag ac gat atc atc atc atc atc atc atc atc atc gtc atc gaa ttc cag gat gct tat gat tat ttt ccg caa tgg gat ccg act 873 gac ggc agc tca gga aac tgg aac tca caa tac gac aac atc aca ata 921 aac ggc ggc acg cat ata tgg att gat cat tgt agt 969 tcc cgt ccg gac agc aca tcg cca aag tat ttc ggc aga aaa tat cag 1017 cac cat gac ggc caa acc gat gct tct aac ggc gct aac tat atc acg 1065 atg tct tac aac tat tat cac gat cat gat tat g gga 1113 tca agc gac agc aaa aca tct gat gac ggc aaa tta aaa atc acg ctc 1161 cat cat aac cgc tat aaa aat atc gtc cag cgc gca ccg aga gtc cgc 1209 ttc ggg cag gtg cac gtt tat ac c agc aca agc 1257 tcc tcg gat tat gcc ttc agc tat gcg tgg gga atc gga aaa tca tct 1305 aaa atc tac gct caa aac aat gtc att gac gtg cct gga ctg tca gcc 1353 gct aaa gag gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc ag gc gc gc gc gc gc gc gc gc gc gc gc gc gc gc ag gct tta tat gac tca 1401 ggc aca ttg ctg aat ggc acg cag atc aac gca tcg gct gca aac ggg 1449 ctg agt tct tct gtc ggc tgg aca ccg tct ctg cac ggc aca atc gat 1497 gct tcc gc gc tc gc tc gc tc gc tc gc tc gc tc gc tc gc gc tca gc gc tc gc gc gca tct caa gcg ggt gcg ggt 1545 aaa cta aat taa gaaatgaaaa aacacaaagg gctgctcacc tttgtgtttt 1597 ttaattaatt aaaatgttta ttaacttagt taaggagtag aatgggaaag gggatcggaa 1657 aacaagtata taggaggaga cctatttatg gcttcagaaa aagacgcagg aaaacagtca 1717 gcagtaaagc tt 1729 <210> 4 <211> 386 <212> DNA <213> Bacillus subtilis <400> 4 gcg gat tta ggc cat caa aca tta gga tca aat gat ggc tgg ggc gcg 48 tac tcg acc ggc aca aca ggc gga tca aaa gct tcg tca tcc cac gtg 96 tat acc gtc agc aac aga aac tac gtt tta ggc aag gac 144 acc aac aca acg cca aaa atc att tat att aag gga acg att gac atg 192 aac gtc gat gac aat ctg aag ccg ctt ggt cta aat gat tat aaa gat 240 cca gag tac gat ttg gac aaa tat ttg aaa gcc tat gac cct agc aca 288 tgg ggc aaa aag gag ccg tcg ggg aca cta gaa gag gg cg gaa gag cg aaa aat caa aaa gca cga gtc atg gtg gat att ccg gca aac 384 ac 386 <210> 5 <211> 20 <212> PRT <213> Bacillus subtilis <400> 5 Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn Asp Gly Trp Gly Ala 1 5 10 15 Tyr Ser Thr Gly 20 <210> 6 <211> 7 <212> PRT <213> Bacillus subtilis <400> 6 Val Asp Ile Pro Ala Asn Thr 1 5 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> unsure <222> 3 <223> n = a, c, g or t. <220> <221> unsure <222> 9 <223> n = a, c, g or t. <220> <221> unsure <222> 12 <223> n = a, c, g or t. <400> 7 gcngayytng gncaycarac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> unsure <222> 6 <223> n = a, c, g or t. <220> <221> unsure <222> 9 <223> n = a, c, g or t. <220> <221> unsure <222> 18 <223> n = a, c, g or t. <400> 8 gtrttngcng gdatrtcnac 20 <210> 9 <211> 29 <212 > DNA <213> Artificial Sequence <220> <223> unsure <400> 9 ggggaattcc tttgcggtgt atgtgttgc 29 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> unsure <400> 10 ccttctagat acttgttttc c 21

──────────────────────────────────────────────────の Continued on the front page (51) Int. Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/26 D06M 16/00 Z D06M 16/00 C12N 5/00 A // D06M 101: 06 F term ( Reference) 4B024 AA20 BA12 CA03 CA07 DA07 EA04 FA18 GA11 GA21 4B050 CC03 CC04 DD02 LL10 4B065 AA19X AA19Y AB01 AC14 AC15 BA01 BA10 CA31 CA60 4L031 AB01 BA33 BA39 CA00

Claims (11)

[Claims] 1. Encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in this sequence and having protopectinase activity gene. 2. The gene according to claim 1, wherein a signal peptide is added. 3. The gene to which the signal peptide has been added comprises a protein having the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added,
The gene according to claim 2, which encodes a protein having protopectinase activity. 4. Base number 358 of the sequence shown in SEQ ID NO: 3
The gene according to any one of claims 1 to 3, which has a nucleotide sequence represented by any one of claims 1 to 1557. 5. The protein produced from a gene,
1) has protopectinase activity; 2) has a molecular weight of about 43,000 on an SDS-polyacrylamide gel electrophoresis gel; 3) has an optimal pH of 8.0; 4) has an optimal temperature of 60 ° C;
And 5) the gene according to any one of claims 1 to 4, having a property of being inhibited by Cu, Hg, Mn, and Zn. 6. A recombinant vector containing the gene according to any one of claims 1 to 5. A transformant comprising the recombinant vector according to claim 6. 8. The transformant according to claim 7, wherein the host of the transformant is a microorganism. 9. A method for producing the transformant according to claim 7 or 8. 10. A method for scouring fibers using the transformant according to claim 7 or 8. 11. A fiber treated by the method of claim 10.