JPH11318457A - Pectate lyase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a pectate lyase gene capable of mass-producing a simple one and useful as a detergent, a food processing agent, a textile treating agent, etc., by encoding a specific amino acid sequence. SOLUTION: This gene is capable of encoding an amino acid sequence represented by formula I or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. The pectate lyase gene is preferably produced by culturing a transformant having the base sequence represented by formula II or a base sequence in which one or several bases are deleted, substituted or added in the base sequence, containing a recombinant vector containing the pectate lyase gene and transforming a host which is a microorganism. The transformant is deposited as Bacillus sp. KSM-P 103 strain (FERM P-15988) or Bacillus sp. KSM-97 strain (FERM P-15985).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding pectate lyase useful as a detergent, a food processing agent, a fiber treatment agent and the like.

[0002]

2. Description of the Related Art Pectic acid lyase (EC 4.2.
2.2), in 1962, Bacillus polymixer ( Baci
llus polymyxa ) and Erwinia Cultura ( Erwinia )
cartovora ), which requires calcium ions for its reaction (Starr & Mora)
n, Science, 135, 920-921, 1962). Since then, many bacteria and fungi have been known to produce pectate lyase (Sakai, et al. Adv. Appl. Mic
robiol. 39, 213-294, 1993). Furthermore, with the development of genetic recombination technology, for example, Erwinia chrysanthe
mi EC16 (Tamaki, et al., J. Bacteriol., 170, 3468-
3478, 1988), Xanthomonas campestrispv. Malvaccarum
B414 (Liao et al., MPMI, 9, 14-21, 1996), Fusar
ium solani f. sp. pisi (Guo et al., J. Bacteriol.,
177, 7070-7077, 1995; Arch.Biochem.Biophys., 33
2, 305-312, 1996, Gonzalez-candels & kolattukudy,
J. Bacteriol., 174, 6343-6349, 1992), Aspergillus
nidulans (Ho et al., Curr. Genet., 27, 142-149, 1
995) and the like, the pectate lyase gene has been cloned, its nucleotide sequence has been determined, and its deduced amino acid sequence has been analyzed.

There are relatively few reports of pectate lyase of Bacillus bacteria, which have been thoroughly studied genetically and biochemically. Starr and Moran are Bacillus polym
Since the discovery of pectate lyase in the culture of yxa , Bac
illus pumilus (Dave & Vaughn, J. Bacteriol., 198,
166-174, 1971), Bacillus subtilis (Chesson & Codn
er, J. Appl. Bacteriol., 44, 347-364, 1978), and the optimal pH produced by a thermophilic Bacillus bacterium is 8.5 to 8.5.
9.3 enzyme (molecular weight unknown) (Karbassi & Luh, J. F.
ood Sci., 44, 1156-1161, 1979). Recently, Sakamoto et al. (Biosci. Biotech. Biochem., 5
8, 353-358, 1994) purifies pectate lyase having protopectinase activity from Bacillus subtilis IFO3134 (protopectinase-N). Bacillus pectate lyase gene has been determined by Bacill
us subtilis SO 113 (Nasser et al., FEBS 335 319-3
26, 1993), Alkali-tolerant Bacillus sp. YA-14 (Kim
et al., Biosci. Biotech. Biochem. 58, 947-949, 199
4) only, and the SO 113 strain and YA-14
The nucleotide sequences of the strains showed 99% homology, and the amino acid sequences were completely identical.

[0004]

Although some of these conventional pectate lyases are available as a part of the product, they are expensive and contain various carbohydrate-degrading enzymes. Is not widely used.

[0005] Accordingly, the present invention provides a gene encoding a pectate lyase useful in a wide range of industries, such as detergents and fiber treatments, in order to enable single and mass production thereof, and a gene containing the gene and a set containing the gene. It is an object to provide a recombinant vector and a transformant containing the same.

[0006]

Means for Solving the Problems Accordingly, the present inventors searched for a microorganism that produces pectate lyase from the natural world,
Various studies have been conducted to establish the gene, its properties and its mass production technology using genetic engineering technology.
Cloning of the gene for a novel pectate lyase produced by Bacillus bacteria isolated from soil,
We succeeded in establishing a production technology by genetic recombination and completed the present invention.

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 a gene encoding pectate lyase having an amino acid sequence in which several amino acids have been deleted, substituted or added. The present invention also provides a recombinant vector containing the above pectate lyase gene and a transformant containing the recombinant vector.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The gene of the present invention encodes 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 are deleted, substituted or added. Has an array. As long as pectate lyase activity is not lost, deletion, substitution or addition (hereinafter, sometimes referred to as mutation) of an amino acid in the amino acid sequence is not particularly limited. In addition, SEQ ID NO: 1
Alternatively, at the N-terminus in the amino acid sequence of SEQ ID NO: 2, 1
~ Several amino acids may be added.

In SEQ ID NO: 1, amino acids 94 to 228
Among the other enzymes, as shown in FIG. 6, pectate lyase PelA derived from Aspergillus nidulans (Meng-Chen Ho, et al., Curr. Gcne
t., 27. 142-149, 1995) has the highest homology, but 4
There is only 3.9% homology. Therefore, when properly aligned between amino acid Nos. 94 to 228 of SEQ ID NO: 1, enzymes having the above homology are included in the enzyme encoded by the gene of the present invention. That is, the homology of the amino acid sequence is the same as amino acid number 9
It is preferably at least 45%, more preferably at least 70%, particularly preferably at least 80%, with respect to 4-228.

In SEQ ID NO: 1, amino acids 1 to 3
When the homology with other enzymes was examined between 02, Glomer
Pectate lyase from ella cingulata (GenBank Accession No. GCU32942) has the highest homology, but has only 39.9% homology between SEQ ID NOs: 14-287. Therefore, amino acids 14 to 2 of SEQ ID NO: 1
Enzymes having more than these homology when properly aligned between 87 are included in the enzymes encoded by the gene of the present invention. That is, the homology of the amino acid sequence is preferably 40% or more, more preferably 70% or more, and particularly preferably 80% or more with respect to amino acid numbers 14 to 287 in SEQ ID NO: 1.

The pectate lyase gene of the present invention may be any as long as it encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a mutant thereof, and the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 or Those having a base sequence in which one or several bases are deleted, substituted or added are preferred.

The pectate lyase encoded by the gene of the present invention is a microorganism belonging to the genus Bacillus isolated from soil (for example, Bacillus sp. Strain KSM-P103 (F.
ERM P-15988) and Bacillus SP KS
M-P7 strain (FERM P-15985) or a mutant thereof can be cultured and collected from the resulting culture.

[0013] The pectate lyase gene of the present invention is, for example, a Bacillus sp. KSM-P103 strain (FER
MP-15988) and Bacillus SP KSM-
It can be cloned from P7 strain (FERM P-15985) and the like. Examples of the cloning means include known methods, for example, a method of cloning a target gene by a shotgun method, a PCR method, and the like.

To prepare a recombinant vector containing the pectate lyase gene, the pectate lyase gene may be incorporated into any vector suitable for expressing the gene in the intended host. Examples of such a vector include pUC18 and pBR32 when Escherichia coli is used as a host.
2, pUC19 and the like. When Bacillus subtilis is used as a host, pUB110 and the like can be mentioned.

Transformation of a host with the thus obtained recombinant vector is carried out by a conventional method, for example, a protoplast method, a competent cell method or the like. The host is not particularly limited, but is preferably a microorganism, and includes Gram-positive bacteria such as Bacillus and Streptomyces; Gram-negative bacteria such as Escherichia coli ; and fungi such as Saccharomyces yeast and Aspergillus mold. Can be

The obtained transformant is inoculated into a medium containing an assimilable carbon source, nitrogen source and other essential nutrients, cultured according to a conventional method, and a method for collecting and purifying general enzymes from the obtained culture solution. Pectate lyase can be obtained by a method according to the above.

[0017]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1: Bacillus sp. KSM-
Culture of P103 strain and Bacillus sp. KSM-P7 strain For culturing of Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain, 50 ml of a liquid medium was added to a Sakaguchi flask and shaking culture was performed at 30 ° C. for 2 days. The medium composition was 3% (w / v) polypeptone S,
0.5% yeast extract, 1% fish meat extract, 0.15% dipotassium phosphate, 0.001% calcium chloride, 0.5%
Pectin, 0.5% sodium carbonate (separate sterilization).

Example 2 Bacillus sp. KSM-
Purification of pectate lyase from P103 strain and Bacillus sp. KSM-P7 strain The culture solution of Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain was centrifuged (10,0
(00 × g, 15 minutes, 4 ° C.), and ammonium sulfate was gradually added to the supernatant (900 ml) so as to be 65% saturated. After standing overnight at 5 ° C, centrifugation (10,000 xg,
The precipitate was collected for 15 minutes at 4 ° C. This was dissolved in a small amount of a basic buffer [10 mM Tris-HCl buffer (pH 7.5) containing 1 mM calcium chloride], and dialyzed against the same buffer overnight. DEAE-Bi obtained by equilibrating the obtained dialysis solution (105 ml) with a basic buffer solution in advance.
o-GelA (Bio-Rad) column (2.5 ×
16 cm). Activity was observed in the fraction eluted by washing with the basic buffer, and this was collected. Next, this non-adsorbed fraction was previously equilibrated with a basic buffer solution.
-Bio-GelA (manufactured by Bio-Rad) column (2.5 x 22 cm). After performing washing and elution with about 300 ml of the basic buffer, the adsorbed protein was eluted by a concentration gradient elution method using a basic buffer (400 ml each) containing 0 to 0.1 M potassium chloride. KSM
The pectate lyase derived from the -P103 strain and the KSM-P7 strain was eluted as a single peak at a potassium chloride concentration of about 70 to 80 mM, and was detected as a homogeneous protein in SDS-PAGE and PAGE by the Taber method. These fractions were collected, concentrated by ultrafiltration (YM3 membrane, manufactured by Amicon), and stored frozen at -20 ° C as a 20% glycerol solution. In the case of the purified pectate lyase enzyme derived from the KSM-P103 strain obtained by the above operation, the activity yield was about 42%, and the purification magnification was about 500 times. On the other hand, in the case of the purified pectate lyase enzyme derived from the KSM-P7 strain, the activity yield was about 25% and the purification magnification was about 180 times. Each purified enzyme was blotted on a ProSorb filter (manufactured by PerkinElmer), and the amino terminal sequence was determined up to the 18th amino acid using a protein sequencer (model 476A, manufactured by Applied Biosystems). As a result, the KSM-P103 strain and K
An enzyme derived from SM-P7 strain and ANFNQQGFSTLN
GGTTGG.

Example 3: Cloning of pectate lyase gene (1) Preparation of chromosomal DNA of Bacillus sp. KSM-P103 and Bacillus sp. KSM-P7 strains Bacillus sp. KSM-P103 and Bacillus sp. The culture solution obtained by shaking culture was centrifuged to collect the cells. From the obtained cells, S
aito and Miura's method (Biochim. Biophys. Act
a, 72, 619-629, 1963).

(2) Preparation of Primers Primers 1 and 2 were synthesized from the amino terminal sequence of the purified enzyme obtained in Example 2 and the amino acid sequence of the pectate lyase conserved region (FIG. 1). (3) Cloning Using these primers 1 and 2, Bacillus sp.
PCR was performed using the chromosomal DNA of the SM-P103 strain (0.5 μg) as a template. The obtained amplified fragment was purified with a PCR fragment purification kit (manufactured by Boehringer Mannheim), and then cloned by introducing it into the SmaI site of plasmid vector pUC19. When the base sequences of the four clones obtained were determined, a part of the target pectate lyase gene sequence was detected, and the amino acid sequence could be estimated (see FIG. 2). Next, inverse PCR was performed to amplify the upstream and downstream regions of the above PCR amplified fragment. The nucleotide sequences shown in FIG. 2, primers 3 and 4, were used. Bacillus sp KSM-P1
03 shares chromosome of the (1μg), pre-Hin dIII
Digestion with phenol / chloroform extraction, T4D
Intramolecular binding (circularized DNA binding) using NA ligase
This was used as a mold. PCR is Long Template System
This was performed using a PCR kit (TaKaRa). As a result, an amplified fragment of about 4.5 kbp was detected. Sequencing between this DNA fragment and primers 1-2 by direct sequencing revealed that the sequence from the N-terminal amino acid sequence to the amino acid before the stop codon (TAA) was determined.
The amino acid sequence and base sequence (SEQ ID NOS: 1 and 3) of a pectate lyase consisting of 02 amino acids were determined. From this sequence, the molecular weight of pectate lyase (secretory mature enzyme) produced by Bacillus sp. KSM-P103 is estimated to be 33,212 Da (about 33 kDa). In a similar manner, 302 amino acids from the N-terminal amino acid sequence of the pectate lyase produced by the Bacillus sp. KSM-P7 strain to the amino acid before the stop codon (TAA).
The amino acid sequence and base sequence of pectate lyase consisting of amino acids (SEQ ID NOs: 2 and 4) were determined. The molecular weight of pectate lyase (secretory mature enzyme) produced by Bacillus sp.
It is estimated to be 3,355 Da (about 33 kDa).

Example 4 Construction of Recombinant Plasmid and Production of Pectate Lyase by Bacillus subtilis First, the N-terminal amino acid Ala of pectate lyase produced by Bacillus sp.
An upstream primer (primer 5, see FIG. 3) was designed so that a signal sequence derived from the host vector used was ligated. As the downstream primer (Primer 6, see FIG. 3), a 26 bp sequence located 73 bp downstream from the termination codon (TAA) of the pectate lyase gene was designed. Used vector pHSP64 (Su
mitomo et al., Biosci. Biotech. Biochem., 56, 872-8.
77, 1992), a PCR-amplified DNA fragment was inserted between the Sal I site located 3 'to the signal sequence coding portion and the Sma I site located 11 bp downstream of the Sal I site.

That is, PCR was performed using primers 5 and 6 and the chromosomal DNA of Bacillus sp. KSM-P103 as a template, and an approximately 1 kb pDNA fragment was amplified. This is digested with Xho I, and Sal I and S
It was ligated with pHSP64 and ligases cut with ma I (see Figure 4). This recombinant plasmid was prepared using pHSP-1.
03PAL.

Next, Bacillus subtilis IS using pHSP-103 PAL.
When W1214 was transformed and cultured in a liquid medium at 30 ° C. for 3 days, a significant amount of pectate lyase was produced extracellularly. In addition, a significant amount of pectate lyase was produced extracellularly by the same method as above using the pectate lyase structural gene derived from Bacillus sp. KSM-P7.

Reference Example 1: Characteristics of recombinant pectate lyase The crude enzyme solutions of two pectate lyases obtained in Example 4 were completely purified according to Example 2. [Standard enzyme activity measurement method] 0.2 ml of 0.5 M glycine-sodium hydroxide buffer (pH 10.5) was added to a test tube.
1 ml of 4 mM calcium chloride solution, 0.2 ml of 1% (w /
v) Polygalacturonic acid aqueous solution (ICN: Lot 1448)
2. Add pH adjusted to 6.8 with sodium hydroxide solution and 1.4 ml of deionized water, incubate at 30 ° C. for 5 minutes, and then 0.1 ml of appropriately diluted enzyme solution (diluted to 1 mM) 50 mM Tris-HCl buffer containing calcium chloride,
(performed at pH 7.5) was added to initiate the reaction. 30
After incubating at 10 ° C. for 10 minutes, 2 ml of 50 mM hydrochloric acid was added to stop the reaction. The amount of unsaturated polygalacturonide formed is 2
The absorbance at 35 nm was measured, and the molecular extinction coefficient of unsaturated digalacturonide was 4600 (Hasegawa & Nagel, J. Foo).
d Sci., 31, 838-845, 1966). The blind test was performed by adding 2 ml of 50 mM hydrochloric acid to the reaction solution treated without adding the enzyme solution, and then adding 0.1 ml of the enzyme solution. One unit (1 U) of the enzyme was defined as an amount capable of producing 1 μmol of unsaturated polygalacturonide equivalent to 1 μmol of unsaturated digalacturonide per minute under the above reaction conditions.

(1) N-terminal amino acid sequence The N-terminal amino acid sequences of these enzymes were determined in the same manner as in Example 2, and both enzymes were found to be ANFNQQG
A sequence containing FSTLNGGTTTGG was detected. (2) pH-activity curves The pH-activity curves of P103 and P7 recombinant pectate lyase were examined. When polygalacturonic acid is used as a substrate, P
The pH-activity curve of 103 recombinant pectate lyase was obtained from the Bacillus sp. KSM-P10 obtained in Example 2.
The pH-activity curves of the pectate lyases derived from the three strains almost completely coincided with each other, and the optimum reaction pH was around pH 10 in a glycine-sodium hydroxide buffer (FIG. 5). Similar results were obtained with the P7 recombinase.

(3) Molecular weight The molecular weight of the obtained recombinant pectate lyase was determined by SDS-
As determined by polyacrylamide electrophoresis (15% acrylamide), the molecular weight of the P7 and P103 recombinant pectate lyases was found to be in the range of 31 to 33 kDa. This value almost coincided with the molecular weight calculated from the deduced amino acid sequence. As a standard protein, SDS-PAGE Molecula manufactured by Biorat was used.
r Weight Standards, Low Range were used. (4) Pectin release from cotton fiber As a substrate, work gloves for quality control (with 40 Smooth bleached fingers)
Cotton, made by Hisada Nakata Shoten)
mg of 0.05 M glycine containing 0.4 mM CaCl _2-
After suspending in a sodium hydroxide buffer (pH 10.5), the two recombinant pectate lyases obtained in Example 4 were added, and the mixture was reacted at 30 ° C. for 1 hour. The reaction-finished solution is centrifuged, and the pectin released in the supernatant is determined by the carbazole sulfate method (Sakai, Methods Enzymol., 161, 335-350, 1988).
Quantified. As a result, when 0.1 U of the enzyme was used,
For all enzymes, 2.3 to 3.6 mg of pectin was released as galacturonic acid per 1 g of cotton fiber. This means that both recombinant pectate lyases act on protopectin on the surface of the cotton fiber, and the absorbance of the supernatant at 235 nm was also increased. Is determined to have protopectinase activity.

[0028]

EFFECT OF THE INVENTION Using the pectate lyase gene of the present invention, pectate lyase useful as a detergent, a food processing agent, a fiber treatment agent and the like can be produced singly and in large quantities.

[0029]

[Sequence List] SEQ ID NO: 1 Sequence length: 302 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Ala Asn Phe Asn Gln Gln Gly Phe Ser Thr Leu Asn Gly Gly Thr Thr 5 10 15 Gly Gly Glu Gly Gly Lys Thr Val Thr Val Lys Thr Gly Asn Glu Leu 20 25 30 Leu Ala Ala Leu Lys Asn Lys Gly Thr Asn Glu Lys Leu Lys Ile Val 35 40 45 Val Asp Gly Thr Ile Thr Pro Ser Asn Thr Ser Ala Asn Lys Ile Asp 50 55 60 Val Lys Asp Thr Asn Asn Val Ser Ile Val Gly Lys Gly Thr Asn Gly 65 70 75 80 Glu Phe Asn Gly Ile Gly Ile Lys Val Trp Arg Ala Asn Asn Ile Ile 85 90 95 Ile Arg Asn Leu Lys Ile His His Ser Lys Ile Gly Asp Lys Asp Ala 100 105 110 Ile Gly Ile Glu Gly Gly Ser Lys Asn Ile Trp Val Asp His Asn Glu 115 120 125 Leu Tyr Asn Thr Leu Asn Ser Gly Lys Asp Asp Tyr Asp Gly Leu Phe 130 135 140 Asp Val Lys Asn Asp Ser Asp Tyr Ile Thr Phe Ser Trp Asn Tyr Val 145 150 155 160 His Asp Ser Trp Lys Thr Met Leu Met Gly Ser Ser Asp Asn Asp Asn 165 170 175 Tyr Asn Arg Lys Ile Thr Phe His Asn Asn Arg Phe Glu Asn Leu Asn 180 185 190 Ser Arg Val Pro Ser Met Arg Phe Gly Glu Gly His Val Tyr Asn Asn 195 200 205 Tyr Tyr Lys Gly Ile Leu Thr Thr Ala Ile Asn Ser Arg Met Gly Ala 210 215 220 Lys Met Arg Ile Glu His Asn Val Phe Glu Asn Thr Asn Asn Ala Ile 225 230 235 240 Gly Ser Trp Asp Ser Ser Gln Val Gly Thr Trp His Val Ile Asn Asn 245 250 255 Ser Tyr Ile Asn Ser Thr Gly Ser Leu Pro Thr Ser Ser Thr Gly Thr 260 265 270 Tyr Asn Pro Pro Tyr Asn Tyr Ser Leu Leu Asn Val Asn Asn Val Lys 275 280 285 Ser Glu Val Ile Ser Asn Ala Gly Val Gly Lys Val Asn Pro 290 295 300

SEQ ID NO: 2 Sequence length: 302 Sequence type: amino acid Topology: linear Sequence type: protein Sequence Ala Asn Phe Asn Gln Gln Gly Phe Ser Thr Leu Asn Gly Gly Thr Thr 5 10 15 Gly Gly Glu Gly Gly Lys Thr Val Thr Val Lys Thr Gly Asn Glu Leu 20 25 30 Leu Ala Ala Leu Lys Ser Lys Gly Thr Asn Glu Lys Leu Lys Ile Val 35 40 45 Val Asp Gly Thr Ile Thr Pro Ser Asn Thr Ser Ala Asn Lys Ile Asp 50 55 60 Val Lys Asp Thr Asn Asn Val Ser Ile Val Gly Lys Gly Thr Asn Gly 65 70 75 80 Glu Phe Asn Gly Ile Gly Ile Lys Val Trp Arg Ala Asla Asn Val Ile 85 90 95 Ile Arg Asn Leu Lys Ile His His Ser Lys Ile Gly Asp Lys Asp Ala 100 105 110 Ile Gly Ile Glu Gly Ala Ser Lys Asn Val Trp Val Asp His Asn Glu 115 120 125 Leu Tyr Asn Thr Leu Asn Ser Asp Lys Asp Asp Tyr Asp Gly Leu Phe 130 135 140 Asp Val Lys Asn Asp Ser Asp Tyr Ile Thr Phe Ser Trp Asn Tyr Val 145 150 155 160 His Asp Ser Trp Lys Thr Met Leu Met Gly Ser Ser Asp Asn Asp Asn 165 170 175 Tyr Asn Arg Lys Ile Thr Phe His Asn Asn Arg Phe Glu Asn Leu Asn 180 185 190 Ser Arg Val Pro Ser Met Arg Phe Gly Glu Gly His Val Tyr Asn Asn 195 200 205 Tyr Tyr Lys Asn Ile Leu Thr Thr Ala Ile Asn Ser Arg Met Gly Ala 210 215 220 Lys Met Arg Ile Glu His Asn Val Phe Glu Asn Thr Lys Asn Ala Ile 225 230 235 240 Gly Ser Trp Asp Ser Arg Gln Val Gly Thr Trp His Val Ile Asn Asn 245 250 255 Ser Tyr Ile Asn Ser Thr Gly Ser Leu Pro Thr Ser Ser Thr Gly Thr 260 265 270 Tyr Asn Pro Pro Tyr Asn Tyr Ser Leu Leu Asn Val Asn Asn Val Lys 275 280 285 Ser Glu Val Val Ser Asn Ala Gly Val Gly Lys Val Asn Pro 290 295 300

SEQ ID NO: 3 Sequence length: 909 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Bacillus sp. KSM-P103 sequence GCCAACTTTA ATCAACAAGG CTTCTCCACA TTAAATGGCG GCACAACAGG CGGTGAAGGC 60 GGTAAAACTG TGACAGTCAA AACTGGAAAT GAATTATTGG CTGCCCTCAA AAATAAAGGA 120 ACAAACGAAA AATTAAAAAT TGTTGTCGAC GGGACGATTA CCCCTTCTAA TACATCAGCC 180 AATAAAATCG ATGTGAAAGA TACAAATAAT GTTTCGATCG TCGGTAAAGG GACGAACGGT 240 GAATTTAATG GGATTGGCAT TAAAGTATGG CGTGCCAATA ACATCATCAT CCGCAACTTG 300 AAAATCCACC ACAGCAAAAT CGGTGACAAA GATGCGATCG GGATCGAAGG CGGTTCTAAA 360 AACATATGGG TCGACCATAA TGAACTGTAC AACACATTGA ATTCTGGGAA GGATGACTAC 420 GATGGTTTAT TTGATGTGAA AAATGACTCT GACTATATTA CCTTCTCGTG GAACTATGTA 480 CATGACAGCT GGAAAACAAT GCTCATGGGC AGCTCCGATA ACGATAACTA CAATCGAAAA 540 ATTACGTTCC ACAACAACCG TTTTGATCTCGATCACGATCACTCGATCTCGATC C ATGTCTATAA CAACTATTAT AAAGGCATCC TGACAACTGC CATTAACTCG 660 CGTATGGGCG CGAAGATGAG AATTGAACAC AACGTATTTG AAAACACCAA TAATGCGATC 720 GGAAGCTGGG ACAGCAGTCA AGTTGGAACA TGGCATGTCA TCAACAACTC GTATATTAAC 780 AGCACAGGCA GTCTGCCAAC GTCTTCAACA GGTACGTATA ACCCTCCTTA TAACTACTCT 840 TTGCTTAACG TGAACAATGT TAAATCAGAA GTGATATCAA ATGCAGGTGT TGGTAAAGTA 900 AATCCTTAA 909

SEQ ID NO: 4 Sequence length: 909 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Bacillus sp. KSM-P7 sequence GCCAATTTTA ATCAGCAAGG ATTCTCCACA TTAAATGGCG GTACAACAGG CGGTGAAGGC 60 GGTAAAACGG TGACGGTGAA AACCGGAAAT GAACTATTAG CTGCCCTCAA AAGCAAAGGA 120 ACAAATGAAA AATTGAAAAT CGTGGTAGAC GGAACCATTA CTCCTTCTAA TACTTCAGCC 180 AATAAAATTG ATGTGAAAGA TACAAACAAT GTCTCCATCG TCGGTAAAGG GACAAACGGC 240 GAATTTAACG GGATTGGTAT TAAAGTATGG CGCGCCAATA ACGTCATCAT CCGCAACCTC 300 AAAATCCATC ACAGCAAAAT CGGTGATAAA GATGCCATCG GAATCGAAGG TGCCTCTAAA 360 AACGTATGGG TCGACCATAA TGAACTCTAC AACACTTTAA ACTCTGATAA AGATGACTAT 420 GATGGGTTAT TCGATGTGAA AAATGACTCT GATTACATTA CGTTCTCATG GAACTATGTC 480 CATGACAGCT GGAAAACGAT GCTGATGGGC AGCTCCGATA ACGATAACTA CAACAGAAAA 540 ATCACGTTCC ACAACAACCG TTTCGATC TGTCTCTCTCTCTCTCTCTCGATC GTATAA CAACTATTAT AAAAACATCC TAACAACAGC GATTAATTCA 660 CGTATGGGCG CGAAAATGAG AATTGAACAC AACGTATTTG AAAACACGAA AAATGCCATC 720 GGCAGCTGGG ACAGCCGTCA AGTCGGTACT TGGCATGTCA TCAACAACTC TTATATTAAC 780 AGCACAGGTA GCCTGCCAAC GTCTTCAACA GGTACGTACA ACCCTCCTTA TAACTACTCT 840 TTGCTTAACG TGAACAATGT GAAATCAGAA GTTGTATCAA ATGCGGGTGT AGGAAAAGTA 900 AATCCTTAA 909

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequences of primers 1 and 2 for pectate lyase gene cloning.

FIG. 2 is a view showing a DNA sequence and a deduced amino acid sequence between primers 1 and 2, and positions of primers 3 and 4.

FIG. 3 is a diagram showing primers 5 and 6 for amplifying the whole region of pectate lyase of the present invention. The amplified fragment (about 1 kbp) generated by the primer for PCR amplification shown in Example 4 was digested with XhoI , and then digested with SalI.
Is coupled to pHSP64 digested with Sma I and.

FIG. 4. Pectate lyase gene (P103-derived pa
1) Introduction into a secretion vector (pHSP64) and the created pectate lyase expression secretion vector pHSP-1
It is a figure showing 03PAL. Amp: Ampicillin resistance marker gene Tet: Tetracycline resistance marker gene

FIG. 5 shows a pH-activity curve of P103 recombinant pectate lyase (Example 4). In a 50 mM glycine-sodium hydroxide buffer solution, the value of pH about 10 showing the maximum activity was 100
The relative activity was expressed as%.

FIG. 6 shows the homology between the amino acid sequences of P103 recombinant pectate lyase and other enzymes. Amino acid numbers were homologous to other amino acid sequences based on SEQ ID NO: 94 to 228. * Indicates a common amino acid residue.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1: 125) (C12N 9/88 C12R 1: 125) (72) Inventor Norihiko Higaki Shellfish in Haga-gun, Tochigi 2606 Kao Co., Ltd., Research Laboratories in Kao Corporation (72) Inventor Toru Kobayashi 2606 Kabane-cho, Kaigacho, Haga-gun, Tochigi Pref. Inside

Claims (5)

[Claims] 1. A pectate lyase gene encoding 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. The pectate lyase gene has a nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence in which one or several bases of the nucleotide sequence are deleted, substituted or added. Item 7. The pectate lyase gene according to Item 1. 3. A recombinant vector containing the gene according to claim 1 or 2. A transformant comprising the recombinant vector according to claim 3. 5. The transformant according to claim 4, wherein the host is a microorganism.