JP2002085064A - NOVEL PLASMID, STRAIN ESCHERICHIA COLI JM83/pKP2 TRANSFORMED THEREWITH, AND PHYTASE PRODUCED THEREFROM - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel plasmid vector for transformation, for the mass production of a phytase serving the role to enhance the phosphorous bioavailability in grains used as monogastic livestock feeds. SOLUTION: This novel plasmid vector is a recombinant vector pKP1 or pKP2. The strain Escherichia coli JM83/pKP2 transformed by the vector, and the phytase produced from the strain are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a strain E. coli JM83 / pK transformed using a novel plasmid.
It relates to P2 and phytase produced therefrom, more specifically for the mass production of novel phytinolytic enzymes that enhance the bioavailability of phosphorus (P) in cereals for monogastric livestock feed. A new recombinant vector pKP1 or pKP2 is determined by genetic analysis using the gene sequence
And Escherichia coli JM83 / pKP2 obtained by transforming Escherichia coli using the above-mentioned recombinant vector pKP1 or pKP2.

[0002]

2. Description of the Related Art Phytic acid-degrading enzymes are known as phytic acid (phytiic acid).
It is an enzyme that decomposes c acid to produce phosphate and phosphate inositol. The phytic acid accounts for 50 to 70% of the total content of phosphorus (P) contained in cereals used as livestock feed.
However, monogastric livestock such as chickens and pigs do not have phytinolytic enzymes that degrade phytic acid in vivo, and therefore have extremely low utilization of vegetable phosphorus. Phyticate, which is not digested, is discharged to a water source or the like and acts as a serious environmental pollutant, and acts as a main cause of green algae in small lakes or red algae in the sea. By the way, phytic acid is insolubilized by chelating with trace amounts of minerals, amino acids, vitamins, etc., which are essential for monogastric livestock, and not only can animals not use the above substances, but also are excreted in feces. Major bioactivators are released as water sources, altering environmental ecosystems and causing severe environmental pollution.

[0003] Therefore, when phytinolytic enzyme is applied to monogastric livestock, the amount of phosphorus (P) can be reduced by increasing the availability of phosphorus (P). It can improve the availability of phosphorus and important trace bioactive substances, reduce the amount of phosphorus emitted as animal dung and thereby reduce the environmental pollution. In particular, since 1996, legislative announcements have been made to regulate the content of phosphorus in animal wastewater released, and in Europe, phytinolytic enzymes must be compulsorily added to feeds of monogastric livestock. Thus, the utility of phytinolytic enzymes in animals can be said to be infinite. In addition, when phytinolytic enzymes are added, trace minerals such as calcium and zinc ions, which bind to phytic acid having an anion and decrease in availability, and increase in bioactive substances such as amino acids and vitamins are increased. As a result, the productivity of livestock can be greatly improved. If the phytinolytic enzyme is added to the feed and applied to monogastric livestock as described above, the availability of the feed can be increased, and the productivity can be improved, the economic benefit can be reduced, and the environmental pollution can be reduced.

Due to the advantages described above, phytinolytic enzymes have been studied mainly in Europe (AH
J. Ullah et al., 1994; KCEnrich, 1994; CS Piddingto
n, 1993), and studies on the effects of phytinolytic enzymes on animals have been advanced (LG Young et al., 1993; XG Lei et al., 1
994; Z. Morez et al., 1994). However, the number of phosphorus that the phytinolytic enzyme cleaves is limited, and most of them are produced by fungi, and there is a problem that the growth period is long and economic production is difficult. In addition, since it is not compatible with the physiology of single stomach livestock, there is a limit as an additive for single stomach livestock.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have monitored the problems of the above-mentioned phytinolytic enzymes, and have shown an activity superior to that of conventional phytinolytic enzymes, and a novel phytinolytic enzyme having different properties. Bacillus (Bac
illus sp.) DS-11 was identified and deposited at the Genetic Resource Center in the Institute of Bioscience and Biotechnology at the Korea Institute of Science and Technology (KCTC 0231BP), which is a patent strain depository in Korea. I applied for a patent [Korean Patent No. 96-6817]. In addition, the characteristics of a novel phytinolytic enzyme produced by the microorganism were examined. According to the results, the activity against heat and pH was excellent, and it was recognized that there was stability against this.

[0006] Accordingly, the present inventors first attempted to mass-produce a phytinolytic enzyme having the above-mentioned excellent properties, first of all, the above-mentioned patented strain Bacillus sp.
p.) The gene coding for phytinolytic enzyme was cloned by DS-11, and the DNA sequencing was examined. As a result, Aspergillus awamori (WO 94-3072A), which has cloned the gene and determined the nucleotide sequence,
Aspergillus ficuum (EP 420358, US 5436156), Asperg
illus niger (EP 420358), Aspergillus tereus (EP 6843)
13) It was found that this was a novel gene sequence completely different from the nucleotide sequence of, for example, U85968 from GenBank of NCBI in the United States.
(January 21, 1997) was given a deposit number. Thereafter, Escherichia coli was transformed with a plasmid vector (pKP1 or pKP2) encoding a phytinolytic enzyme of Bacillus genus DS-11 DS-11 (Bacillus sp. DS-11), and the transformed new strain was used. Escherichia coli JM83 / pKP2 was deposited at the Genetic Resource Center in the Institute of Bioscience and Biotechnology at the Korea Institute of Science and Technology, a patent strain depository in Korea (K
CTC 0308BP; January 28, 1997).

Accordingly, the present invention provides a plasmid vector for transformation, pKP1 and pKP2, and a novel Escherichia coli JM83 / pKP2 (KCTC 0308BP) transformed therewith, for producing phytinolytic enzyme in large quantities. Another object is to provide a method for mass-producing phytinolytic enzyme from the above strain.

[0008]

The present invention relates to the genus Bacillus (Bacillu).
s sp.) It relates to a novel phytinolytic enzyme produced by DS-11, and the DNA encoding it, as shown in Sequence Listing 1.
It is characterized by a base sequence and an amino acid sequence as shown in Sequence Listing 2. The present invention also includes a plasmid pKP1 or pKP2 containing a DNA base sequence as shown in Sequence Listing 1 linked to express phytinolytic enzyme from Escherichia coli. The present invention also provides a novel strain of Escherichia coli JM83 / pKP2 (KCTC 0308BP) transformed into Escherichia coli by plasmid pKP1 or pKP2, which characteristically contains SEQ ID NO: 1 encoding phytinolytic enzyme.
Including.

The present invention will be described in more detail as follows. The present invention produces a novel recombinant DNA expression vector, pKP1 or pKP2, by inserting a gene encoding a phytinolytic enzyme produced by Bacillus genus DS-11 into a plasmid vector pUC19, and expressing the recombinant DNA. The vector was cloned into Escherichia coli JM-83 and cultured, and colonies with confirmed titers were selected and cultured to produce phytinolytic enzymes in large quantities.
A Separate only the expression vector pKP1 or pKP2 and DNA
The nucleotide sequence was confirmed. Hereinafter, the present invention will be described in detail step by step.

[Production of novel plasmids pKP1 and pKP2] (1) Determination of N-terminal amino acid sequence SDS-PAGE (SDS-pol) was applied to the purified phytinolytic enzyme protein.
yacrylamide gel electrophoresis)
Transferred to PVDF membrane (Bio-Rad Lab). Subsequently, 10 mM CAPS (3-cyclohexylamino-1-propanesu) containing 10% methanol was used.
lfonic acid) buffer solution, pH 11.0, electroblotting at 4 ° C, 400 mA for 45 hours
(electroblotting) was performed. Cut only the desired protein band and use the protein / peptide sequencer [Applied Bios
ystems model 476A Protein / Peptide Sequencer (Applie
d Biosystems Ins., CA, USA)]. N-terminal amino acid sequence of purified phytinolytic enzyme protein; Ser-Asp-Pro-Tyr-His-Phe-Thr-Val-Asn-Ala-Ala-X-Glu-
Thr-Glu

(2) Amino-acid of the internal peptide (Amino-acid
Determination of the sequence of purified inner phytinolytic enzyme in 1% (w /
v) converted to a concentration of about 100 times as much CNB as mass
The reaction was carried out for 24 hours at room temperature after adding r. Subsequently, the reaction was stopped by adding 100-fold water, and electrophoresis was performed as in (1) above to determine the amino acid sequence of the internal peptide. N-terminal amino acid sequence of protein fragment of phytinolytic enzyme cleaved with CNBr; Ala-X-Asp-Asp-Glu-Tyr-Gly
-Ser-Ser-Leu-Tyr

(3) Oligonucleotide probe (oligonu
Oligonucleotide probe is designed based on the amino acid sequence determined from (1) and (2), and a DNA synthesizer (Appl.
ied Biosystems ABI 380B DNA synthesizer). Oligonucleotides synthesized by the above method as a primer (primer), DS-11 chromosomal DNA (chromosomal
DNA) as template DNA and tag DNA polymerase
(polymerase) and dNTPs under the following conditions under PCR (pol
ymerase chain reaction). Denaturation: 1 minute at 95 ° C Annealing: 1 minute at 50 ° C Polymerization: 1 minute at 72 ° C Post-elongation: 7 minutes at 72 ° C Perform PCR under the above conditions After, 1.5% agarose gel electrophoresis (agarose gel electrophoresis) was performed to 600 bp
PCR products were found. This PCR product was recovered from the gel and used as a probe.

Oligonucleotide probe based on the N-terminal amino acid sequence; Amino acid sequence: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Ser-Asp-Pro-Tyr-His-Phe-Thr-Val-Asn -Ala-Ala-X-Glu-Thr-Glu Possible codons: 5 'GAT-CCT-TAT-CAT-TTT 3' CCCCCGA

Oligonucleotide probe based on the N-terminal amino acid sequence of the internal protein fragment; amino acid sequence: 1 2 3 4 5 6 7 8 9 10 11 Ala-X-Asp-Asp-Glu-Tyr-Gly-Ser-Ser -Leu-Tyr Possible codons: 3 'CTA-CTA-CTT-ATA-CCA 5' GGCGGTC

(4) Hybridization of DS-11 genomic DNA Chromosomal DNA derived from Bacillus sp. DS-11
(chromosomal DNA) using the Marmur method (Marmur J. 1961, Mo
l Biol. 3, 208). In order to confirm that the probe produced from the above (3) is suitable for selection of a genomic library (genomic library), after performing agarose gel electrophoresis by cutting genomic DNA with some restriction enzymes, Transferred to Nylon membrane. Subsequently, the DIG DNA labeling and detection kit [DIG
DNA Labeling and detection kit (Boehringer Mannhei
m product)] and the 600 bp DNA fragment synthesized from (3) above,
Southern hybridization (southerher
n hybridization). As a result, 2.2 kb when using HindIII, 4 kb when using ClaI,
When PstI was used, a signal could be confirmed at 6 kb. Therefore, the HindIII enzyme was used when producing a genomic library of Bacillus DS-11.

(5) Selection of Gene Encoding Phytin Degrading Enzyme Gene Chromosomal DNA (chromosomes) of Bacillus sp. DS-11
mal DNA) was cut with HindIII to select a 3-5 kb DNA fragment. Subsequently, after ligation to vector pUC19 which had been digested with HindIII and treated with phosphatase (CIP), competent E. coli J
M83 was transformed. Ampicilli (ampicilli)
n; 100 μm / ml) at 37 ° C from LB (Luria-Bertani)
After culturing for 16 hours, nylon membrane (Nylon mem
brane). In addition, colony hybridization (colonyhybri
dization) to select a colony showing a signal, Bacillus sp. (Bacillus sp.) DS-11 phytinolytic enzyme gene (Phytase gene) to confirm that the inserted successfully Fiske (Fiske) The titer of phytinolytic enzyme was measured by the method (Fiske CH and Subbarow Y.
P., J. Biol. Chem. 1925, 66, 375). As a result, 10,000
Two colonies showing a signal could be obtained from these colonies. This was cultured and the plasmid was separated. As a result, a 4.9 kb plasmid containing a 2.2 kb insert was identified, and the plasmid was named pKP1. In addition, it was confirmed that the phytinolytic enzyme gene was successfully inserted into pKP1 through titration of the phytinolytic enzyme.

(6) Restriction enzyme mapping (Gene Mapping) and subcloning (Subcloning) As a result of cutting 4.9 kb pKP1 with various restriction enzymes, 2.
EcoRI, BamHI, NdeI, H within 2 kb insert DNA
It was found to have restriction sites for incII and EcoRV. Plasmid subcloning was performed to look for only the genes required to express the titer of the enzyme (FIG. 1a). pKP1 and pUC19 were cut with HindIII and NdeI, respectively, and ligated, and this was used for E. coli JM.
Transformation into 83 resulted in 4.4 kb pKP2 with 1.7 kb insert DNA (FIG. 1b).

[Method for Producing Transformed Strain] Chromosomal DNA of Bacillus sp. DS-11
Was cut with HindIII to select a 3-5 kb DNA fragment. Then, cleave by HindIII and phosphatase
Ligation of the vector pUC19 treated with [hosphatase (CIP)] to produce a novel plasmid pKP1 or pKP2, which was used to transform competent E. coli JM83 as a host microorganism to express phytinolytic enzymes. Converted. The novel strain obtained by the transformation in this manner is E. c
oli JM83 / pKP2 and deposited it with the Genetic Resource Center in the Institute of Biotechnology at the Korea Institute of Science and Technology, a depositary organization for patent strains in the Republic of Korea (January 28, 1997). 0308BP was given. Bacteriological, cultural and microbiological characteristics of the transformed new strain were examined and found to be the same as E. coli except for the ability to produce phytinolytic enzymes.

[Separation / Purification Method of Phytin-Degrading Enzyme Produced by the Transformed Novel Strain] The above-mentioned transformed novel strain E. coli JM83 / pKP2 is contained in 100 μg / ml of ampicillin. After culturing at 37 ° C in the obtained LB liquid medium, the microorganism was collected by centrifugation. The collected microorganism was washed with a phosphate buffer solution (phosphate phosphate) containing 10 mM EDTA.
buffer, 10 mM, pH 7.0).
450) for 1 hour to destroy microorganisms. Subsequently, the disrupted microorganism was centrifuged again, and the supernatant was used as a crude enzyme solution, and 50% acetone-saturated protein was purified by FPLC (fast protein liquid chromatography; phlc).
Nyl sepharose CL-4B open column and Resource S supe
rose 12HR 10/30 column).
Before manipulating the gene, the same phytinolytic enzyme produced by Bacillus sp. DS-11 could be isolated.

[Titration of phytinolytic enzyme produced by the transformed new strain] (1) Titer measurement of phytinolytic enzyme The above transformed novel strain E. coli JM83 / pKP2 was
LB (Lu) containing 100 μg / ml ampicillin
ria-Bertani) plates were cultured at 37 ° C. for 16 hours, and then transferred to a nylon membrane. After performing colony hybridization (colony hybridization) with the DNA probe synthesized from the above (3) to select colonies showing a signal, Bacillus ssp.
p.) DS-11 phytase gene (Phytase gene)
Fiske (F
iske) titer of phytinolytic enzyme was measured (Fisk
eC.H. and Subbarow YP, J. Biol. Chem. 1925, 66,
375). As a result, only transformed cells having full enzyme activity were selected.

(2) Comparison of activity, stability and molecular weight of phytinolytic enzyme to heat and pH The phytinolytic enzyme produced by the transformed new strain Escherichia coli JM83 / pKP2 was produced from the original parent strain. To confirm whether the enzyme is the same as phytinolytic enzyme, the activity and stability against pH were compared. First, in order to measure heat stability, the mixture was left at each temperature for 10 minutes in the same manner, and the remaining activity was measured.
As shown in FIG.3a, when calcium ion (Ca ²⁺ ) was not added, the activity decreased from 40 ° C., but when 5 mM calcium ion was added, the activity was stable up to 70 ° C. and 75 ° C. However, it maintained 50% activity.

In addition, the activity of phytinolytic enzyme is measured at various pH values.
As shown in FIG. 3b, the optimum pH of the two phytinolytic enzymes was 7.0. Also, pH
The phytinolytic enzyme was allowed to stand at various pHs for 1 hour in order to measure its stability against phytinolytic enzyme. As a result, it was confirmed that the enzyme had a considerable enzymatic activity even under acidic conditions of pH 4 or less. Thus, it is determined that the stomach is very stable even under acidic conditions. In addition, the molecular weights of the two phytinolytic enzymes were both 43,000 daltons. From the above results, it was determined that the phytinolytic enzyme produced by the transformed cells was the same as that of the parent strain (Bacillus sp. DS-11).

[Determination of DNA base sequence coding for phytinolytic enzyme] In order to determine the sequence of a 1.7 kb insert DNA (insert DNA) in pKP2, a cut fragment of any size was determined based on a restriction enzyme map. After obtaining a clone (deletion subclone), the entire nucleotide sequence of 1.7 kb DNA was obtained using a forward primer and a reverse primer. Using a program called MacMolly 3.5, search for the open reading frame (ORF) of phytinolytic enzyme consisting of 1149 nucleotides (383 amino acids),
(Bacillus sp.) Of phytinolytic enzyme isolated from DS-11
It was confirmed that the amino acids corresponded to the N-terminal amino acids (15) (Fig.
2). In addition, the above phytinolytic enzyme is a
It was considered to be a new phytinolytic enzyme derived from a microorganism having a nucleotide sequence completely different from that of phytinolytic enzyme produced by a strain of s sp. According to the amino acid sequence analysis, the sporulation regulating protein (sporulat) of Bacillus subtilis
It was found that 80% of the 175 amino acids from the C-terminal part were identical to the operon gene under the control of ion regulatory protein).

[0024]

According to the present invention, recombination for transformation is performed using the DNA base sequence and amino acid sequence determined by the above-described method.
By preparing a DNA expression vector, it can be used to transform phytinolytic enzymes having excellent activity and characteristics into other organisms having a high growth rate and easy management. This makes it possible to mass-produce phytinolytic enzymes that are economical.

[Sequence List] SEQUENCE LISTING <110> Korea Institute of Science and Technology; Daesung Microbiological Labs. Co., Ltd. <120> A strain E.coli JM83 / pKP2 transformed with a novel plasmid and phytase produced therefrom <130> X1H -1430 <160> 2 <210> 1 <211> 1152 <212> DNA <213> Bacillus sp. DS-11 <220> <221> CDS <222> 377 ... 1526 <221> sig peptide <222> 377 ... 466 <221> mat peptide <222> 467 ... 1526 <400> 1 atgaatcatt caaaaacact tttgttaacc gcggcagccg gattgatgct cacatgcggt 60 gcggtttctt ctcaggccaa acataagctg tctgatcctt atcattttac cgtgaatgcg 120 gcggcggaaa cggagccggt tgatacagcc ggtgatgcag ctgatgatcc tgcgatttgg 180 ctggacccca agaatcctca gaacagcaaa ttgatcacaa ccaataaaaa atcaggctta 240 gccgtgtaca gcctagaggg aaagatgctt cattcctatc ataccgggaa gctgaacaat 300 gttgatatcc gatatgattt tccgttgaac ggaaaaaaag tcgatattgc ggcggcatcc 360 aatcggtctg aaggaaagaa taccattgag atttacgcca ttgacgggaa aaacggcaca 420 ttacaaagca ttacggatcc aaaccgcccg attgcatcag caattgatga agtatacggt 480 ttcagcttgt accacagtca aaaaacagga aaat attacg cgatggtgac aggaaaagaa 540 ggcgaatttg aacaatacga attaaatgcg gataaaaatg gatacatatc cggcaaaaag 600 gtaagggcgt ttaaaatgaa ttctcagaca gaagggatgg cagcagacga tgaatacggc 660 agtctttata tcgcagaaga agatgaggcc atctggaagt tcagcgctga gccggacggc 720 ggcagtaacg gaacggttat cgatcgtgcc gatggcaggc atttaacccc tgatattgaa 780 ggactgacga tttactacgc tgctgacggg aaaggctatc tgcttgcctc aagccagggt 840 aacagcagct atgcgattta tgaaagacag ggacagaaca aatatgttgc ggactttcag 900 ataacagacg ggcctgaaac agacggcaca agcgatacag acggaattga cgttctgggt 960 ttcgggctgg ggcctgaata tccgttcggt ctttttgtcg cacaggacgg agagaatata 1020 gatcacggcc aaaaggccaa tcaaaatttt aaaatggtgc catgggaaag aatcgctgat 1080 aaaatcggct ttcacccgca ggtcaataaa caggtcgacc cgagaaaaat gaccgacaca 1140 agcggaaaat aa 1152 <210> 2 <211> 383 <212> PRT <213> Bacillus sp. DS-11 <400 > 2 Met Asn His Ser Lys Thr Leu Leu Leu Thr Ala Ala Ala Gly Leu Met 1 5 10 15 Leu Thr Cys Gly Ala Val Ser Ser Glu Ala Lys His Lys Leu Ser Asp 20 25 30 Pro Tyr His Phe Thr Val Asn Ala Ala A la Gln Thr Gln Pro Val Asp 35 40 45 Thr Ala Gly Asp Ala Ala Asp Asp Pro Ala Ile Trp Leu Asp Pro Lys 50 55 60 Asn Pro Glu Asn Ser Lys Leu Ile Thr Thr Asn Lys Lys Ser Gly Leu 65 70 75 80 Ala Val Tyr Ser Leu Gln Gly Lys Met Leu His Ser Tyr His Thr Gly 85 90 95 Lys Leu Asn Asn Val Asp Ile Arg Tyr Asp Phe Pro Leu Asn Gly Lys 100 105 110 Lys Val Asp Ile Ala Ala Ala Ser Asn Arg Ser Gln Gly Lys Asn Thr 115 120 125 Ile Gln Ile Tyr Ala Ile Asp Gly Lys Asn Gly Thr Leu Glu Ser Ile 130 135 140 Thr Asp Pro Asn Arg Pro Ile Ala Ser Ala Ile Asp Gln Val Tyr Gly 145 150 155 160 Phe Ser Leu Tyr His Ser Glu Lys Thr Gly Lys Tyr Tyr Ala Met Val 165 170 175 Thr Gly Lys Gln Gly Gln Phe Gln Glu Tyr Gln Leu Asn Ala Asp Lys 180 185 190 Asn Gly Tyr Ile Ser Gly Lys Lys Val Arg Ala Phe Lys Met Asn Ser 195 200 205 Glu Thr Gln Gly Met Ala Ala Asp Asp Gln Tyr Gly Ser Leu Tyr Ile 210 215 220 Ala Gln Gln Asp Gln Ala Ile Trp Lys Phe Ser Ala Gln Pro Asp Gly 225 230 235 240 Gly Ser Asn Gly Thr Val Ile Asp Arg Ala Asp Gly Ar g His Leu Thr 245 250 255 Pro Asp Ile Gln Gly Leu Thr Ile Tyr Tyr Ala Ala Asp Gly Lys Gly 260 265 270 Tyr Leu Leu Ala Ser Ser Glu Gly Asn Ser Ser Thr Ala Ile Tyr Gln 275 280 285 285 Arg Glu Gly Glu Asn Lys Tyr Val Ala Asp Phe Glu Ile Thr Asp Gly 290 295 300 Phe Gln Thr Asp Gly Thr Ser Asp Thr Asp Gly Ile Asp Val Leu Gly 305 310 315 320 Phe Gly Leu Gly Pro Gln Tyr Pro Phe Gly Leu Phe Val Ala Glu Asp 325 330 335 Gly Gln Asn Ile Asp His Gly Glu Lys Ala Asn Glu Asn Phe Lys Met 340 345 350 Val Pro Trp Gln Arg Ile Ala Asp Lys Ile Gly Phe His Pro Glu Val 355 360 365 Asn Lys Glu Val Asp Pro Arg Lys Met Thr Asp Arg Ser Gly Lys 370 375 380 383

[Brief description of the drawings]

FIG. 1a is a drawing showing the subcloning and restriction map of pKP1.

FIG. 1b is a drawing showing the subcloning and restriction map of pKP2.

FIG. 2 is a drawing showing a deduced nucleotide sequence of a phytinolytic enzyme (Phytase) DS-11;

FIG. 3a. Transformed strain E. coli JM83 / pK
3 is a drawing showing the relative activity of phytinolytic enzyme (Phytase) DS-11 produced by P2 to heat.

FIG. 3b. Transformed strain E. coli JM83 / pK
Phytase DS-11 produced by P2
3 is a drawing showing relative activity with respect to pH.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI theme coat ゛ (Reference) (C12N 1/21 (C12N 9/16 B C12R 1:19) C12R 1:19) (C12N 9/16 C12N 15/00 ZNAA C12R 1:19) C12R 1:07) (72) Inventor Kim Yeong-dong, Rongju 2-dong, Nam-gu, Busan, Republic of Korea (No address) Donggil Apartment 2 Building 903 No. 903 (72) Inventor Kim Toru Kim Gwang South Korea Daejeon Metropolitan City, Yuseok-gu, Uoseki-dong 99 Hanbit Apartment 123 Building No. 102, No. 72 (72) Inventor Park Katsuharu Korea South Korea Daejeon Metropolitan City, Yuseong-gu, Jwyan-dong (No address) Namsan Villa 2 Building 201 No. (72) Inventor Li Dongkei South Korea Seoul Seonghyeon-dong, Yongsan-gu, Seoul (No address) No. 206 New Dong-A Apartment No. 206 (72) Inventor Li Jing-ki South Korea Expo Apartment 407 Building No. 504 F-term (reference) 4B024 AA10 AA20 BA11 CA03 DA06 EA04 GA11 4B050 CC03 DD02 LL02 LL05 4B065 AA15Y AA26X AB01 AC14 BA02 CA31 CA43

Claims (4)

[Claims] 1. A plasmid having the DNA base sequence shown in SEQ ID NO: 1 in the sequence listing. 2. A strain of Escherichia coli JM83 / pKP2, which has been transformed with a plasmid having the DNA base sequence shown in SEQ ID NO: 1 in the sequence listing. 3. A phytinolytic enzyme produced by the strain Escherichia coli JM83 / pKP2. 4. The phytinolytic enzyme according to claim 3, which has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.