WO1998044125A1

WO1998044125A1 - A STRAIN E.COLI JM83/pKP2 TRANSFORMED WITH A NOVEL PLASMID AND PHYTASE PRODUCED THEREFROM

Info

Publication number: WO1998044125A1
Application number: PCT/KR1998/000056
Authority: WO
Inventors: Tae Kwang Oh; Young Ok Kim; Hyung Kwoun Kim; Seung Chun Park; Dong Kyoo Lee; Jung Kee Lee
Original assignee: Daesung Microbiological Labs Co Ltd; Korea Institute of Science and Technology KIST
Current assignee: Daesung Microbiological Labs Co Ltd; Korea Institute of Science and Technology KIST
Priority date: 1997-03-27
Filing date: 1998-03-21
Publication date: 1998-10-08
Anticipated expiration: 1999-09-27
Also published as: KR100206453B1; US6277623B1; ATE254175T1; DK1084257T3; DE69819789D1; DE69819789T2; KR19980074925A; EP1084257B1; EP1084257A1

Abstract

This invention relates to a novel strain E.coli JM83/pKP2 transformed by a novel plasmid and phytase produced therefrom, and more particularly, to the strain E.coli JM83/pKP2 transformed with a novel recombinant vector pKP1 or pKP2, so prepared by a gene manipulation, through elucidating the gene sequence intended for the mass production of a novel phytase serving the role to enhance the phosphorous bioavailability in grains used as livestock feeds.

Description

A STRAIN E.coli JM83/pKP2 TRANSFORMED WITH A NOVEL PLASMID AND PHYTASE PRODUCED THEREFROM

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to a strain E.coli JM83/pKP2 transformed with a novel plasmid and phytase produced therefrom and, more particularly, to a strain E.coli JM83/pKP2 transformed with a novel recombinant vector pKPl or pKP2, so prepared by a gene manipulation, through elucidating the gene sequence intended for the mass production of a novel phytase serving the role to enhance the phosphorous bioavailability in grains used as livestock feeds.

Description of the Prior Art

Phytase is an enzyme which degrades phytic acid into phosphate and phosphate inositol. 50 ~ 70% of phosphate in grain used as livestock feeds exists in the form of phytic acid, but phytase is not present in monogastric animals such as hens and hogs, thus resulting in low phosphate availability. Further, indigested phytic acid phytate released to a water source has become one of the serious environment contamination sources and causes eutrophication in small lakes or tides. Further, monogastric animals can not utilize phytic acid in their intestine due to its chelation with a trace amount of minerals, amino acids and vitamins which are essential for the metabolism of livestock. These formed water-insoluble and indigestible chelate- complexes released in the form of feces are responsible for the change of the environmental ecosystem, thus inducing a serious environmental contamination. - In view of these situations, the application of phytase into the livestock feeds can reduce the supply of inorganic phosphate due to an increase of phosphate bioavailabiltity in livestock, thus leading to economic benefits. In addition, the improved availability of phosphate and other bioactive substances may also contribute much to the reduction of the environmental contamination.

In particular, the utilization of phytase in livestock is very important in that the law regulating the amount of phosphate in animal waste was established in 1996 in Korea and, in addition to that, it has been mandatory to add phytase in the feeds of animals in the European countries. Further, when phytase is added to the feeds, it may greatly improve the productivity of livestock by enhancing the availability of some bioactive substances such as vitamins and amino acids, including some trace elements such as calcium and zinc ions whose activity is reduced by chelation with negatively charged phytate. As such, the use of feeds containing phytase in livestock can enhance the availability of feeds and reduce the environmental contamination caused by phosphate.

From the aforementioned benefits, the intensive studies with respect to phytase including the effects of phytase on animals (L.G. Young et al., 1993; X.G. Lei et al., 1994; Z. Morez et al., 1994) have been performed mainly in Europe (A.H.J. Ullah et al., 1994; K.C.Enrich, 1994; C.S. Piddington, 1993). However, since phytase can cleave a limited number of phosphate only and is mostly produced by molds which have slow growth rate, it is not economical in terms of mass production. In addition, it is difficult to use the phytic acid as an additive for monogastric animals since it is undesirable for their physiological characteristics. The inventor, et al. have performed intensive studies for overcoming the above problems associated with phytase. As a result, a novel strain Bacillus sp. DS-11 producing phytase with an excellent activity and different characteristics over the conventional phytase was identified and deposited to the Korean Collection for Type Cultures within the Korea Research Institute of Bioscience and Biotechnology affiliated with the Korea Institute of Science and Technology (KCTC 023 IBP), the Korean Patent Strain Depository Institute. The above patent application was filed with the

Korean Industrial Patent Office (The Korean Patent Appl. No.: 96-6817). Hence, various characteristics on a novel phytase produced from the microorganism were o

investigated and, as a result, the novel phytase proved to be excellent on heat and pH with better stability.

From the above results, the inventor et al. sequenced the DNA by cloning some phytase-coding gene in a strain Bacillus sp. DS-1 1 under the patent application so as to ensure the mass production of a novel phytase having the above excellent characteristics. As a result, the phytase-coding gene sequence of Bacillus sp. DS-

1 1 was recognized to be a novel one, being entirely different from that of Aspergillus awamori( 0 94-3072A), Aspergillus ficuum (EP 420358, US 5436156), Aspergillus niger(EP 420358) and Aspergillus terreus(EP 684313) among the genes cloned hitherto. Thus, its accessory No. U85968(dated January 21 , 1997) was given from GenBank of NCBI in the U.S.A.

Next, the inventor et al. transformed E.coli with the plasmid vector (pKPl or pKP2) encoding the phytase gene of Bacillus sp. DS-1 1 , and the transformed strain E.coli JM83/pKP2 was deposited at the Korean Collection for Type Cultures within the Korea Research Institute of Bioscience and Biotechnology affiliated with the Korea Institute of Science and Technology (KCTC 0308BP dated January 28, 1997), the Korean Patent Strain Depository Institute.

SUMMARY OF THE INVENTION Therefore, an object of this invention is to provide a plasmid vector pKPl and pKP2 for transformation intended for mass production of phytase, a transformed strain E.coli JM83/pKP2(KCTC 0308BP) herewith, and a process of mass production of phytase from said strain.

DESCRIPTION OF THE DRAWINGS

Fig. la shows the subcloning and mapping of pKPl by restriction enzyme; Fig. lb shows the subcloning and mapping of pKP 2 by restriction enzyme; Fig. 2 shows the base sequence and the estimated amino acid sequence of phytase DS-11 ;

Fig. 3a shows the relative activity of phytase DS-1 1, produced from a transformed strain E.coli JM83/pKP2, on heat;

Fig. 3b shows the relative activity of phytase DS-11 , produced from a transformed strain E. coli JM83/pKP2, on pH.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a novel phytase from Bacillus sp. DS1 1 and characterized by DNA base sequence of the sequence table 1 or amino acid sequence of the sequence table 2.

Also, this invention includes plasmid pKPl or pKP2 containing DNA of sequence table 1, which is ligated in such a manner and expressed in E.coli.

Further, this invention includes a novel strain E.coli JM83/pKP2 (KCTC 0308BP) transformed with plasmid pKPl or pKP2 containing the phytase-coding gene of the sequence table 1.

This invention is explained in more detail as set forth hereunder.

According to this invention, the phytase-coding gene obtained from Bacillus sp. DS-1 1 is inserted into a plasmid pUC19 vector to prepare a novel recombinant DNA expression vector pKPl or pKP2. After culturing E.coli JM-83 cloned by recombinant DNA expression vector, some colonies with effective expression potency are selected and then used for the mass production of phytase via cultivation of such colonies. Further, only pKPl or pKP2, the recombinant DNA expression vector, is isolated from the colonies to determine its DNA sequence.

This invention is explained in more detail by the following steps.

Preparation of Novel Plasmid pKPl and pKP2 (1) Sequencing of N-terminal amino acid

Purified phytase protein was applied to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane(Bio-Rad Lab). Then, the electroblotting was performed using 10 mM CAPS(3-cyclohexylamino-l- propanesulfonic acid) buffer solution containing 10 % methanol under pH 1 1.0, 4 ^°C and 400 raA for 45 hours. After cleaving the desired protein band only, it was analyzed by the Edman method using a protein/peptide sequencer [Applied Biosystems model 476 A Protein/Peptide Sequencer( Applied Biosystems Ins., CA, USA)]. N-terminal amino acid sequence of purified phytase protein:

Ser-Asp-Pro-Tyr-His-Phe-Thr-Val-Asn-Ala-Ala-X-Glu-Thr-Glu

(2) Amino acid sequencing of inner peptide Purified phytase protein was added to 70 % formic acid to 1 % (w/v) concentration, and with the addition of about 100-fold mass of CNBr, the mixture was reacted at room temperature for 24 hours. Then, 100-fold water was added to the reacting solution, and the reaction was discontinued. Using the same procedure as described in the above (1), electrophoresis was carried out to determine the amino acid sequence of inner peptide.

N-terminal amino acid sequence of internal protein fragments of phytase cleaved with CNBr;

Ala-X-Asp-Asp-Glu-Tyr-Gly-Ser-Ser-Leu-Tyr

(3) Preparation of oligonucleotide probe Oligonucleotide probe was designed based on the amino acid sequence obtained in the procedure as described in the above (1) and (2), and synthesized with

DNA synthesizer(Applied Biosystems ABI 380B).

With oligonucleotide, so synthesized by the above method as a primer and chromosomal DNA of DS-11 as template DNA as well as Taq DNA polymerase and dNTP in use, polymerase chain reaction(PCR) was carried out under the following conditions:

(2) Annealing: 50 ^°C for one minute (3) Polymerization: 72 ^°C for one minute

(4) Post-elongation: 72 ^°C for 7 minutes

Under the above conditions, the PCR was carried out and followed by 1.5 % agarose gel electrophoresis to obtain 600-bp PCR product. After recovering the PCR product from the gel, it was used as a probe.

Oligonucleotide probe based on 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 combination of codons: 5' GAT - CCT - TAT - CAT - TTT 3'

C C C C C

G A Oligonucleotide probe based on N-terminal amino acid sequence of internal protein fragments;

Amino acid sequence :

1 2 3 4 5 6 7 8 9 10 1 1 Ala-X-Asp-Asp-Glu-Tyr-Gly-Ser-Ser-Leu-Tyr

Possible combination of codons: 3' CTA - CTA - CTT - ATA - CCA 5' G G C G G

T C (4) Hybridization of DS-11 genomic DNA

Chromosomal DNA derived from Bacillus sp. DS-11 was isolated by the Marmur method(Marmur J. 1961, Mol Biol. 3, 208). To ascertain whether the oligonucleotide probe prepared by the above (3) was appropriate in the screening of genomic library, genomic DNA cleaved with several restriction enzymes was applied to agarose gel electrophoresis and then transferred to the nylon membrane. Then, with DIG DNA labeling and detection kit (Boehringer Mannheim, Germany)] as well as 600-bp DNA fragments as a probe, so synthesized from the above (3), southern hybridization was performed. As a result, it was confirmed that when HmdIII, Cla

I and Pstl were applied, the gene showed a positive signal at 2.2 kb, 4 kb and 6 kb, respectively. When the genomic library of Bacillus sp. DS-1 1 was prepared, therefore, restriction enzyme HmdIII was employed.

(5) Screening for the phytase-coding gene

Chromosomal DNA of Bacillus sp. DS-1 1 was cleaved with HmdIII and then, 3-5 kb DNA fragments were screened. Such DNA fragments were also cleaved with HmdIII, ligated to vector pUC19 treated with phosphatase (CIP) and introduced into the competent E.coli JM83. Such transformed strain was cultured in

LB(Luria-Bertani) plate containing 100 /zg/mf, of ampicillin at 37 ^°C for 16 hours and transferred to the nylon membrane. Further, the strain was under colony hybridization with DNA oligonucleotide probe, so synthesized from the above (3), to select some colonies representing the signal. In order to identify whether phytase gene of Bacillus sp. DS-11 was properly introduced into the host, the phytase activity was measured by the Fiske method (Fiske C. Η. and Subbarow Y. P., J.Biol. Chem. 1925, 66, 375). As a result, 2 colonies having the signal could be obtained among

10,000 colonies. They were cultured and then plasmids, 4.9-kb in size joined by 2.2-kb insert DNA, were isolated. And such plasmid was named as pKPl . In addition, it was ascertained that the pKPl contained phytase gene properly inserted through measuring the expression potency of phytase.

(6) Mapping and subcloning using a restriction enzyme

As a result of cleaving 4.9-kb pKPl with several restriction enzymes, it was confirmed to be some restriction sites of Ec RI, BamHl, Ndel, Hindi and EcoRV within 2.2-kb insert DNA. To find out the genes only necessary for the expression of enzyme potency, the subcloning of the pKPl plasmid was carried out (Fig. la). pKPl and pUC19 were cleaved with HmdIII and Ndel, respectively, joined each other. Such plamid vector was introduced into E.coli JM83 so that E.coli JM83 with 4.4-kb pKP2 containing 1.7kb-insert DNA might be obtained(Fig. lb).

Transformation Process of Strain Chromosomal DNA of Bacillus sp. DS-11 was cleaved with HmdIII and then,

3-5 kb DNA fragments was selected. Such DNA fragments were also cleaved with

HmdIII, ligated to vector pUC19 treated with phosphatase (CIP) to obtain a novel plasmid pKPl or pKP2. To express such plasmid into phytase, it was introduced into the competent E.coli JM83 as a host. Thus, the transformed strain, was named as E.coli JM83/pKP2 and deposited to the Korean Collection for Type Cultures within the Korea Research Institute of Bioscience and Biotechnology affiliated to the Korea Institute of Science and Technology dated January 28, 1997 (the accession No.: KCTC 0308BP).

The bacteriological, cultural and microbiological characteristics of the transformed strain were studied, and all results were the same as that of E.coli except for the production capability of phytase.

Isolation and Purification of Phytase Produced from the Transformed Strain

The novel strain E.coli JM83/pKP2, so transformed, was cultured in LB liquid medium containing 100 μglml of ampicillin at 37 ^°C , centrifuged and recovered. The recovered microorganism was dissolved in the Tris buffer solution (10 mM, pΗ 7.0) containing 5 mM CaCl2 and sonicated for 1 hour using Sonifier 450. Then, the sonicated microorganism was re-centrifuged, and its supernatants were used as crude enzyme solution. The protein saturated with 50 % acetone was isolated- on Fast Protein Liquid Chromatography (FPLC consisting of open column of phenyl sepharose CL-4B and Resource S superose 12ΗR 10/30 column), the same enzyme as phytase produced from Bacillus sp. DS-11 prior to gene manipulation could be isolated. Measurement of Phytase Potency Produced from the Transformed Strain]

(1) Measurement of phytase potency

The novel strain E.coli JM83/pKP2, so transformed, was cultured in LB agar (Luria-Bertani) plate containing 100 βg wi of ampicillin at 37^°C for 16 hours and transferred to the nylon membrane. The strain was applied to colony hybridization with DNA oligonucleotide probe, so synthesized in the above (3), so as to examine the colonies representing the signal. To ascertain whether phytase-coding gene of

Bacillus sp. DS-11 was properly introduced into E.coli JM83, the phytase potency was measured by the Fiske method(Fiske C. H. and Subbarow Y. P., J.Biol. Chem. 1925, 66, 375). As a result, the transformed strains having complete enzymatic activity were selected.

(2) Comparison on activity and stability of phytase on heat and pH including its molecular weight

To ascertain whether phytase produced from the transformed strain E.coli JM83/pKP2 was the same as that phytase produced from the original strain, the activity and stability on heat and pH of phytase were compared. To measure its stability on heat, each phytase was left at predetermined temperature for 10 minutes in the same method and then its residual activity measured. As shown in Fig. 3a. when calcium ion (Ca 2+ ) was not added into the phytase-containing solution, the activity of phytase began to reduce at 40 ^°C , while in case of adding 5 mM calcium ion, it was stabilized up to 70 ^°C and its activity was maintained by 50% even at 90 ^°C .

Also, Fig. 3b shows the phytase activity depends on pH and the optimum pH of both phytases is 7.0. Further, to identify its stability on pH, each phytase was left at different values of pH for 1 hour and followed to measure its residual activity, respectively. Even at acidic condition of less than pH 4, both phytases showed significant enzymatic activity and thus, it was considered that they may be stabilized in the stomach.

Besides, both phytases have the same molecular weight of 43,000 Dalton. From the above results, it was considered that phytase produced from the transformed strains was the same as one produced from the original one {Bacillus sp. DS-11).

DNA Sequencing of Phytase-coding Gene

To sequencing 1.7-kb insert DNA within pKP2, after deletion subclones in several different sizes were obtained based on restriction site. The DNA fragment of the total 1.7-kb was prepared from them with PCR using forward and reverse primers. And then, the open reading frame (ORF) of phytase consisting of 1 149 nucleotides (383 amino acids) was sequenced using MacMolly 3.5 program and as a result, it was ascertained that the above phytase coincided with N-terminal amino acid of phytase (15 amino acids) isolated from Bacillus sp. DS-11 strain (Fig. 2). Further, it was considered that this was a novel phytase, being entirely different from that produced from the conventional Aspergillus sp. strains. As a result of analyzing its amino acid sequence, 80%o between 175 amino acids of C-terminal of this invention and gene of operon regulated by the Sporulation Regulatory Protein of Bacillus subtilis was coincided.

Sequence Table 1

Sequence length : 1 149 Type of sequence: Nucleic acid Number of chain: Double helix Shape : Linear

Sequence type : Genomic DNA Origin :

Name of species : Bacillus sp.

Name of strain: DS-1 1 Features of sequence:

Signal representing the features : CDS

Location of presence: 377..1526

Method to determine the features : E

Signal representing the features : sig peptide

Location of presence: 377..466 Method to determine the characteristics : E

Signal representing the characteristics : mat peptide Location of presence: 467..1526

Method to determine the characteristics: E

Sequence 1

10 20 30 40 50 60

ATGλλTCλTT CλλλλλCλCT TTTGTTλλCC GCβGCλGCCG GATTGATGCT CACλTGCGGT GCGGTT CTT CTCλGGCCλλ ACλ λAGCTβ TCTQλTCCTT ATCATTTTAC CGTOλΛTGCβ GCGGCββλλλ CGGΛGCCGGT TQATACAGCC GβTQATOCAβ CTGATOATCC TGCGATTTGG CTGGλCCCCλ Aβλλ CCTCλ GλλCAGCλλλ TTGλTCλCλλ CCλλTλλλλλ ATCAGGCTTA GCCGTG ACλ GCCXλOλOGO AAAOATSCTT CATTCCTATC ATACCGGGAA βCTOλACAAT GTTOATATCC GATATGATTT TCCσTTGAλC GGλλλλλλλG TCGATλT OC GGCGGCATCC AATCGGTCTG AAGGAAAGAA TACCATTQAG ATTTACGCCA TTGλCGGβλλ AλλCGGCACλ TTΛCλλλGCλ TTλCGGλTCC λλλCCGCCCG λTTGCλTCλG CλλTTGλTGλ AGTATΛCGGT TTCAGCTTGT ACCλCλGTCA AλλλλCλGβλ AAATATTACQ CGATGGTGAC λGGλλλλGAλ GGCGλλTTTG AACλλTλCGλ λTTλλλTGCG GA AAAAA G GATACATATC CGGCλλλλλΘ GTAAGGGCGT TTAAAATGAA TTCTCAGACA QAAGGGATGG CAGCAGACGA TGAATACGGC AGTCTT AXA TCGCAGAAGA AGATGAGGCC Λ CTGGλλGT TCλβCGCTGλ GCCGGλCGGC GGCAGTAACG GAACGGTTAT CGATCGTGCC GλTGGCλGGC λTTTλλCCCC TGΛTATTOAA GGACTGACGA TTTACTACGC TGCTGACGGG AAAGGCTATC TGCTTGCCTC λAGCCλGGGT AλCAGCλβCT ATGCGATTTλ TGAAAGACAG GGACAGAACA AATATGTTT GGACTTTCAG ATλλCAGACG GGCCTGλλλC AGACGGCACA AGCGATACAG ACGGAATTGA CG TCTGGGT TTCGGGCTGG GGCCTGΛλTλ TCCGTTCGGT CTTTTTGTCG CλCλGGλCGG AGAGAATATA GATCACGGCC λλλλGGCCλλ TCΛλλλTTTT AAAATGGTGC CλTGGαλAAG AATCGCTGAT AλλATCGGCT TTCλCCCGC GGTCAATλλλ CAGGTCGACC CGAGAAAAAT GλCCGλCλCλ AGCGGλλλAT AA

Sequence Table 2

Sequencing length : 383 Sequencing form : amino acid Shape : Linear Sequence type : protein

Sequence

1 2 3 4 5 6 7 8 9 10

Met Asn His Ser Lys Thr Leu Leu Leu Thr

Ala Ala Ala Gly Leu Met Leu Thr Cys Gly

Ala Val Ser Ser Glu Ala Lys His Lys Leu

Ser Asp Pro Tyr His Phe Thr Val Asn Ala

Ala Ala Gin Thr Gin Pro Val Asp Thr Ala 5

Gly Asp Ala Ala Asp Asp Pro Ala lie Trp

Leu Asp Pro Lys Asn Pro Glu Asn Ser Lys

Leu He Thr Thr Asn Lys Lys Ser Gly Leu

Ala Val Tyr Ser Leu Gin Gly Lys Met Leu

His Ser Tyr His Thr Gly Lys Leu Asn Asn 10

Val Asp lie Arg Tyr Asp Phe Pro Leu Asn

Gly Lys Lys Val Asp De Ala Ala Ala Ser

Asn Arg Ser Gin Gly Lys Asn Thr lie Gin

He Tyr Ala He Asp Gly Lys Asn Gly Thr

Leu Glu Ser lie Thr Asp Pro Asn Arg Pro 15

He Ala Ser Ala Ue Asp Gin Val Tyr Gly

Phe Ser Leu Tyr His Ser Glu Lys Thr Gly

Lys Tyr Try Ala Met Val Thr Gly Lys Gin

Gly Gin Phe Gin Glu Tyr Gin Leu Asn Ala

Asp Lys Asn Gly Tyr De Ser Gly Lys Lys 20

Val Arg Ala Phe Lys Met Asn Ser Glu Thr

Gin Gly Met Ala Ala Asp Asp Gin Tyr Gly

Ser Leu Tyr He Ala Gin Gin Asp Gin Ala lie Trp Lys Phe Ser Ala Gin Pro Asp Gly

Gly Ser Asn Gly Thr Val He Asp Arg Ala 25

Asp Gly Arg His Leu Thr Pro Asp He Gin

Gly Leu Thr De Tyr Tyr Ala Ala Asp Gly

Lys Gly Tyr Leu Leu Ala Ser Ser Glu Gly

Asn Ser Ser Tyr Ala ue Tyr Gin Arg Glu

Gly Glu Asn Lys Tyr Val Ala Asp Phe Glu 30 lie Thr Asp Gly Phe Gin Thr Asp Gly Thr

Ser Asp Thr Asp Gly He Asp Val Leu Gly

Phe Gly Leu Gly Pro Gin Tyr Pro Phe Gly

Leu Phe Val Ala Glu Asp Gly Gin Asn lie

Asp His Gly Glu Lys Ala Asn Glu Asn Phe 35

Lys Met Val Pro Trp Gin Arg lie Ala Asp

Lys He Gly Phe His Pro Glu Val Asn Lys

Glu Val Asp Pro Arg Lys Met Thr Asp Arg

Ser Gly Lys This invention has the advantages of economy with respect to the preparation of phytase in a large-scale since a recombinant DNA expression vector is prepared using the sequences of DNA and amino acid in such a manner as elucidated in the above and may be introduced into other living organisms having a rapid growth rate and easily regulatable to produce phytase having excellent activity and characteristics.

Claims

CLAIMSWhat is claimed is :

1. A plasmid having the DNA sequence of sequence table 1

2. E.coli JM83/pKP2 transformed with the plasmid having the DNA sequence of sequence table 1.

3. Phytase produced from the strain E. coli JM83/pKP2.

4. Phytase according to claim 3, wherein said phytase is the amino acid sequence of sequence table 2.