KR20040003100A - Amino acid sequences of phytase from Pseudomonas syringae and gene sequences of it thereof - Google Patents

Abstract

PURPOSE: Amino acid sequences of phytase derived from Pseudomonas syringae MOK1 and nucleotide sequences encoding the same are provided, thereby hydrolyzing phytate in plant fodder to separate inorganic phosphate, improving the use of phosphate, and decreasing the amount of phosphate in the feces of domestic animals in order to minimize environmental pollution. CONSTITUTION: Phytase derived from Pseudomonas syringae MOK1 (KFCC-11192) has the amino acid sequence of SEQ ID NO: 2. A gene encoding the phytase derived from Pseudomonas syringae MOK1 (KFCC-11192) has the nucleotide sequence of SEQ ID NO: 1. A method for sequencing the amino acid sequence of the phytase derived from Pseudomonas syringae MOK1 (KFCC-11192) comprises the steps of: extracting and purifying total DNA from Pseudomonas syringae MOK1 (KFCC-11192); analyzing the internal amino acid sequence from the extracted and purified MOK1 phytase; constructing degenerate primers A and B using the N-terminal and internal amino acid sequence of the MOK1 phytase; carrying out PCR using the degenerate primers A and B to obtain a DNA fragment having 691 kb; cloning the 691 kb DNA fragment to pGEM-T easy vector and sequencing it; constructing inverse PCR primers A and B; and carrying out inverse PCR using the inverse PCR primers A and B to obtain the total amino acid sequence of the MOK1.

Description

Amino acid sequences of phytase from phytase from Pseudomonas syringae and gene sequences of it interacting with Pseudomonas syringae (Pseudomonas syringae and gene sequences of it)

The present invention relates to an enzyme that hydrolyzes an organic protein and releases it into an inorganic protein, and more particularly, a novel phytase derived from Pseudomonas syringae MOK1 (KFCC-11192). And genes encoding the same.

Phytate (myo-inositol hexakisphosphate) is the main phosphate storage compound present in the seeds of higher vegetation such as cereals or legumes used as feedstock for livestock. It exists in the form of organic phosphate, which is a combination of myo-inositol and phosphate, and the use of phosphorus from this compound is very important for livestock (Lolas, GM and Markakis, P. (1997) Phytase of navy beans.J. Food Sci 42, 1094-1097). On the other hand, phytase is an enzyme that hydrolyzes phytate, which is an organic phosphate, and finally releases myo-inositol and inorganic phosphate. do. However, the digestive tract of monogastric animals such as pigs and chickens lacks the activity of phytase, which degrades the phosphate form of phosphorus present in plant feeds, which not only reduces the availability of phosphorus but also degrades and uses it. Untreated phytates are excreted as livestock meals in densely populated livestock farms and flow into sewage, causing a rapid increase in phosphorus concentrations, causing environmental pollution problems such as eutrophication. It also acts as an antinutritional factor that degrades their digestibility by combining proteins with metal ions such as Ca, Zn and Fe that are nutritionally important (Sharma, CB, Goel, M and Irshad, M (1978) Myo -inositol hexakisphosphate as a potential inhibitor of α-amylase of different origins.Phytochemistry 17, 201-204 .; Graf, E (Ed.) (19 86) Phytic acid.Chemistry and Applications.Pillsbury, Pilatus Press, Minneapolis, MN.). Therefore, the addition of phytase in plant feed will ultimately maximize the utilization of phosphorus from phytate, resulting in improved productivity of livestock, as well as reducing the amount of phosphorus excreted in flour, thereby minimizing environmental pollution and further improving plant feed quality. have.

Strains with phytase activity in microorganisms are mostly A. terreus (Yamada, K., Minoda, Y and Yamamoto, S (1986) Phytase from Aspergillus terreus.Part I. Production, purification and some general prorperties of the enzyme.Agric. Chem. 32, 1275-1282), A. ficuum (Gibson, D (1987) Production of extracellular phytase from Aspergillus ficuum on starch media.Biotechnol. Lett. 9, 305-310.), A. niger (Shie, TR and Ware, JH (1968) Survey of microorganisms for the production of extracellular phytase.Appl. Microbiol. 6 1348-1351.) Fungi and Escherichia coli (Greiner, R) from Aspergillus sp. , Konietzny, U and Jany, KID (1993) Purification and characterization of two phytase from Escherichia coli.Arch.Biochem.Biophys.303, 107-113), Bacillus subtilis (Powar, VK and Jagannathan, V. (1982) Purification and properties of phytate-specific phosphatase from Bacillus subtilis.J. Bacteriol. 151, 1102-1108. In particular, studies are being actively conducted to clone phytase genes from these strains by biotechnology to analyze sequencing, characterize the genes, and mass-produce phytase from industrially useful foreign hosts. , Already most A. niger (Ehrlich, KC, Montalbano, BG, Mullaney, EJ, Dishinger, HC and Ullah, AHJ (1993) Identification and cloning of a second phytase gene (phy B) from Aspergillus niger. Bichem. Biophys. Res. Commun. 195 53-57.), A. fumigatus (Passamontes, L., M. Haiker, M. Wyss, and M. Tessier. 1997. Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus . ... appl Environ Microbiol 63: .. 1696~1700), A. terrus, Myceliophthora thermophila (Mitchell, DB, K. Vogel, B. Weimann, L. Pasamontes, and APGM van Loon 1997. The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila Microbiology 143:... 245~252) There have been many reports on the cloning and identification of phytase gene sequences derived from fungal strains In bacteria, only two strains of the genus Bacillus sp. And E. coli are present.

In the present invention, a novel phytase gene was cloned from the bacterial Pseudomonas syringae MOK1 strain (KFCC-11192) and its nucleotide sequence was identified. Mass expression was intended to be used as feed additives for livestock.

Therefore, an object of the present invention is to identify the phytase gene sequence derived from Pseudomonas syringae MOK1 (KFCC-11192) strain.

In addition, another object of the present invention is to identify the amino acid sequence of the strain-derived phytase.

The above object of the present invention is to extract and purify total gene DNA from Pseudomonas syringae MOK1 (KFCC-11192) strain, and after analyzing the amino acid sequence, degenerate primers using the determined amino acid sequence primers were prepared and PCR reactions were performed, followed by cloning of the complete MOK1 phytase gene using Inverse PCR technique to reveal the sequence.

1 is a photograph showing electrophoresis of the result of PCR amplification using degenerate primers.

The present invention comprises the steps of extracting and purifying the phytase total gene DNA from Pseudomonas syringae MOK1 (KFCC-11192) strain; Analyzing an internal amino acid sequence from the extracted and purified MOK1 phytase; Based on the N-terminal and internal amino acid sequences of the extracted and purified MOK1 phytase, a degenerate primer A.B was prepared, and a 691bp DNA fragment amplified by performing a PCR reaction was pGEM-T easy. performing TA cloning and sequencing by vector; Complete MOK1 phytase gene cloning and sequencing using the Inverse PCR technique.

Hereinafter, the present invention will be described in detail.

The phytase producing strain of the present invention uses Pseudomonas syringae MOK1 strain (KFCC-11192) deposited in the Korean spawn association, and the transformed strain according to gene manipulation is Escherichia coli (E). coli) using the TOP 10F 'strain. Gene cloning and sequencing vectors use the plasmid pGEM-T easy vector (promega). DNA sequencing is analyzed using an ABI 377 automated DNA sequencer using a M13 forward / reverse primer and an internal synthetic primer as a Big dye terminator cycle reaction kit (Perkin Elmer Co., USA).

On the other hand, extraction of total gene DNA from Pseudomonas syringae MOK1 (KFCC-11192) strain was performed by Marmur, J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms J. Mol. Biol. 3 208-218).

In order to analyze the internal amino acid sequence from the purified MOK1 phytase, we cut the purified MOK1 phytase enzyme into small peptide fragments, and as a result, obtained the amino acid sequence of Gln-Gln-Val-Ser-Gln-Leu.

Based on the N-terminal and internal amino acid sequences of the purified MOK1 phytase, the following degenerate primers A. B are prepared, respectively.

Primer A: 5'-GC (C / G) GA (C / T) GG (C / T) TA (C / T) GT (C / G) TT (A / G) GA-3 '

(20 mer)

Primer B: 3'-GT (C / T) GT (CT) CA (C / G) AG (C / G) GT (C / T) AA (C / T) GT-5 '

(20 mer)

The primers A and B were then subjected to the following PCR reaction to obtain amplified 691 bp DNA fragment, which was then TA cloned into the pGEM-T easy vector and sequenced. .

Since the MOK1 phytase isolated and purified above was about 45000 Da, the 691 bp DNA fragment sequenced did not include the full-length MOK1 phytase gene sequence. Therefore, the inverse PCR primers A and B were respectively determined as follows. To make.

Inverse primer A (24 mer)

5'-AAGGCTTGCCGCTGGACCAGGTTG-3 '

Inverse primer B (24 mer)

5'-CGACCGGTGACCTTGGCCAGTTTG-3 '

Thereafter, the total gene DNA was cleaved with a restriction enzyme not included in the already known 691 bp DNA sequence, followed by self-ligation using a ligase. PCR is performed as a template and the amplified bands are sequenced to obtain the nucleotide sequence of the complete MOK1 phytase gene as shown in SEQ ID NO: 1 in the Sequence Listing.

Example 1 Gene Isolation of Pythaase Enzyme Derived from Pseudomonas syringae MOK1 (KFCC-11192)

The phytase-producing strain of the present invention was used with the Pseudomonas syringae MOK1 strain (KFCC-11192), which was deposited from the Korean spawn association, and the transformed strain according to gene manipulation was Escherichia coli ( E. coli) TOP 10F 'strain was used. As a vector for gene cloning and sequencing, a plasmid pGEM-T easy vector (promega) was used. DNA sequencing was analyzed using an ABI 377 automated DNA sequencer using a M13 forward / reverse primer and an internal synthetic primer as a Big dye terminator cycle reaction kit (Perkin Elmer Co., USA).

On the other hand, extraction of total gene DNA from Pseudomonas syringae MOK1 (KFCC-11192) strain was performed by Marmur, J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms J. Mol. Biol. 3 208-218).

The purified MOK1 phytase enzyme, 400 pmole, was subjected to electrophoresis on a 10% (w / v) SDS-polyacryl amide gel to analyze internal amino acid sequences from the purified MOK1 phytase, followed by the separation of phytase bands. The cut portions were cut with a knife, transferred to a 1.5 mL eppendorf tube, and treated with protease trypsin to cut MOK1 phytase into small peptide fragments. The cleaved fragments were separated by reverse-phase HPLC, and the amino acid sequence of the separated fragments was analyzed by automatic proteinsequencer (Prosise 491, ABI), and the amino acid sequence of the cleaved fragment was Gln-Gln-Val-Ser-Gln- Appeared as Leu.

Based on the N-terminal and internal amino acid sequences of the purified MOK1 phytase, the following degenerate primers A and B were prepared, respectively.

Primer A: 5'-GC (C / G) GA (C / T) GG (C / T) TA (C / T) GT (C / G) TT (A / G) GA-3 '

(20 mer)

Primer B: 3'-GT (C / T) GT (CT) CA (C / G) AG (C / G) GT (C / T) AA (C / T) GT-5 '

(20 mer)

The following PCR reactions were carried out with primers A and B. The concentration of each primer was 100 pmole, and the total gene DNA of Pseudomonas syringae MOK1 (KFCC-11192) strain to be used as a template was 90ng. The reaction was carried out in a total volume of 50 μl, and PCR was performed under conditions of denaturation at 94 ° C. for 20 seconds, primer annealing at 60 ° C. for 30 seconds, and elongation at 72 ° C. for 30 seconds in a total of 35 cycles. The reaction was carried out. As a result, a specific amplified 691 bp DNA fragment was obtained (FIG. 1). This amplified 691bp DNA fragment was TA cloned into pGEM-T easy vector and sequencing was performed.

Since the MOK1 phytase isolated and purified above was about 45000 Da, the 691 bpDNA fragment sequenced did not include the full-length MOK1 phytase gene sequence. Thus, the inverse PCR primers A and B were respectively determined as follows. Produced.

Inverse primer A (24 mer)

5'-AAGGCTTGCCGCTGGACCAGGTTG-3 '

Inverse primer B (24 mer)

5'-CGACCGGTGACCTTGGCCAGTTTG-3 '

Thereafter, the total gene DNA was cleaved with a restriction enzyme not included in the already known 691 bp DNA sequence, followed by self-ligation using a ligase. As a template, denatured at 94 ° C. for 20 sec at a total of 50 μl in volume, primer annealing at 65 ° C. for 30 sec, and elongation condition at 72 ° C. for 1 min 30 sec. PCR and sequencing the amplified bands were able to identify the nucleotide sequence of the complete MOK1 phytase gene (SEQ ID NO: 1).

The phytase expressed from the Pseudomonas syringae MOK1 (KFCC-11192) strain derived from the phytase coding gene derived from the strain of the present invention hydrolyzes the phytate of plant feed, thereby releasing inorganic phosphorus, thereby increasing the utilization efficiency of livestock It is a very useful invention for the feed industry because it not only improves the productivity of the animal but also reduces the amount of phosphorus excreted in livestock, thereby minimizing environmental pollution and further improving the quality of plant feed.

Claims (2)

A phytase having the amino acid sequence set forth in SEQ ID NO: 2 of the Sequence Listing derived from Pseudomonas syringae MOK 1 (KFCC-11192) strain. The gene base sequence set forth in SEQ ID NO: 1 of the Sequence Listing encoding the amino acid sequence of claim 1.