KR100581318B1 - A novel phytase gene derived from Penicillium oxalicum and a Pichia pastoris transformant producing the phytase - Google Patents

Abstract

The present invention is a method for searching for enzyme genes from various fungi using degenerate primers and polymerase chain reaction (PCR) methods and the phytze gene of Penicillium oxalicum KCTC 6440 obtained using the above method. And it relates to a Peachia pastoris strain transformed with a recombinant vector comprising the gene and a recombinant phytase enzyme produced by the strain.

The transformed strain of the present invention and the phytease enzyme produced therefrom exhibit very high titers, thus providing a useful value as a feed additive.

Fightz, phytase, penicillium oxalicum, pichia pastoris GS115

Description

A novel phytase gene derived from Penicillium oxalicum and a Pichia pastoris transformant producing the phytase}

Figure 1 is an electrophoresis picture of performing a touchdown PCR with a degenerate primer (Py-1 / Py-4).

Figure 2 is agarose gel electrophoresis picture after inverse PCR of the POX gene.

Figure 3 is a genomic DNA sequence of the penicillium oxalicum Fightes gene (POX).

4 is a schematic diagram of the phytez expression vector pPICZαA-POX.

FIG. 5 is the amino acid sequence of recombinant phytez expressed in Pchia pastoris GS115 / pPICZαA-POX.

FIG. 6A shows the production of recombinant phytes via fed-batch fermentation in transformed Peachia Storris GS115 / pPICZαA-POX.

FIG. 6B shows the production of recombinant phytes through fed-batch fermentation in Pchia Pastoris KM71 / pPICZαA-POX.

Figure 7 shows the time-phase expression of protein expression of the fed-batch fermentation of recombinant phytze produced in transformed Peachia pastoris GS115 / pPICZαA-POX.

FIG. 8A shows the optimal pH of the recombinant phytez produced in the transformed Peachia Pastoris GS115 / pPICZαA-POX.

FIG. 8B shows the optimal temperature of recombinant phytez produced in the transformed Peachia Pastoris GS115 / pPICZαA-POX.

The present invention is a method for searching for enzyme genes from various fungi using degenerate primers and polymerase chain reaction (PCR) methods and the phytze gene of Penicillium oxalicum KCTC 6440 obtained using the above method. And it relates to a Peachia pastoris strain transformed with a recombinant vector comprising the gene and a recombinant phytase enzyme produced by the strain.

The technical field to be solved by the present invention is to develop a technique for screening enzymes, especially industrial enzymes, digestive enzymes that can be used in livestock, in particular, phytze enzymes with novel fungi from various molds. It is to develop a production system to secure them and produce them in a form that can be used for livestock raising.

The main storage source of cereals (Pi), which is a major nutrient for livestock feed, is phytic acid, which binds to various minerals (copper, zinc, magnesium, iron and calcium) or forms a protein-phytic acid-mineral complex. It exists in tin form.

In the case of a unit animal, since there is no phytase enzyme that breaks down it, it is impossible to use the phytintaine present in grain feed, so additional supplementation of phosphorus is inevitable and added in the form of inorganic phosphorus such as calcium phosphate (Dicalcium phosphate). It raises feed costs and combines with various proteins, amino acids and essential minerals to act as anti-nutrient factors, reducing digestibility of feed and providing environmental pollution due to phosphorus excretion. To partially solve this problem, microbial-derived phytase enzymes are added to the feed. Several enzyme-producing companies produce and supply fungi-derived phytes, which dominate the market and many laboratories and companies are working to find new and better enzymes. More than 15 fungal phytoses are currently registered in the Gene Bank and will continue to grow. AJ310697, AJ310698, AJ310699, AJ310700].

Enzyme screening method including the conventional phytze is obtained by measuring the enzyme activity after culturing in a selection medium that can determine the enzyme activity by separating the microorganisms and by analyzing some amino acid sequence of the protein to obtain a difference from the existing or strains with high enzyme expression Cloning the gene from the gene is time consuming, and when the gene is separated and analyzed for sequencing or amino acid sequence and compared with the data of the gene bank, it is often difficult to be recognized for its novelty due to its high homology with the existing one. .

In order to solve the problems described above, the present inventors have attempted to develop a method for easily screening an enzyme, in particular, a Pitez enzyme, in which novelty is recognized, by combining various known techniques. The high enzyme activity was screened from various fungal species purchased from strain banks to provide an excellent phytase enzyme.

Accordingly, an object of the present invention is to provide a method for obtaining a high activity of the phytase enzyme gene from an unspecified number of fungal strains and a method for mass-producing a selected gene in Peachia yeast.

The technical problem of the present invention as described above, using the degenerate primer and PCR (polymerase chain reaction) method to search for enzyme genes from a variety of fungi, Penicillium oxalicum KCTC using the method 6440) of the pie Handcrafted gene (POX) screening, and transformed with a recombinant vector containing the gene blood Chiapas Laboratories for GS115 / pPICZαA-POX (Pichia pastoris GS115 / pPICZαA-POX) obtaining KCTC 10386BP strain, and the Achievement was achieved by producing a recombinant Pytease enzyme from the strain.

The present invention relates to a method for searching for enzyme genes from various molds by using a degenerate primer and a polymerase chain reaction (PCR) method, and the phytze oil of Penicillium oxalicum KCTC 6440. The present invention relates to a Peachia pastoris strain transformed with a recombinant vector comprising the former and the genes, and to a recombinant phytase enzyme produced by the strain.

The present invention obtains genomic DNA from various fungi and prepares degenerate primers by selecting regions having high homology from the base sequences of existing fungi derived phytase as templates. step; Selecting a gene different from the existing Pitez by obtaining a gene fragment through PCR (PCR) using the primer and comparing the nucleotide sequence with a nucleotide sequence registered in a Gene Bank; Obtaining the nucleotide sequence of the entire phytez gene from the novel phytez gene fragment obtained above by using an Inverse-PCR method; Conjugating the enzyme gene to a Peachia expression vector and transforming the Pychia bacteria to express Paetez; Selecting a POX gene derived from penicillium oxalicum KCTC 6440 strain among several tetez genes by selecting a gene having a high titer by identifying the characteristics of the expressed tetez; Preparing a recombinant vector pPICZα A-POX comprising the novel Paitez gene (POX) derived from the penicillium oxalicum; Transforming the Peachia Pastoris GS115 or KM71 strain with the vector, and finally selecting the Peachia Pastoris GS115 / pPICZαA-POX strain as a strain having excellent Pitez expression ability; And expressing Fightes in the strain.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 method for securing novel fungal phytez gene using PCR technique

First Step: Searching for the Paetez Gene by Degenerate PCR

Multiple alignment of known fungi-derived Fetez gene sequences was used to prepare oligonucleotide primer pairs as shown in Table 1 using nucleotide sequences of high homology, and a plurality of fungal genomes (genome) ) Was subjected to PCR. The Paetez gene used for multiple alignment was selected to have a homology of less than 90%, except for overlapping with known genes. GeneBank Accession Numbers: L02421 ( Aspergillus niger awamori ), M94550 (A. niger), AB042805 (A. oryzae), U59804 (A. fumigatus), U59805 (A. terreus), U59802 (Talaromyces thermophilus), U59803 (Emericella nidulans)].

Degenerate Primers Used to Detect Moldy Genes Sequence Forward primer, Py-1, SEQ ID NO: 1 5'-TGGGGHCAVTAYKCGCCNTWCTTYTC-3 '(26 mer) Reverse primer, Py-4, SEQ ID 2 5'-GTTDCCBSCRCCRTRGCCGTAGTAYTT-3 '(27 mer) Note) Mixed base code: B (G, T, C); D (G, A, T); H (A, T, C); K (G, T); N (A, C, G, T); R (A, G); S (G, C); Y (C, T); V (A, C, G); W (A, T)

Fungal cells incubated in PD (Potato dextrose) medium for 3 days were recovered, washed and lyophilized using MiraCloth, and then crushed with Zircornium bead and treated with CTAB buffer. After determining the amount of purified fungal genome, an appropriate amount (50 ng) was used for the PCR reaction. PCR was performed under the following conditions by a touch-down method to maximize the selection potential.

I. deaturation at 95 ° C. for 2 minutes; II. 8 cycles (annealing temperature lowered by 2 ° C per cycle)-(1) Denaturation at 95 ° C for 30 seconds, (2) Annealing at 65 ° C to 50 ° C for 45 seconds, (3) Extension at 72 ° C. for 1 minute; III. 30 cycles— (1) denaturation at 95 ° C. for 30 seconds, (2) annealing at 50 ° C. for 45 seconds, (3) extension at 72 ° C. for 1 minute; IV. Store at 4 ° C. for 7 minutes at 72 ° C.

As a result of performing PCR under the above conditions and confirming by agarose electrophoresis, PCR products of approximately 785 bp were identified in various fungal genomes including Penicillium oxalicum KCTC 6440 (see FIG. 1). . After ligation of this PCR product to the pGEM-T vector (Ligation conditions: ligation containing 1.5uL PCR product / 0.5uL T-vector / 2.5uL 2X ligase buffer / 0.5uL T4 DNA ligase [Promega]) The mixture was left at 4 degrees for 16 hours) and transformed into E. coli TOP10F 'strain. Determining the nucleotide sequence of the 785 bp gene fragment and comparing it to that of the GenBank through NCBI Blast search to define a novel homology of 90% or less. Then, 10 novel Fetez gene fragments were obtained.

2nd step: Securing the nucleotide sequence of whole phytez gene through Inverse PCR technique

Inverse PCR is a very powerful PCR technique that can determine the sequence of the entire gene in a short time and in a short time based on the sequence of the gene fragment. Genomic DNA of Penicillium oxalicum KCTC 6440 in the 10 phytez gene fragments secured by the novel gene in the first step was BamHI, BglII, EcoRI, HindIII, PstI, SalI, XbaI, XhoI. Each was cut with a restriction enzyme of (enzyme treatment condition: 1ug DNA / 1X buffer / 50uL / reaction for 16 hours at optimal temperature), and purified (PCR purification kit, Qiagen) to make the DNA concentration 2 ng / μl. Self ligation. The above fragments of DNA are converted from linear DNA to circular DNA. The ligation mixture was purified using a PCR purification kit (Qiagen) and used as a template, and primers were used in Table 2 below.

Sequence Forward primer, INV-1 SEQ ID NO: 3 5'-CAAGACTTGGACAAAGGTGAGCTCGC-3 '(26mer) Reverse primer, INV-2 SEQ ID NO: 4 5'-TTCACCGACACCGAGTGGTCCCAG-3 '(24mer)

Conditions for inverse PCR were as follows: PCR reaction composition: 5 uL ligated DNA / 0.4 uM INV-1 & -2 primer / 0.4 mM dNTP / 1X buffer / 1.25 U Taq polymerase / 25 uL volume].

I. deaturation at 94 ° C. for 1 minute; II. 10 cycles (annealing temperature increased by 1 ° C. per cycle) —Dentation at 94 ° C. for 15 seconds, Annealing at 60 ° C. for 30 seconds, Extension at 68 ° C. for 4 minutes; III. 20 cycles (increase elongation time per cycle by 10 seconds)-15 seconds of denaturation at 95 ° C., annealing at 60 ° C. for 30 seconds, extension at 68 ° C. for 4 minutes; IV. Extension at 68 ° C. for 20 minutes.

The product obtained through the PCR process was confirmed by agarose electrophoresis. Among the eight restriction enzyme treatment groups, inverse PCR products appeared only in four restriction enzyme treatment groups such as BamHI, BglII, PstI, and SalI (see FIG. 2). The four PCR products were cloned into pGEM-T (Promega ™) vector to determine the base sequence. As a result, the entire nucleotide sequence of the Paetis gene (hereinafter referred to as "POX") derived from penicillium oxalicum was determined (see FIG. 3).

As a result of comparing the entire amino acid sequence of the Penicillium oxalicum-derived Paitez gene with other fungal Paetez genes, the most homologous was Aspergillus niger PhyA (Acesion No. M94550). The identity is 58.8% and the homology is 68.8%. As was published as No. die pie Handcrafted a recent patent (P. hordei, Accesion No. AX0851913) belonging to the genus Solarium penny chamber which was confirmed novelty show 52.1% identity and 61.5% homologous when compared POX pie Te jeuwa. Penicillium sp. , Accesion No. AE48448, has a lower homology of 35.5%.

The above method is a novel application from various fungal strains by using a combination of known methods such as preparing a degenerate primer, performing a touchdown PCR, a clone of a PCR product, sequencing, sequencing, a primer for inverse PCR, and performing an inverse PCR. Invented a new method of searching for the Pietez gene and obtaining the entire gene. Through this method, a novel gene that was previously unknown from various fungal strains was found. To present.

Example 2 Preparation of Recombinant Vectors and Transgenic Strains for Fightetz Expression

Step 1: PCR amplification and cloning of the Paetez gene

Based on the phytez gene of the penicillium oxalicum obtained in Example 1, the nucleotide sequence of POX, a primer capable of obtaining only a mature gene portion to be expressed in the Pchia strain was prepared as follows. .

The POX4 primers produced a recognition sequence of EcoRI, a restriction enzyme present in the cloning site of pPICZαA, a Peachia pastoris vector, at the 5 'end, and was also made in accordance with the POX open reading frame (ORF). POX5 primers were also prepared in accordance with ORF, and a stop codon was inserted at the 5 'end.

Primer used for cloning penicillium oxalicum phytez gene (POX) Sequence Forward primer, POX4 SEQ ID NO: 5 5'-TT GAATTC CGTACCTGTGATACCGGTGACGGTGGC-3 ' Eco R I Reverse primer, POX5 SEQ ID NO: 6 5'-T TCA CTTTGAAGAAACGCCACATT-3 'Stop Codon

Using the above-described POX4 and POX5 as a primer, the penicillium oxalicum genome (genomic DNA) as a template, the annealing temperature is 55 ℃, the product obtained by performing PCR for a total of 30 cycles is inserted into the pGEM-T cloning vector The sequence was checked again.

Step 2: ligation into the vector pPICZαA

The mature POX gene cloned into the pGEM-T vector was digested with the restriction enzymes EcoRI and NotI, and then inserted into the same restriction enzyme site present in the multiple cloning site of pPICZαA, a vector for the expression of Peachia pastoris ( See FIG. 4). The ligation product was mixed in 40 μl of E. coli TOP10F 'competent cells, and placed in a 0.2 cm cuvette for 5 minutes on ice. Electroporation was performed under the conditions of 2.5 KV and 5 ms using an electroporator manufactured by Bio-Rad.

After electroporation, the transformed cells were low pH LB agar medium (Low salt Luria-Bertani Agar Medium; pH 7.5 using 10 g tryptone, 5 g sodium chloride, 5 g yeast extract, 15 g agar, sodium hydroxide (NaOH) per liter of medium). Incubation + 25 μg / mL Zeocin ™ was incubated. After plasmid DNA was extracted from the selected transformants, the recombinant plasmid pPICZα A-POX vector capable of expressing phytez was confirmed by final DNA sequencing (see FIGS. 4 and 5).

Third Step: Transformation of Pchia Pastoris GS115 or KM71

Vector DNA was purified by Promeaga's Wizard plasmid purification Kit from E. coli Top10F '/ pPICZαA-POX cells incubated for 16 hours. 50 ㎍ purified DNA was linearized by treatment with BglII restriction enzyme to increase the conversion efficiency, and then the same volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) solution as the restriction enzyme reaction solution. The protein was removed by treatment, and precipitated by treatment with 1/10 volume of 3M sodium acetate, pH 5.2 solution and 2 times volume of ethanol.

Host strain Pychia Pastoris GS115 (Invitrogen, catalog No. C181-00) or KM71 (Invitrogen, catalog No. C183-00) yeast is 10 g per 1 liter of medium Inoculated into 50 mL of yeast extract, 20 g peptone, 20 g glucose (Dextrose), pH 5.8, shake incubated at 200 rpm for 30 hours at 30 ° C., and 0.5 mL of 500 mL YPD medium (2 L flask). ) Was inoculated and shaken at 300 nm at 200 rpm until absorbance (OD600) reached 1.3. After centrifugation of the culture solution at 1,500 × g for 5 minutes at 4 ° C., the pellets are suspended with 500 mL of cooled sterile distilled water, centrifuged under the same conditions, and the same process is repeated with 250 mL of cooled sterile distilled water. Competent cells were prepared by suspending the final cells with sterile distilled water cooled to a final volume of 1.5 mL. 80 μl of the competent cells and 10 μg of linear DNA were mixed, placed in a 0.2 cm electroporation cuvette, and allowed to stand on ice for 5 minutes, followed by transformation by electroporation at 1500 volts and 200 μs. Immediately after electroporation, 1 mL of 1 M sorbitol is poured and transferred to a sterile 15 mL conical tube and shaken for 1 hour at approximately 150 rpm in a 30 ° C. incubator. At the end of the culture, transformed cells were transformed into YPDS-zeocin agar medium (YPD + 1M Sorbitol + 100 ㎍ / mL Zeocine, pH 6.5) at 100 to 300 μl and colonies were formed at 30 ° C. for about 2 to 4 days. Incubate until

Step 4: Screening of Pchia Pastoris Transformants

Selection of recombinant phytez expressing strains among the zeocin resistant strains was performed using MMH-Phytate agar medium. The composition and preparation method (per 1 liter of medium) of the MMH-Phytate agar medium used in the present invention are as follows.

20 g of agar (Bacto Agar) is dissolved in 750 mL of distilled water and autoclaved. After sterilization, cool to 60 ℃ in a constant temperature water bath. 5 mL of 2% myo-Inositol steam sterilization solution, 15 mL of 20% calcium chloride (CaCl ₂ ) steam sterilization solution, 30 mL of 20% sodium phytic acid (Na-Phytate), pH 6.0 filtered sterilization solution Are added sequentially while stirring to react. As sodium phytic acid is added, a white precipitate develops. The solution is slowly and consistently added so that the precipitate is fine and uniform in size. 100 mL of 10X YNB (13.4% Yeast Nitrogen Base with amino acids), 2 mL of 500X Biotin (0.02% Biotin), and 100 mL of 10X Methanol (5% Methanol) solution are added with stirring. Colonies formed in YPD-zeocin agar medium were transferred to MMH-Phytate agar medium with 0.2 mL pipette tips. As a result, a clear ring occurs due to the action of the phytées, and as a size of the clear ring, a strain having excellent phytez expression ability was selected.

Step 5: Investigate whether Pyte Pastoris pPICZαA-POX expresses phytez expression

The strains selected in the fourth step were subjected to a phytase assay through a flask liquid culture. A selection strain was inoculated into a 500 mL baffle flask containing 50 mL of GMMY medium (Invitrogen) and incubated for 24 hours. 1 mL of the culture was taken as a sample and 50X methanol was added to a final 0.5% concentration to induce the expression of Paitez. After the first induction of methanol expression, methanol addition was repeated every 24 hours until 72 hours. Samples were subjected to Pitez enzyme titer analysis and protein quantification with a supernatant taken by centrifugation at 12,000 x g for 5 minutes, and the cell weight was measured by pellets.

The Pitez enzyme titer assay was performed by adding the supernatant of the culture sample to a substrate solution in which 0.25% sodium phytic acid (Sigma) was dissolved in 0.1 M sodium citrate, pH 5.5 buffer, and adding this solution to 37 mL. After reacting for 30 minutes at 1 ° C., 1 mL of 5% trichloroacetic acid solution was added to terminate the reaction. 100 μl of the finished sample was diluted in 900 μl of distilled water, and then, 1 mL of color developing reagent (0.5% ammonium molybdate: 0.6M sulfuric acid: 2% ascorbic acid) was added and the color reaction was performed at 50 ° C. for 10 minutes. Was performed, and the inorganic phosphate liberated by absorbance at 820 nm was quantified. 1 unit of Fightez enzyme titer was defined as the amount of enzyme that liberates 1 mole of inorganic phosphoric acid for 1 minute under the above conditions.

Protein was quantified by absorbance at 595nm using the Protein Assay Kit of Bio-Rad. 1 mL broth was centrifuged at 12,000 x g for 10 minutes, the pellets were collected and dried at 80 ° C. for 2 hours, and then dried cell weight was measured.

Example 3 Production and Characterization of Recombinant Fightz

First step: production of recombinant phytez in a 7 liter fermenter

As a strain having excellent phytez expression ability through a flask experiment, a Peachia pastoris GS115 / pPICZαA-POX strain was finally selected. The transformed strain blood Chiapas pastoris GS115 / pPICZαA-POX (Pichia pastoris GS115 / pPICZαA-POX) is a Korea Research Institute of Bioscience and Biotechnology Genetic Resources Center (KCTC) to give the KCTC-10386BP accession number and deposit on November 27, 2002 received.

The strains were cultured in a laboratory fermenter and examined for their properties. Fermentation experiments used in the present invention were carried out according to the Peachia fermentation guidelines of Invitrogen ™. Selected strains were inoculated in 100 ml of MGY medium, shaken at 30 ° C. for 20 hours, and then inoculated in 2 L fermentation medium. After 24 hours of glycerol batch fermentation, oil culture was carried out with glycerol for about 4 hours, and methanol oil culture for about 70 hours was performed. The medium was added on the basis of dissolved oxygen (DO). Samples were taken at approximately 12 hour intervals, and analyzed for Paitez enzyme titers, protein concentrations, and cell weights.

After fermentation is complete, phytase enzyme titer is 470 U / mL, protein concentration is 1.1 g / L, dry cell weight (WCW) is 350g / L, dry cell weight (DCW) Was analyzed at the 90 g / L level (see FIG. 6). In addition, as a result of analyzing the fermentation broth by polyacrylamide gel electrophoresis (SDS-PAGE) containing an SDS as an anionic detergent, it was confirmed that it has a molecular weight of 64.6 KDa (see Fig. 7).

Zymogram experiment was performed to confirm the enzymatic activity by reacting gels subjected to electrophoresis with PSS solution (0.2% α-naphtyl phosphate, 0.1% Fast Garnet GBC, 50 mM sodium acetate solution, pH 5.2). As a result, phosphatase activity was confirmed at the same position expected in SDS-PAGE (see FIG. 7). Since phytase is also a kind of phosphatase, it is reasonable to assume that the zymogram experiment is due to the activity of phytase.

Second Step: N-terminal Amino Acid Sequencing of Recombinant Fightz

In the SDS-PAGE analysis, almost no protein band other than Paitez was seen. After SDS-PAGE, the protein was transferred to the PVDF membrane by electrophoresis, and only the Paitez band was cut to analyze the N-terminal amino acid sequence.

Molecular Weight and N-terminal Amino Acid Sequence of the Recombinant POX Fightase Enzyme Molecular Weight N-terminal amino acid sequence DNA Analysis Theory Estimates 49 kDa Glu-Ala-Glu-Ala-Glu-Phe-Arg-Thr-Cys-Asp Experiment result 64.6 kDa Glu-Ala-Glu-Ala-Glu-Phe-Arg-Thr-X * -Asp * Likely to be cysteine or modified amino acid

According to the results, the recombinant Pytease produced in the Pchia pastoris GS115 / pPICZαA-POX strain was normally expressed, and the expected Kex 2 signal cleavage site (Glu-Lys-Arg * Glu-Ala-Glu-Ala; * Was cleaved accurately and secreted extracellularly (see FIGS. 5 and 7). The difference between the theoretical molecular weight (49 kDa) and the actual molecular weight (64.6 kDa) means that about 25% of the sugar chains were glycosylated. The amino acid sequence of the recombinant Paitez expressed in the Pchia pastoris GS115 / pPICZαA-POX strain is shown in FIG. 5. The underlined portion is the secretion signal of the alpha mating factor inherent in the pPICZαA vector, which is cleaved by Kex2 to secrete the mature POX protein.

Step 3: Enzymatic Characterization of Recombinant Fightz

The effect of the pH of the recombinant Paitez on the enzyme activity was investigated by preparing glycine (pH 2.0 to 3.5), sodium citrate (pH 4.0 to 6.5), trisate (pH 7.0 to 8.5) buffers at 0.2 M concentration. As a result of the experiment, Pytha Pastoris produced Recombinant Paitez had an optimal pH at pH 5.5 and showed 60% of the total activity in the range of pH 4.5 to pH 6.6.

The optimum temperature of the recombinant Fightez was investigated in the range from 4 ° C to 80 ° C. As a result of the experiment, the recombinant Paitez produced by Pchia Pastoris showed the optimum temperature at 50 ° C and showed 50% of the activity from about 30 ° C to 60 ° C. The experiment measured the Pytease enzyme titer by the method described in the fifth step of Example 2.

Enzyme Activity of Recombinant Phytez Expressed in P. pastoris pPICZαA-POX division Protein concentration (mg / ml) Enzyme Titer (U / ml) Specific activity (U / mg) Supernatant 1.1 470 427 Note) The reaction was performed at pH 5.5 and 37 ℃ for 30 minutes, and 1 unit is defined as the amount of enzyme capable of separating 1 μmol of inorganic phosphorus per minute.

The enzyme titer of the crude enzyme solution was 427 units / mg, which was very high compared to the known fungi Fightez. The highest enzyme titer known to date was 400-1200 Unit / mg of phytze isolated from four fungal fungi ( Peniophora lycii, Agrocybe pediades, Ceriporia sp., Trametes pubescens ) belonging to basidiomycetes . Appl Environ Microbiol 67 : 4701-7 (2001). Natuphos (Netherlands BASF Co., Ltd.), which has the highest market share in the phytez market, is a phytze derived from Aspergillus Niger and has low enzymatic activity of 100 units / mg. Other fungal Fighters titers vary from 8-190 units / mg. Therefore, the POX fightes according to the present invention can be classified into novel fightes having a high activity similar to the enzyme activity of the basidiomycete described above.

As described through the above examples, the present invention was designed and demonstrated very efficiently to search for a novel Pytease enzyme gene from a number of fungal strains. A vector system expressing a large amount of Tez gene, pPICZαA-POX, was constructed and transformed into a Pichia pastoris yeast strain, thereby selecting a strain that produces a recombinant phytase enzyme having a high titer. In the present invention, the method for finding the new gene may be usefully applied to the selection of other enzymes as well as phytez. The recombinant phytez also reduces the amount of phosphorus excretion in the manure of the unit animal when used as a feed additive. The present invention is expected to have an effect of improving the digestibility is a very useful invention in the livestock industry and environmental conservation industry.

<110> Choongan Co., Ltd <120> A novel phytase gene derived from Penicillium oxalicum and a          Pichia pastoris transformant producing the phytase <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for degenerate PCR <400> 1 tgggghcavt aykcgccntw cttytc 26 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for degenerate PCR <400> 2 gttdccbscr ccrtrgccgt agtaytt 27 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Inverse PCR <400> 3 caagacttgg acaaaggtga gctcgc 26 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Inverse PCR <400> 4 ttcaccgaca ccgagtggtc ccag 24 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer for POX cloning <400> 5 ttgaattccg tacctgtgat accggtgacg gtggc 35 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for POX cloning <400> 6 ttcactttga agaaacgcca catt 24 <210> 7 <211> 1942 <212> DNA <213> phytase derived from Penicillium oxalicum <400> 7 gagaattgat tcatcttccg ggatcctctc gtttccttct ccccttttct tgatttcctt 60 tcgccatgca cgatccgcgg tgacaaacca cactatccga cgcacgcgcg ggaatatcat 120 atcctcaact accgcatctt ggctaccgcg gctggctgga aaatgacaga tctttaccga 180 ggagacatgt cccatctggt cgaaaagcag gaccttttga gtatctctcc atattctgga 240 agttatgacc accactcctc tcacaggaaa cctcgttcct tggcgaccag atgtttgaca 300 ttgctcttcg ctctggtcct actcgtggcg gggtacgtga tcgctggcca cggactctca 360 ctctcgcgag aggtggctac gaatgatgaa gcccgagccc gtccaggttc agacgcgtcg 420 aacataccgg caaggcccgc agtcaaacag gtctcacatc gtgttcggac ctgtgacacc 480 gtcgacggtg gctatcaatg tttcccgcag ctgtcccatc gatggggcca gtattcgcca 540 tacttttctc ttgccaacac tggccttccc agtgaagtgc ccgagaagtg cgagctcacc 600 tttgtccaag tcttgtctcg tcatggcgca cggtatccca cggcgtccaa gagcaaaaag 660 tacaaatctc tcatccaggc catccaagcc aacgcgacag cctacaatgg tcaaagtgca 720 ttcctgcgtg cctacaatta tacgttggga agcgaggatt tgacctcctt tggcgagcac 780 caaatgatca actccgggat caagttctac cagcgctatg cggccctgac tcgcgaccac 840 gtccccttta tccgctcttc agactcatcg cgcgtggtcg cctcgggtca actgttcatt 900 caaggctatg aacaatccaa ggcacaagat tgtgatgcgg atcacagtca agatcacgca 960 gccatcaatg tgctaatctc cgaagccccc ggtgccgaca acaccctcaa tcgcaatacc 1020 tgcgcggcct ttgaagccga taagctcggc gaccaggtct cggcgaaata cacggctctc 1080 attgcacccc ctatggtcca acgcctacac catgatctcc caggcgtcac cctcaccaac 1140 gaccaagtca tatacctcat ggacatgtgc gcctacgaca ccgtctcaag caccccaggc 1200 gcaacctccc tctcgccctt ctgcgccctc ttcaccgaca ccgagtggtc ccagtacaac 1260 taccttcagt ccctcggcaa atactacggt tacggcgccg gcaaccccct cggtccaacc 1320 caaggcgtgg gattcatcaa cgagctcatc gcccgcatga ctcattcccc cgtccacgac 1380 cacaccacca gcaatcgcac cctcgacgca ccaggcgcag actcgttccc caccaaccgc 1440 accctctacg cggatttcac ccacgataac ggcatgatcc ccatcttctt cgccctgggc 1500 ttgtacaatg gatcagaccc gctgccgctc gatcgcatcg tgcctgccac acaggcggat 1560 ggatactctg ccgcgtgggc ggtgccattt gcggcgcgcg cctatatcga aatgatgcag 1620 tgtggtcggg acaccgaacc cctggtgcgg gtgttgatca atggtcgagt ggcgccgctc 1680 aagggatgca acgtggatca actgggtcga tgcaagagga gtgattttgt gaatgccttg 1740 agttttgctc aagatggggg caactgggcg aaatgtggcg tttcttcaaa gtgaagaatt 1800 gaaaatgttc ggaatccctt tctctctctc tcttcaagtc gggtcaattt atttattttg 1860 agtcgtggct cgtggcttgt ggctcgtgcg gacgcgaatt gggggattat accatatcat 1920 tactcgatga ttataaaacg tc 1942

Claims (4)

delete The phytze gene (POX) derived from Penicillium oxalicum KCTC6440 represented by SEQ ID NO: 7 obtained by PCR technique using a primer set of SEQ ID NOs: 1 and 2. Claim 2 of the transformed blood with a recombinant vector containing the gene Chiapas pastoris GS115 / pPICZαA-POX (Pichia pastoris GS115 / pPICZαA-POX) KCTC 10386BP. Recombinant phytase enzyme, characterized in that expressed from the strain of claim 3.

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