KR20040018844A - Isolation and purification of hydrophobin protein from Tricholoma matsutake including useful tradition filamentous fungi, its gene sequence and a composition containing the same - Google Patents

Abstract

PURPOSE: An isolation and purification method of hydrophobin protein from Tricholoma matsutake including useful tradition filamentous fungi, a gene sequence thereof and a composition containing the protein and gene sequence are provided. The hydrophobin has hydrophilic and hydrophobic converting properties, so that it can be useful in cosmetic industry, brewing industry and medicine industry. CONSTITUTION: An isolation and purification method of hydrophobin protein from Tricholoma matsutake including useful tradition filamentous fungi comprises the steps of: adding 60% ethanol into a fruit body of Tricholoma matsutake, pulverizing it, and centrifuging the pulverized solution; adding distilled water into the supernatant and subjecting the mixture to dialysis for overnight; centrifuging the remaining solution at 4 deg. C and freeze-drying the supernatant; dissolving the freeze-dried substance in 25 ml of 0.05M sodium-phosphate buffer solution(pH 6.0) and 1M sodium solution and mixing them; centrifuging the mixture at room temperature; and washing the precipitated hydrophobin containing portion with 1M sodium buffer solution, 80% ethanol and a mixed solution of chloroform and methanol, and drying it. A gene encoding the hydrophobin protein from Tricholoma matsutake has the nucleotide sequence set forth in SEQ ID NO: 4.

Description

Isolation and purification of hydrophobin protein from Tricholoma matsutake including useful tradition filamentous fungi, its gene sequence and a composition containing the same}

The present invention relates to the isolation and purification of hydrophobins from tricholoma matsutake, including useful native filamentous fungi, to gene sequences thereof, and to compositions comprising the same. The present invention relates to separating and purifying hydrophobins from Tricholoma matsutake and obtaining a gene sequence of hydrophobins through gene amplification using specific primers and providing a composition comprising the same.

Hydrophobin is a small molecule protein produced in filamentous fungi (de Vries et al., 1993; Wessels 1997), the first reported by the identification of four hydrophobin genes in the fungus Schzophyllum commune (Lugones et al. , 1998; Schuren et al., 1990; van Wetter et al., 1998 & 2000), several researchers have isolated various types of hydrophobins from different organisms.

Hydrophobin is one of the cell wall proteins involved in cell-cell and cell-surface contact in streptococcus and basidiomycetes, creating a hydrophobic layer on the surface of the cell to propagate mycelia from the hydrophilic medium into the air. It plays an essential role in this process, including desiccation tolerance, including spore formation, growth of mycelium, aggregation of hyphae, host-surface adhesion, infection structure and fruiting body formation. (Kershaw and Talbot, 1998; Wessels 1996, 1997, 1999; Wessels et al. 1995; Wosten and Wessels, 1997; Wosen et al. 1994, 1999). In addition, these hydrophobins in mycorrhizal fungi are known to play an important role when mycelia adhere to the roots of symbiotic or parasitic plants (Mankel et al. 2002). Currently, studies on the function and utility of hydrophobin found in exogenous mycorrhiza are actively conducted, and it is expected to play an important role in mycorrhiza formation process such as contact of mycelia on the root surface of plants and infiltration of plant root cells by mycelia. .

Hydrophobins are largely divided into class I and class II. As a result of comparing the amino acid sequences of the hydrophobin proteins investigated to date, hydrophobins show different bio-physical properties depending on the hydrophobin protein (Wessels 1994, 1997). In addition, it has a characteristic self-fusion property, so that a thin protein monolayer film is formed at the interface between the liquid phase and the gas phase, thereby changing the hydrophobic surface to be hydrophilic and the hydrophilic surface to be hydrophobic. These physicochemical properties of hydrophobin imply various applications in medicine and industry, and it is necessary to separate different hydrophobin proteins in nature, which show the most effective properties depending on the purpose of use.

Recently, hydrophobins separated from existing hydrophobins have different physicochemical properties to separate new hydrophobins that can be used for other purposes, and increase the expression rate of existing hydrophobin proteins to increase production capacity and genetic engineering. Research on hydrophobin has been actively conducted in various fields such as improved physical properties.

Accordingly, the inventors of the present invention are to provide a method for separating and purifying a novel hydrophobin different from the existing hydrophobins from the trichophyte mycorrhiza ( Tricholoma matsutake ) which interacts with pine roots in view of the above. .

Another object of the present invention is to provide a nucleotide sequence of a gene encoding a novel hydrophobin protein isolated from pine mushroom.

The object of the present invention is to isolate and purify novel hydrophobins from pine mushrooms ( Tricholoma matsutake ) that grow and form exogenous mycorrhiza in pine roots, and to generate specific primers capable of selectively amplifying the genes encoding the hydrophobins. The development was performed by amplifying the gene encoding the hydrophobin through PCR and cloning to achieve the gene sequence of the hydrophobin.

Hereinafter, the specific configuration of the present invention will be described in detail.

Figure 1 shows a 25 kDa hydrophobin protein electrophoresis picture isolated and purified from the fruiting body of Tricholoma matsutake.

Figure 2 shows the SDS-PAGE results of hydrophobin protein extracted and purified separately from the fruiting body, mustard and bag of Matsutake mushroom, M is the electrophoresis standard, 1 is fruiting body, 2 is fresh, 3 is bag Respectively extracted and purified.

Figure 3 shows the results of multiple-alignment of the amino acid sequence of the hydrophobin gene of basidiomycete using the Clustal X program.

Figure 4 shows the amino acid sequence of the hydrophobin gene of Aspergillus nidulans .

Figure 5 shows the nucleotide sequences of the specific primers hydAnA and B developed for the amplification of the hydrophobin gene.

Figure 6 shows the results of electrophoresis after amplifying the DNA of the pine mushroom fruiting body using specific primers hydAnA and hydAnB.

Figure 7 shows the nucleotide sequence of the hydrophobin gene isolated from pine mushroom.

The present invention comprises the steps of separating and purifying the novel hydrophobin from the pine mushroom ( Tricholoma matsutake ) to grow and form exogenous mycorrhiza in the pine root; Preparing a specific PCR primer capable of selectively amplifying the gene encoding the hydrophobin; PCR amplification of the hydrophobin coding gene using the primer; Comprising the sequence analysis of the hydrophobin coding gene.

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 Isolation and Purification of Novel Hydrophobins from Matsutake Mushrooms

The fruiting body of Matsutake mushroom used in the present invention was harvested from nearby Yasan in Gachang, Daegu Metropolitan City. Immediately after harvesting, the resultant was washed five times with distilled water to remove contaminants and soils attached to the pine mushroom and stored at -70 ° C. When the frozen mushrooms were used, they were lyophilized for 2 days and only fruiting bodies were used. The freeze-dried fruiting body was put in 60% ethanol, ground in a high speed mixer for 30 seconds, centrifuged at 10,000 x g for 30 minutes, and then dialyzed overnight by adding 50-fold distilled water to the yellow supernatant obtained above. The solution remaining after dialysis was centrifuged at 12,000 × g for 1 hour at 4 ° C., then the pellet was discarded and the supernatant was lyophilized. The frozen material was dissolved in 25 mL of 1 M salt solution in 0.05 M sodium-phosphate buffer (pH 6.0) and mixed vigorously for 5 minutes. The mixture was centrifuged at 10,000 xg for 1 hour at room temperature, the supernatant containing the transparent strip attached to the wall of the centrifuge tube was discarded, and only the portion containing the hydrophobin submerged at the bottom was washed once with 3 mL of 1 M salt buffer. Twice with 2 mL of 80% ethanol, washed twice with 2 mL of chloroform / methanol (2: 1, v / v) mixture and dried.

In order to identify the hydrophobin protein separated and extracted by the above method, the obtained dry sample was subjected to SDS-sample buffer (SDS-sample buffer: 2% SDS, 0.05 M Tris / HCl, pH 6.8, 10% (v / v)). Glycerol) and analyzed on 12.5% polyacrylamide gel. After electrophoresis, proteins were stained using Coomassie Blue.

As shown in FIG. 1, the hydrophobin protein isolated and purified from the pine mushroom fruiting body of the present invention showed a single band at 25 kDa.

Figure 2 shows the fruiting body of pine mushrooms by part and shows the difference in the type and expression of hydrophobin protein present in each part by SDS-PAGE, 1 is fruiting body, 2 freshly, 3 is extracted and purified from the bag Hydrophobin. Single bands were shown at 25 kDa each, and there was no significant difference in the degree of expression.

Example 2 Hydrophobin Gene Amplification by PCR from DNA Extracted from Matsutake Mushroom

In order to amplify the hydrophobin gene from DNA isolated from pine mushroom fruiting bodies, 18 hydrophobin genes reported from 7 basidiomycetes were arranged. As a result of arranging the amino acid sequence of the hydrophobin gene, a very well-conserved amino acid sequence was found, but a region to be used for constructing a specific PCR primer capable of specifically amplifying the hydrophobin gene was not found (FIG. 3). ).

Thus, to produce a variety of genetic studies a large amount of genetic information that specific primers to the multi-phase aligned (multiple-alignment) after the selection of the amino acid sequence of the hydro pobin gene in the fungus Aspergillus (Aspergillus), which is reported to The conserved region was found and used for the preparation of a specific primer to be used for amplification of the hydrophobin gene derived from pine mushroom (FIG. 4).

Figure 4 shows the Aspergillus nidulan strain The hydrophobin amino acid sequence and the production region of the selected specific primers are shown, and FIG. 5 shows the specific primer sequences of the hydrophobin genes prepared therefrom, which are named hydAnA and hydAnB, respectively. As a primer, hydAnB was used as a primer for downstream amplification.

PCR amplification of Tricholoma matsutake DNA was carried out using PROGENE (Techne Cambridge Ltd. Duxford). In the PCR reaction, each primer concentration was used at 100 pmol and using amplification buffer (5 mM Tris-HCl, pH 8.0, 10 mM) using 0.2 M dNTP, 1.5 mM MgCl ₂ , 0.5 unit of Taq DNA polymerase. NaCl, 0.01 mM EDTA, 0.1 mM DTT, 5% glycerol and 0.1% Triton X-100) were mixed and calibrated with 200 μl of the whole with sterile water. The PCR program repeated 42 cycles of 30 seconds at 95 ° C, 1 minute at 55 ° C, and 30 seconds at 72 ° C. Gene amplification of hydrophobin under the above PCR conditions confirmed the PCR fragment of about 280 bp as shown in FIG. The PCR fragment of about 280 bp produced was cloned into the host by linking it with a pGEMT-easy vector and the nucleotide sequence of the hydrophobin gene was determined using an OmniBase DNA Cycle Sequencing System.

As shown in FIG. 7, the nucleotide sequence of the hydrophobin gene of pine mushroom obtained from PCR amplification is composed of 284 nucleotides. The nucleotide sequence was searched in the genetic database of the National Center for Biotechnology Information (NCBI), and was identified as a hydrophobin gene, but showed a very low sequence homology compared to the hydrophobin gene reported in other strains. .

As described above, the present invention has the effect of providing a method for separating and purifying a novel hydrophobin protein from pine mushrooms containing useful native filamentous fungi. In addition, the present invention has the effect of providing a gene nucleotide sequence encoding a hydrophobin protein derived from pine mushroom and the nucleotide sequence is very low compared to the nucleotide sequence of hydrophobin derived from conventionally known strains It is a very useful invention in the cosmetic industry, beer industry and medical industry because it has the homology and can use the specific properties in the hydrophilicity and hydrophobicity of the hydrophobin of the present invention.

Claims (5)

60% ethanol was added to the fruiting body of Tricholoma matsutake , ground in a high speed mixer for 30 seconds, and centrifuged at 10,000 xg for 30 minutes. 50 times distilled water was added to the yellow supernatant obtained above and dialyzed overnight, The solution remaining after the dialysis was centrifuged at 12,000 × g for 1 hour at 4 ° C., then the pellet was discarded and the supernatant was lyophilized, The frozen material was dissolved in 25 mL of 1 M salt solution in 0.05 M sodium-phosphate buffer (pH 6.0) and mixed vigorously for 5 minutes, The mixture was centrifuged at 10,000 x g for 1 hour at room temperature, After centrifugation, wash only the portion containing the hydrophobin subsided once with 3 mL of 1 M salt buffer, twice with 2 mL of 80% ethanol, and 2 mL of chloroform / methanol (2: 1, v / v) mixture. Method of obtaining a hydrophobin derived from pine mushroom, characterized in that it comprises a step of washing after washing twice. 60% ethanol was added to the fruiting body of Tricholoma matsutake , ground in a high speed mixer for 30 seconds, and centrifuged at 10,000 xg for 30 minutes. 50 times distilled water was added to the yellow supernatant obtained above and dialyzed overnight, The solution remaining after the dialysis was centrifuged at 12,000 × g for 1 hour at 4 ° C., then the pellet was discarded and the supernatant was lyophilized, The frozen material was dissolved in 25 mL of 1 M salt solution in 0.05 M sodium-phosphate buffer (pH 6.0) and mixed vigorously for 5 minutes, The mixture was centrifuged at 10,000 x g for 1 hour at room temperature, After centrifugation, wash only the portion containing the hydrophobin subsided once with 3 mL of 1 M salt buffer, twice with 2 mL of 80% ethanol, and 2 mL of chloroform / methanol (2: 1, v / v) mixture. Hydrophobin derived from Matsutake mushroom, which is obtained from the process of washing with water twice and drying. Specific primers according to SEQ ID NOS: 2 and 3 for mass production of a gene encoding hydrophobin derived from pine mushroom including useful native filamentous fungi. A base sequence of a gene encoding a hydrophobin derived from a pine mushroom, including the useful native filamentous fungus described in SEQ ID NO: 4, characterized by being amplified using the specific primers described in SEQ ID NOs: 2 and 3. A composition for food, liquor, medical use and cosmetics, comprising a hydrophobin protein derived from pine mushroom, including the useful native filamentous fungus, and a nucleotide sequence of a gene encoding the same as an active ingredient.