KR102026016B1 - Mutant Porphyra yezoensis 500GEAA comprising higher essential amino acids content, method of preparing thereof and method of preparing glutamate using the same - Google Patents

Abstract

The present invention relates to a mutant radiation pattern laver 500GEAA having an increased essential amino acid content, a method for preparing the same, and a method for producing an essential amino acid using the same. Therefore, it can be effectively used for the essential amino acid production method.

Description

Mutant Porphyra yezoensis 500GEAA comprising higher essential amino acids content, method of preparing etc and method of preparing glutamate using the same

The present invention relates to a mutant radiation pattern laver 500GEAA having an increased essential amino acid content, a method for preparing the same, and a method for producing an essential amino acid using the same. It relates to a preparation method thereof and a method for preparing an essential amino acid using the mutant radiation pattern laver.

Porphyra is a species of algae belonging to the red algae. It is an edible seaweed belonging to the genus.

Seaweed is a red algae mainly grown and used as food in Korea, Japan, and China, and wild products are attached to the rocks of the sea, and the farmed species are mainly the seaweeds ( Porphyra yezoensis ) and sesame ( Porphyra tenera ). About 50 species grow, and in Korea, round squirrel ( Porphyra suborbiculata ) and long snail ( Porphyra) The ten paper distribution such pseudolinearis), base pattern Seaweed (Porphyra seriata).

In 2011, the amount of seaweed produced was 220 billion won, accounting for the highest percentage of seaweeds (58% of total seaweeds). In view of the amount of production and the scale of production, seaweed is a very important product of aquaculture fish farms. The seaweed industry accounts for a great deal of geography, economy and industry, but the seaweed industry is very small.

Kim's seedling production increased slightly from year to year at 2.84 million boxes in 2009, from 7 billion won to 3.67 million boxes in 2012, and 9.2 billion won. In the production of all seedlings, general laver (charm and radiant laver) accounted for about 40% of the total laver seedlings.

The total amount of seaweed seedling production amounted to W7 ~ 9bn, which is very small in the total seaweed farming industry (220 billion won), but it is very important in the industrial position because seaweed seedling production directly affects the seaweed farming industry.

Recently, the government selected Kim as one of the top ten export strategic items, formulated a plan to foster the preoccupation of the world market, and proposed the International Standard for Kim to the Asian Food Standards Committee in 2010. In addition, in 2011, the 34th CODEX General Assembly decided to establish Kim's standards with the consent of 184 member countries around the world.

Accordingly, Korea can take the lead in the process of enacting Kim's international standards in the future, and will help Kim maintain the international export market by encouraging Kim's international standards to be the same as domestic standards. It is expected.

Currently, about 25 to 40% of the total seedling production of ginseng is a foreign seed (hybrid mixed with Japanese), which may cause payment problems. In order to solve this problem, the development of new seed varieties is difficult to be carried out by small Kim Jongmyo producers, and it is a time for the development of excellent substitute seeds at the national level.

However, the research on the breeding of Kim seeds is very insignificant. Studies on the improvement of seaweed varieties in Korea include aquaculture characteristics of large young seaweed, large radiation pattern seaweed, adaptation test for derivation of new species by sea area, and low salt resistant varietal improvement by tissue culture of radioactive seaweed. There is, however, did not lead to commercialization or industrialization.

The Mokpo branch of Namhae Fisheries Research Institute conducted varieties for the seaweeds and radiant seaweeds, which have been farmed in the South Sea area since 1994, and the seaweeds native to Korea's coast. (Namhae Fisheries Research Institute, 2004, Regional Coastal Seaweed Breeding and Technology Development in the South Coast, 2003 Business Report, pp 571-600).

In Japan, breeding research for the improvement of laver varieties has been undertaken since the 1960s, especially large seaweed laver in 1962 and large radish laver in 1967 by farmers (Miura, A., PF). Fuand JA Shin, 1992. lnterspecific cross experiments between Porphyra yezoensis Ueda and P. tene Kjellman (Bangiales, Rhodophyta) by using pigmentation variants. J. Tokyo Univ. Fish., 79, 103-120.). In addition, protoplast cell fusion research was conducted to develop new varieties, but it was not grown as a practical breed.

Essential amino acid, the source of the rich taste we feel in food, was originally extracted from kelp and the like. Essential amino acids are known to have a neurotransmitter function in the body of animals, and are not only the main component of monosodium glutamic acid, a salt for seasoning, but also essential for the production of folic acid.

Therefore, if cultivated P. yezoensis and cultivated varieties to obtain mutant radiation pattern with high essential amino acid content and provide the method and use of such mutant radiation pattern seaweed, the added value is higher than other varieties. Are expected to prefer.

Accordingly, the present inventors confirmed that the essential amino acid content of the radial patterned laver treated with ethyl methanesulfonate was significantly superior to the radial patterned laver not treated with ethyl methanesulfonate.

Accordingly, an object of the present invention is a mutant radiation pattern seaweed 500GEAA ( Porphyra) having increased essential amino acid content by ethyl methanesulfonate treatment. yezoensis 500GEAA).

It is another object of the present invention to provide a method for preparing a mutant radiation patterned seaweed 500GEAA having an increased essential amino acid content.

Still another object of the present invention relates to a method for preparing essential amino acids using mutant radiation patterned seaweed 500GEAA.

The present invention relates to a mutant radiation pattern laver 500GEAA ( Porphyra yezoensis 500GEAA) having increased essential amino acid content, a method for preparing the same, and a method for producing an essential amino acid using the same. It can be usefully used for the preparation of essential amino acids.

The present inventors obtained spores released from the radial patterned laver treated with ethyl methanesulfonate, and measured the essential amino acid content of the mutant radiant laver obtained by culturing the spores. In contrast, it was confirmed that the essential amino acid content is significantly superior.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a mutant radiation pattern lame 500GEAA deposited with accession number KCTC13428BP with increased essential amino acid content.

Essential amino acids include tryptophan, lysine, methionine, valine, leucine, isoleucine, threonine, threonine, and phenylalanine.

Tryptophan is a natural relaxant that helps insomnia by inducing normal sleep. It helps treat migraines, strengthen the immune system, and reduce the risk of spasms in the arteries and heart. It also works with lysine to reduce cholesterol levels.

Lysine aids in proper calcium intake and the formation of collagen, which makes up cartilage and connective tissue, and contributes to the production of antibodies, hormones and enzymes. Deficiency can lead to fatigue, weakness, excitement, eye redness, growth retardation, hair loss, and anemia.

Methionine is a major source of sulfur that prevents abnormalities in hair, skin and nails. It also helps lower cholesterol levels by increasing lecithin production in the liver. It functions to reduce liver fat, protects the kidneys, and acts as a natural chelating agent for heavy metals. Regulating ammonia formation reduces irritation of the bladder and affects hair follicles to promote hair growth.

Phenylalanine produces norepinephrine in the brain, which is a chemical that transmits signals between nerve cells and the brain. Reduces fasting pain, acts as an antidepressant and helps improve memory.

Threonine is a major component of collagen, elastin and enamel proteins. It helps to prevent the liver from accumulating fat, regulates the digestive tract and intestinal function more smoothly, and assists metabolism and assimilation.

Valine controls mental vitality and helps maintain a calm mental state.

Leucine and isoleucine provide raw materials for the manufacture of other essential biochemical components in the body.

The mutant radiation pattern laver may be one obtained as spores released from Porphyra yezoensis treated with ethyl methanesulfonate.

The ethyl methanesulfonate may be to be treated at a concentration of 1 to 5 mM, 2 to 5 mM or 3 to 5 mM, for example, to be treated at a concentration of 4 to 5 mM, but is not limited thereto. no. When the ethyl methanesulfonate is treated at a concentration of 1 mM or less, the frequency of occurrence of mutations may not be sufficient, and when treated at a concentration exceeding 5 mM, the survival rate of seaweed may be significantly reduced.

The essential amino acid content of the mutated radioactive seaweed is 15.00 to 20.00 wt%, 15.00 to 18.00 wt%, 16.00 to 25.00 wt%, 16.00 to 20.00 wt%, or 16.00 to 18.00% by weight, for example, 17.00 to 18.00% by weight, but is not limited thereto.

The threonine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.60 wt%, 2.80 to 4.00 wt% or 2.80 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The valine content of the mutant radiation pattern laver may be 2.00 to 3.50% by weight, 2.00 to 3.20% by weight, 2.00 to 3.00% by weight, 2.50 to 3.50% by weight or 2.50 to 3.20% by weight, for example, 2.50 to 3.00% by weight It may be%, but is not limited thereto.

The methionine content of the mutant radiation pattern laver may be 0.90 to 2.00% by weight, 0.90 to 1.80% by weight, 0.90 to 1.50% by weight, 1.00 to 2.00% by weight, or 1.00 to 1.80% by weight, for example, 1.00 to 1.50% by weight. It may be%, but is not limited thereto.

The isoleucine content of the mutated radiation pattern laver may be 1.20 to 2.00 wt%, 1.20 to 1.80 wt%, 1.20 to 1.50 wt%, 1.25 to 2.00 wt% or 1.25 to 1.80 wt%, for example, 1.25 to 1.50 wt% It may be%, but is not limited thereto.

The leucine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.50 wt%, 3.00 to 4.00 wt% or 3.00 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The phenylalanine content of the mutant radiation pattern laver may be 1.90 to 3.00 wt%, 1.90 to 2.80 wt%, 1.90 to 2.50 wt%, 2.00 to 3.00 wt% or 2.00 to 2.80 wt%, for example, 2.00 to 2.50 wt% It may be%, but is not limited thereto.

The lysine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.50 wt%, 3.00 to 4.00 wt% or 3.00 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

Another embodiment of the present invention is a method for preparing a mutant radiation patterned seaweed 500GEAA deposited with accession number KCTC13428BP with an increased essential amino acid content, comprising the following steps:

A culture step of treating ethyl methanesulfonate by radiation pattern laver; And

A screening step of screening radiant laver with high essential amino acid content.

The ethyl methanesulfonate may be to be treated at a concentration of 1 to 5 mM, 2 to 5 mM or 3 to 5 mM, for example, to be treated at a concentration of 4 to 5 mM, but is not limited thereto. no. When the ethyl methanesulfonate is treated at a concentration of 1 mM or less, the frequency of occurrence of mutations may not be sufficient, and when treated at a concentration exceeding 5 mM, the survival rate of seaweed may be significantly reduced.

The culturing step may be performed under MGM (Modified Grund Media) medium conditions.

The MGM medium is C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA), FeSO ₄ · 7H ₂ O, Na ₂ HPO ₄ · 12H ₂ O, NaNO ₃ , MnCl ₂ · 7H ₂ O and It may be one containing cyanocobalamin (Cyanocobalamin).

In one embodiment of the present invention, the MGM medium is a sterilized seawater 1 liter C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA) 3.72 g, FeSO ₄ · 7H ₂ O 0.28 g, Na ₂ HPO ₄ .12H ₂ O 10.74 g, NaNO ₃ 42.5 g, MnCl ₂ · 7H ₂ O 0.019197 g, cyanocobalamin (Cyanocobalamin; Vitamin B ₁₂ ) can be used as one containing.

The culturing step is 10 to 50 umol / m ² s, 10 to 40 umol / m ² s, 10 to 30 umol / m ² s, 10 to 20 umol / m ² s, 20 to 50 umol / m ² s, 20 It may be carried out at a brightness of from 40 umol / m ² s or 20 to 30 umol / m ² s, for example, may be performed at a brightness of 20 umol / m ² s, but is not limited thereto. The best growth was observed when the laver was cultured under luminous conditions of 20 umol / m ² s.

The culturing step may be performed in a light: dark cycle of 12h: 12h, but is not limited thereto.

The culturing step may be performed at a temperature of 10 to 25 ℃, 10 to 20 ℃ or 15 to 25 ℃, for example, 15 to 20 ℃, but is not limited thereto.

The essential amino acid content of the mutated radioactive seaweed is 15.00 to 20.00 wt%, 15.00 to 18.00 wt%, 16.00 to 25.00 wt%, 16.00 to 20.00 wt%, or 16.00 to 18.00% by weight, for example, 17.00 to 18.00% by weight, but is not limited thereto.

The threonine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.60 wt%, 2.80 to 4.00 wt% or 2.80 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The valine content of the mutant radiation pattern laver may be 2.00 to 3.50% by weight, 2.00 to 3.20% by weight, 2.00 to 3.00% by weight, 2.50 to 3.50% by weight or 2.50 to 3.20% by weight, for example, 2.50 to 3.00% by weight It may be%, but is not limited thereto.

The methionine content of the mutant radiation pattern laver may be 0.90 to 2.00% by weight, 0.90 to 1.80% by weight, 0.90 to 1.50% by weight, 1.00 to 2.00% by weight, or 1.00 to 1.80% by weight, for example, 1.00 to 1.50% by weight. It may be%, but is not limited thereto.

The isoleucine content of the mutated radiation pattern laver may be 1.20 to 2.00 wt%, 1.20 to 1.80 wt%, 1.20 to 1.50 wt%, 1.25 to 2.00 wt% or 1.25 to 1.80 wt%, for example, 1.25 to 1.50 wt% It may be%, but is not limited thereto.

The leucine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.50 wt%, 3.00 to 4.00 wt% or 3.00 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The phenylalanine content of the mutant radiation pattern laver may be 1.90 to 3.00 wt%, 1.90 to 2.80 wt%, 1.90 to 2.50 wt%, 2.00 to 3.00 wt% or 2.00 to 2.80 wt%, for example, 2.00 to 2.50 wt% It may be%, but is not limited thereto.

The lysine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.50 wt%, 3.00 to 4.00 wt% or 3.00 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The culturing step may be a method for preparing a mutant radiation pattern laver, comprising the following steps:

A first culture step of culturing the radial patterned laver treated with ethyl methanesulfonate;

A second culture step of selecting and culturing the radiation pattern laver surviving in the first culture step; And

The third culture step of culturing the spores released by the radiation pattern laver in the second culture step.

The ethyl methanesulfonate may be to be treated at a concentration of 1 to 5 mM, 2 to 5 mM or 3 to 5 mM, for example, to be treated at a concentration of 4 to 5 mM, but is not limited thereto. no. When the ethyl methanesulfonate is treated at a concentration of 1 mM or less, the frequency of occurrence of mutations may not be sufficient, and when treated at a concentration exceeding 5 mM, the survival rate of seaweed may be significantly reduced.

In one embodiment of the present invention, the first culturing step may be performed at 15 ° C. for 12 weeks, but is not limited thereto.

In one embodiment of the present invention, the second culture step may be performed at 10 to 15 ℃ for 12 weeks, for example, may be performed at 15 ℃ for 12 weeks, but is not limited thereto.

In one embodiment of the present invention, the third culture step may be performed at 10 to 15 ℃ for 12 weeks, for example, may be performed at 15 ℃ for 12 weeks, but is not limited thereto.

Release of the spores may be induced by raising the culture temperature of the radiation pattern laver to 18 to 20 ℃, for example, may be raised to 20 ℃ ℃, but is not limited thereto. At this temperature, the release of spores may be carried out rather than the growth of the leaves.

Another aspect of the invention is a process for preparing an essential amino acid comprising the following steps:

A culture step of culturing the mutant radiation pattern laver 500GEAA deposited with accession number KCTC13428BP; And

Extraction step of extracting the essential amino acid from the radiation patterned seaweed 500GEAA.

The culturing step may be performed under MGM medium conditions.

The medium is C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA), FeSO ₄ · 7H ₂ O, Na ₂ HPO ₄ · 12H ₂ O, NaNO ₃ , MnCl ₂ · 7H ₂ O and cya It may include nocobalamin (Cyanocobalamin).

In one embodiment of the present invention, the MGM medium is a sterilized seawater 1 liter C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA) 3.72 g, FeSO ₄ · 7H ₂ O 0.28 g, Na ₂ HPO ₄ .12H ₂ O 10.74 g, NaNO ₃ 42.5 g, MnCl ₂ · 7H ₂ O 0.019197 g, cyanocobalamin (Cyanocobalamin; Vitamin B ₁₂ ) can be used as one containing.

The culturing step is 10 to 50 umol / m ² s, 10 to 40 umol / m ² s, 10 to 30 umol / m ² s, 10 to 20 umol / m ² s, 20 to 50 umol / m ² s, 20 It may be carried out at a brightness of from 40 umol / m ² s or 20 to 30 umol / m ² s, for example, may be performed at a brightness of 20 umol / m ² s, but is not limited thereto. The best growth was observed when the laver was cultured under luminous conditions of 20 umol / m ² s.

The culturing step may be performed in a light: dark cycle of 12h: 12h, but is not limited thereto.

The culturing step may be performed at a temperature of 10 to 25 ℃, 10 to 20 ℃ or 15 to 25 ℃, for example, 15 to 20 ℃, but is not limited thereto.

The essential amino acid content of the mutated radioactive seaweed is 15.00 to 20.00 wt%, 15.00 to 18.00 wt%, 16.00 to 25.00 wt%, 16.00 to 20.00 wt%, or 16.00 to 18.00% by weight, for example, 17.00 to 18.00% by weight, but is not limited thereto.

The threonine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.60 wt%, 2.80 to 4.00 wt% or 2.80 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The valine content of the mutant radiation pattern laver may be 2.00 to 3.50% by weight, 2.00 to 3.20% by weight, 2.00 to 3.00% by weight, 2.50 to 3.50% by weight or 2.50 to 3.20% by weight, for example, 2.50 to 3.00% by weight It may be%, but is not limited thereto.

The methionine content of the mutant radiation pattern laver may be 0.90 to 2.00% by weight, 0.90 to 1.80% by weight, 0.90 to 1.50% by weight, 1.00 to 2.00% by weight, or 1.00 to 1.80% by weight, for example, 1.00 to 1.50% by weight. It may be%, but is not limited thereto.

The isoleucine content of the mutated radiation pattern laver may be 1.20 to 2.00 wt%, 1.20 to 1.80 wt%, 1.20 to 1.50 wt%, 1.25 to 2.00 wt% or 1.25 to 1.80 wt%, for example, 1.25 to 1.50 wt% It may be%, but is not limited thereto.

The leucine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.50 wt%, 3.00 to 4.00 wt% or 3.00 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The phenylalanine content of the mutant radiation pattern laver may be 1.90 to 3.00 wt%, 1.90 to 2.80 wt%, 1.90 to 2.50 wt%, 2.00 to 3.00 wt% or 2.00 to 2.80 wt%, for example, 2.00 to 2.50 wt% It may be%, but is not limited thereto.

The lysine content of the mutant radiation pattern laver may be 2.50 to 4.00 wt%, 2.50 to 3.80 wt%, 2.50 to 3.50 wt%, 3.00 to 4.00 wt% or 3.00 to 3.80 wt%, for example, 3.00 to 3.50 wt% It may be%, but is not limited thereto.

The present invention relates to a mutant radiation pattern laver 500GEAA having an increased essential amino acid content, a method for preparing the same, and a method for producing an essential amino acid using the same. Therefore, it can be effectively used for the essential amino acid production method.

1 is a graph showing the essential amino acid content of mutant radiation pattern laver according to an embodiment of the present invention.
Figure 2 is a mutant radiation patterned seaweed 500GEAA according to an embodiment of the present invention ( P. yezoensis 500GEAA) is a photograph confirming the sequence variation compared to the radiation pattern laver not treated with ethyl methanesulfonate.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Mutant Radiation Stripe 500GEAA ( P. yezoensis Manufacturing of 500GEAA)

Radial laver ( P. yezoensis ) was used from the Seaweed Research Center of the National Fisheries Research and Development Institute. Modified Grund Media (MGM) medium is 1.72 g of C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA), 0.28 g of FeSO ₄ · 7H ₂ O, Na ₂ HPO _{4 in} 1 liter of sterile seawater. 10.74 g of 12H ₂ O, 42.5 g of NaNO ₃ , 0.019197 g of MnCl ₂ · 7H ₂ O, and 1 mg of cyanocobalamin (Cyanocobalamin; Vitamin B ₁₂ ) were used.

Radiated laver is placed in Modified Grund Media (MGM) medium, and then 100 ml of MGM medium with radiant laver is placed in a plant cultured dish (100 x 40 mm) at a volume of 20 umol / m ² s. In a 12:12 h light: dark cycle, the temperature was maintained at 15 ° C. for 12 weeks.

Ethyl methanesulfonate was treated for 1 hour to 1, 2, 3, 4, and 5 mM concentrations of the radiation patterned laver in culture in MGM medium, and then, each washed sample was washed again in MGM medium. The luminous viability was analyzed by cell counting under a microscope after 12 weeks of incubation for 12 weeks at a light: dark cycle of 12h: 12h at a light intensity of 20 umol / m ² s or more.

Radiation strips surviving after treatment with ethyl methanesulfonate were transferred to MGM medium and incubated for 12 weeks at a light: dark cycle of 12h: 12h at a brightness of 20 umol / m ² s and maintaining a temperature of 15 ° C. The release was induced by raising the incubation temperature of the seaweed to 20 ° C., and the resulting spores were transferred to fresh MGM medium at a light: dark cycle of 12 h: 12 h at a light intensity of 20 umol / m ² s or more, maintaining the temperature at 15 ° C. for 12 weeks. Incubated for The control spine laver was prepared in the same manner as the above, but the ethyl methanesulfonate treatment was not performed.

Among the radiation pattern seams obtained by culturing the radiation pattern seams surviving after the ethyl methanesulfonate treatment in MGM medium, the radiation pattern seaweeds having higher essential amino acid content than the radiation pattern seams not treated with ethyl methanesulfonate were selected and mutated. Patterned laver 500GEAA ( P. yezoensis 500GEAA) was named.

Test Example 1: Confirmation of sequence variation of mutant radiation pattern laver

Sequence variation was compared using randomly amplified polymorphic DNAs (RAPDs).

Example 1 into the respective genomic DNA 1 uL obtained in ^{AccuPower ⓡ PCR PreMix (Eurofins Genomics)} , by the addition of 20 primers (10 pmol / ul) 1 uL with Buffer (DEPC-water) 7 uL shown in Table 1 PCR was performed. The reaction conditions were denatured at 95 ° C. for 5 minutes, amplified for 35 cycles at 95 ° C. for 30 seconds, at 60 ° C. for 30 seconds, and at 72 ° C. for 30 seconds, and then at 75 ° C. for 7 minutes. I was.

Through this, the genetic pattern of the mutant radiation pattern 500 500AAAA can be identified by screening the radiation patterned laver which is not treated with ethyl methanesulfonate and the radiation patterned laver with high essential amino acid content.

SEQ ID NO: designation Sequence (5 '-> 3') One Primer OPA-01 CAGGCCCTTC 2 Primer OPA-02 TGCCGAGCTG 3 Primer OPA-03 AGTCAGCCAC 4 Primer OPA-04 AATCGGGCTG 5 Primer OPA-05 AGGGGTCTTG 6 Primer OPA-06 GGTCCCTGAC 7 Primer OPA-07 GAAACGGGTG 8 Primer OPA-08 GTGACGTAGG 9 Primer OPA-09 GGGTAACGCC 10 Primer OPA-10 GTGATCGCAG 11 Primer OPA-11 CAATCGCCGT 12 Primer OPA-12 TCGGCGATAG 13 Primer OPA-13 CAGCACCCAC 14 Primer OPA-14 TCTGTGCTGG 15 Primer OPA-15 TTCCGAACCC 16 Primer OPA-16 AGCCAGCGAA 17 Primer OPA-17 GACCGCTTGT 18 Primer OPA-18 AGGTGACCGT 19 Primer OPA-19 CAAACGTCGG 20 Primer OPA-20 GTTGCGATCC

As can be seen in Figure 2, when comparing the gene amplification characteristics of the mutant radiation patterned seaweed 500GEAA that selected the radiation patterned seaweed with high essential amino acid content by the above method compared to the radiation patterned seaweed without treatment with ethyl methanesulfonate, SEQ ID NO: 10 In the case of using the primers 11, 13, it could be seen that there was a difference in the nucleotide sequence between the DNA of the mutated radiation patterned kimchi and 500GEAA which was not treated with ethyl methanesulfonate. In particular, it was newly confirmed that the primer can be used to distinguish it from the radiation pattern laver not treated with ethyl methanesulfonate.

Test Example 2: Determination of the Essential Amino Acid Content of Mutant Radiation Strip Kim 500GEAA

The measuring method of essential amino acid content is as follows. 10 g of dried radiant laver samples were extracted three times at 80 ° C. with 150 mL of 70% ethanol. In order to remove the fat-soluble component, the volume of hexane corresponding to at least two times of the ethanol was added and extracted, and the hexane fractionation layer was removed by using a pipette, and the aqueous layer was concentrated under reduced pressure at 45 ° C. to prepare a sample. It was.

250 mL of glass prep reagent (Uriprep, Waterbury, CT, USA) and 10 mL of sample were added to a 50 mL volumetric flask and centrifuged and filtered through a 0.2 um syringe filter. It was diluted about 40-fold with lithium diluent (Lithium diluent, pH 2.36) and 10 uL was injected into an HPLC analyzer (Agilent 1100 Series, Agilent Technologies Inc., Santa Clara, CA, USA).

For amino acid analysis, a cation exchange column (3 × 250 mm, 8 um, Pickering Laboratories Inc.) and a PINNACLE PCX reactor (Pickering Laboratories Inc.) were used and analyzed at a flow rate of 0.3 mL / min. Column and reactor temperatures were analyzed while maintaining at 40 ° C. and 130 ° C., respectively (Grunau JA, Swiader JM. 1992. Chromatography of 99 amino acids and other ninhydrin-reactive compounds in the Pickering lithium gradient system.J Chromatography A 594: 165- 171.).

The peak corresponding to the essential amino acid in the amino acid analysis result was confirmed to derive the content value of the essential amino acid, and is shown in Table 2 below.

Essential amino acid
content Untreated radial pattern
(g / 100g) Radiation seaweed 500GEAA
(g / 100g) Threonine 2.3828 3.3941 Valine 1.9943 2.6753 Methionine 0.8288 1.0382 Isoleucine 1.0360 1.3177 Leucine 2.7713 3.4739 Phenylalanine 1.8907 2.2361 Lysine 2.3310 3.0746 total 13.2349 17.2098

As can be seen in Figure 1, the essential amino acid content of the mutant radiation patterned seaweed 500GEAA was 17.21 (g essential amino acid / 100 g seaweed sample), the essential amino acid content obtained from the control radiation patterned seaweed is 13.24 g compared to 100 g of the radiation patterned seaweed. The essential amino acid content of the mutant radiation patterned seaweed showed 29.98% higher value than the control radiation patterned seaweed.

Korea Research Institute of Bioscience and Biotechnology KCTC13428BP 20171214

<110> Industry-University Cooperation Foundation Chonnam University <120> Mutant Porphyra yezoensis 500 GEAA comprising higher essential          amino acids content, method of preparing background and method of          preparing glutamate using the same <130> PN170294 <160> 20 <170> KopatentIn 2.0 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 1 caggcccttc 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 2 tgccgagctg 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 3 agtcagccac 10 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 4 aatcgggctg 10 <210> 5 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 5 aggggtcttg 10 <210> 6 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 6 ggtccctgac 10 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 7 gaaacgggtg 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 8 gtgacgtagg 10 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 9 gggtaacgcc 10 <210> 10 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 10 gtgatcgcag 10 <210> 11 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 11 caatcgccgt 10 <210> 12 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 12 tcggcgatag 10 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 13 cagcacccac 10 <210> 14 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 14 tctgtgctgg 10 <210> 15 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 15 ttccgaaccc 10 <210> 16 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 16 agccagcgaa 10 <210> 17 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 17 gaccgcttgt 10 <210> 18 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 18 aggtgaccgt 10 <210> 19 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 19 caaacgtcgg 10 <210> 20 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 20 gttgcgatcc 10

Claims (16)

Mutant radiation patterned seaweed 500GEAA ( Porphyra yezoensis 500GEAA) deposited with accession number KCTC13428BP with increased essential amino acid content. The mutant radiation patterned seaweed of claim 1, wherein the mutant radiation patterned seaweed is obtained as spores released from Porphyra yezoensis treated with ethyl methanesulfonate. The mutant radiation patterned seaweed of claim 1, wherein the essential amino acid content of the mutant radiation patterned seaweed is 15.00 to 25.00 wt%. Method for the preparation of mutant radiation patterned seaweed 500GEAA (porphyra yezoensis 500GEAA) deposited with accession number KCTC13428BP with an increased essential amino acid content, including the following steps:
A culture step of culturing the treated with ethyl methanesulfonate to the radiation pattern laver ( Porphyra yezoensis ); And
A screening step of selecting a radiation patterned seaweed with higher essential amino acid content than the radiation patterned seaweed treated with ethyl methanesulfonate from the cultured radiation patterned seaweed. The method of claim 4, wherein the culturing step is performed under Modified Grund Media (MGM) medium conditions. The method of claim 5, wherein the MGM medium is C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA), FeSO ₄ · 7H ₂ O, Na ₂ HPO ₄ · 12H ₂ O, NaNO ₃ , MnCl ₂ · 7H ₂ O and cyanocobalamin (Cyanocobalamin) comprising a method for producing a mutant radiation pattern laver. The method of claim 4, wherein the culturing step is performed at a luminous intensity of 10 to 50 umol / m ² s. The method of claim 4, wherein the culturing step is performed in a light: dark cycle of 12h: 12h. The method of claim 4, wherein the culturing step is performed at a temperature of 10 to 25 ° C. 6. The method of claim 4, wherein the essential amino acid content of the mutant radiation patterned laver is 15.00 to 25.00 wt%. The method of claim 4, wherein the culturing step comprises the following steps:
A first culture step of culturing the radial patterned laver treated with ethyl methanesulfonate;
A second culture step of selecting and culturing the radiation pattern laver surviving in the first culture step; And
The third culture step of culturing the spores released by the radiation pattern laver in the second culture step. Preparation method of essential amino acid comprising the following steps:
A culture step of culturing the mutant radiation pattern laver 500GEAA ( Porphyra yezoensis 500GEAA) deposited with accession number KCTC13428BP; And
Extraction step of extracting the essential amino acid from the radiation patterned seaweed 500GEAA. The method of claim 12, wherein the culturing step is performed under Modified Grund Media (MGM) medium. The method of claim 13, wherein the medium is C ₁₀ H ₁₄ O ₆ N ₂ Na ₂ · 2H ₂ O (Na ₂ EDTA), FeSO ₄ · 7H ₂ O, Na ₂ HPO ₄ · 12H ₂ O, NaNO ₃ , MnCl ₂ 7H ₂ O and cyanocobalamin (Cyanocobalamin) comprising a method for producing an essential amino acid. The method of claim 12, wherein the culturing step is performed at a luminous intensity of 10 to 50 umol / m ² s. The method according to claim 12, wherein the essential amino acid content of the mutant radiation pattern laver is 15.00 to 25.00% by weight.

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