Streptomyces coelicolor mutant strain, beta-agarase production method using same and new agaro-oligosaccharide preparation method using same

文档序号：816797 发布日期：2021-03-26 浏览：19次中文

阅读说明：本技术 天蓝色链霉菌突变菌株，利用其的β-琼脂糖酶生产方法及利用其的新琼寡糖制备方法 (Streptomyces coelicolor mutant strain, beta-agarase production method using same and new agaro-oligosaccharide preparation method using same ) 是由李济贤金恩妵李娟嬉于 2019-09-10 设计创作，主要内容包括：本发明提供一种通过紫外线照射而使存在于野生型天蓝色链霉菌(Streptomyces coelicolor)A3(2)菌株的DagB基因的碱基序列发生点突变的天蓝色链霉菌(Streptomyces coelicolor)A3(2)-M22-2C43菌株。根据本发明的天蓝色链霉菌(Streptomyces coelicolor)A3(2)-M22-2C43菌株几乎不表达DagBβ-琼脂糖酶或着表达几乎没有β-琼脂糖酶活性的DagB突变酶,因此没有必要额外地从培养液中对DagA酶进行分离提纯,并且,通过利用天蓝色链霉菌(Streptomyces coelicolor)A3(2)-M22-2C43菌株的培养液或其上层液,可以制备新琼四糖(neoagarotetraose)或新琼六糖(neoagarohexaose)的含量相对比新琼二糖(neoagarobiose)高的新琼寡糖。(The present invention provides a Streptomyces coelicolor A3(2) _ M22-2C43 strain in which the nucleotide sequence of the DagB gene present in a wild-type Streptomyces coelicolor A3(2) strain is point-mutated by ultraviolet irradiation. The Streptomyces coelicolor A3(2) _ M22-2C43 strain according to the present invention hardly expresses DagB β -agarase or a DagB mutant enzyme having almost no β -agarase activity, and therefore, it is not necessary to additionally isolate and purify DagA enzyme from the culture broth, and a neoagarotetraose (neoagarotetraose) or neoagarohexaose (neoagarohexaose) having a higher content than neoagarobiose (neoagarobiose) can be prepared by using the culture broth of Streptomyces coelicolor A3(2) _ M22-2C43 strain or a supernatant thereof.)

1. A Streptomyces coelicolor strain A3(2) _ M22-2C43 with the preservation number of KCCM 12577P.

2. The Streptomyces coelicolor strain A3(2) _ M22-2C43 with accession number KCCM12577P as claimed in claim 1,

comprises a DagB gene modified by gene mutation, the DagB gene consisting of the base sequence of SEQ ID NO. 2.

3. The Streptomyces coelicolor strain A3(2) _ M22-2C43 with accession number KCCM12577P as claimed in claim 2,

the DagB mutant enzyme expressed by the modified DagB gene consists of the amino acid sequence of SEQ ID No. 6.

4. A method for producing a β -agarase, comprising:

(a) a step of inoculating the Streptomyces coelicolor A3(2) _ M22-2C43 strain having a accession number of KCCM12577P according to any one of claims 1 to 3 to a liquid medium containing galactose as a carbon source, and obtaining a culture solution by culturing;

(b) a step of obtaining a supernatant by centrifuging the culture solution.

5. A method for producing a β -agarase, comprising:

(b) a step of obtaining a supernatant by centrifuging the culture solution;

6. The process for producing beta-agarase according to claim 4,

the concentration of galactose in the liquid medium is 0.5% (w/v) to 4% (w/v).

7. The process for producing beta-agarase according to claim 4,

the culture temperature of the streptomyces coelicolor A3(2) _ M22-2C43 strain with the preservation number of KCCM12577P is 25-35 ℃, and the culture stirring speed is 200-300 rpm.

8. The process for producing beta-agarase according to claim 4,

the culture time of the Streptomyces coelicolor A3(2) _ M22-2C43 strain with the preservation number of KCCM12577P is 40-150 hours.

9. The process for producing beta-agarase according to claim 5,

the ammonium sulfate was added to make the protein saturation concentration of the supernatant 45% to 70%.

10. A method for preparing new agaro-oligosaccharide is characterized by comprising the following steps:

(a') a step of preparing a culture broth or a supernatant of the culture broth of the Streptomyces coelicolor A3(2) _ M22-2C43 strain deposited No. KCCM12577P according to any one of claims 1 to 3; and

(b') a step of enzymatically reacting agar or agarose with β -agarase present in a culture solution of Streptomyces coelicolor A3(2) _ M22-2C43 strain or a supernatant of the culture solution.

Technical Field

The present invention relates to a Streptomyces coelicolor mutant strain, a method for producing Beta-agarase (Beta-agarase) using the same, and a method for preparing neoagarosaccharides (neoagarase) using the same, and more particularly, to a Streptomyces coelicolor mutant strain mainly expressing DagA enzyme compared to a parent strain, a method for mass-producing DagA enzyme in Beta-agarase using the same, and a method for preparing neoagarotetraose (neoagarotetraose) or neoagarotetraose (neoagarotetraose) from agar or agarose, the neoagarotetraose having a higher content than neoagarobiose (neoagarobiose).

Background

Agar has been widely used for a long time as a representative polysaccharide (derived from marine algae) used in food additives, pharmaceuticals, cosmetics, livestock feeds, industrial materials, and the like, and is one of relatively abundant aquatic resources having an annual production amount of about 2000 to 5000 tons in korea. However, in actual use, only a part of the total product is simply processed and used as an inexpensive raw material, and the rest is mostly discarded, so that it is in a situation where the added value is very low as compared with the amount of the enriched resource. Therefore, in fact, research on new uses of abundant domestic agar and improvement of added value thereof are urgently needed.

Agar is mostly composed of polysaccharides except for small amounts of proteins, ashes, and fats, and the polysaccharides constituting the agar include agarose (agarose) which is a neutral polysaccharide and agar gel (agaropectin) which is an acidic polysaccharide. Agarose has agarobiose (agarobiose) in which D-galactose (D-galactose) and 3, 6-lacto-L-galactose (3,6-anhydro-L-galactose) are bound in the form of β -1,4 as monomers, and has a strong gel formation ability because agarobiose has a linear structure in which linkage is repeated in the form of α -1,3 bonds. In contrast, agar gel, like agarose, has agar disaccharide as a monomer, but since agar gel contains an acidic group pattern such as a sulfate group, gelation ability is weak. Among them, agarose is decomposed into neoagarobiose (neoagarobiose) through neoagarotetraose (neoagarotetraose) by β -agarase (β -agarase) acting on β -1,4 bonds, and is further decomposed into D-galactose (D-galactose) and 3, 6-endo-L-galactose (3,6-anhydro-L-galactose) finally by α -agarase (α -agarase) acting on α -1,3 bonds. In addition, agarose can be broken down into agarobiose (agarobiose) by dilute acids or alpha-agarase. In general, a neoagaroose is an oligosaccharide obtained by hydrolyzing agar or agarose with β -agarase (β -agarase) and binding 2 to 10 monosaccharides such as neoagarobiose (neoagarobiose), neoagarotetraose (neoagarotetraose), neoagarohexaose (neoagarohexaose), and neoagarooctaose (neoagarooctaose). The term "agar oligosaccharide" means an oligosaccharide obtained by hydrolyzing agar or agarose with a dilute acid or α -agarase, wherein 2 to 10 monosaccharides such as Agarobiose (Agarobiose), Agarotetraose (Agarotetraose), Agarohexaose (Agarohexaose), and Agarooctaose (Agarooctaose) are bonded to each other. The agaro-oligosaccharide may exhibit different physiological activities due to the structural difference between the 3, 6-lacto-L-galactose (3,6-anhydro-L-galactose) as a non-reducing end and the D-galactose (D-galactonase) as a non-reducing end.

On the other hand, Streptomyces coelicolor A3(2), which is an actinomycete, is known to produce a. beta. -agarase (β -agarase) which decomposes agar or agarose (agarose) in the form of extracellular (extracellularly secreted) protein (Stanier et al, 1942, bacteriological impurities; Hodgson and Chater,1981, J.Gen.Microbiol.) and which is encoded by a DagA gene or a DagB gene. Of β -agarases (. beta. -agarases) produced by Streptomyces coelicolor A3(2), DagA enzyme mainly decomposes agar or agarose (agarose) to produce DP4 (neoagarotetraose) and DP6 (neoagarobiose), and DagB enzyme mainly decomposes agar or agarose (agarose) to produce DP2 (neoagarobiose). In addition, it has been reported that DP4 (neoagarotetraose) and DP6 (neoagarase) in a β -agarase (β -agarase) reaction product of agar or agarose (agarose) have higher anti-obesity, anti-diabetic, metabolic diseases such as hyperlipidemia, anti-cancer, and immunity-enhancing effects than DP2 (neoagarase), and thus the DagA gene has an important position in research on production of agarase by actinomycetes. In particular, Streptomyces coelicolor is the most widely used strain in molecular biology research, and the sequence of its chromosomal DNA was analyzed in 2002 by Sanger centre (Sanger centre) in UK and has been published (Bantley et al, 2002, Nature).

Regarding the preparation and use of new agaro-oligosaccharides, korean patent laid-open No. 10-0794593 discloses a marine bacterial strain SL-5(Thalassomonas sp. SL-5) KCCM 10790P having agar decomposition ability and a method for preparing new agaro-oligosaccharides selected from one or more of the group consisting of new agarobiose, new agarotetraose and new agarohexaose using β -agarase produced by the above strain. Also, Korean laid-open patent publication No. 10-1072503 discloses a Curvularia sp.SL-12KCCM 10945P and a method for preparing neo-agaro-oligosaccharides using β -agarase (β -agarase) produced by the above-mentioned strain, wherein the neo-agaro-oligosaccharides are one or more selected from the group consisting of neo-agarobiose, neo-agarotetraose, and neo-agarohexaose. Also, korean granted patent No. 10-1303839 discloses a β -agarase isolated from a pseudoalteromonas (Psuedoalteromonas sp) strain and a method for preparing a new agaro-oligosaccharide using the same, wherein the new agaro-oligosaccharide is one or more selected from the group consisting of new agarotetraose and new agarohexaose. Also, korean patent No. 10-1295659 discloses a β -agarase isolated from a strain of saccharophaga (saccharomyces phagus sp.) and a method for producing neoagaro-oligosaccharides selected from one or more of the group consisting of neoagarotetraose and neoagarohexaose using the same. Also, korean laid-open patent publication No. 10-1212106 discloses a method for producing neoagarobiose by reacting β -agarase isolated from a Saccharophagus (saccharomyces phagus sp.) strain with one or more substrates selected from the group consisting of agar, neoagarotetraose and neoagarohexaose. Also, korean laid-open patent publication No. 10-1206006 discloses an agarase high-producing strain mbrc-1 strain KCCM 11151P (Flammeovirga sp. mbrc-1 KCCM 11151P) having agarolytic activity and a method of reacting β -agarase (β -agarase) produced by the above strain with agar to produce neoagaro-oligosaccharide, wherein the neoagaro-oligosaccharide is one or more selected from the group consisting of neoagarobiose, neoagarotetraose, and neoagarohexaose. Also, Korean patent laid-open publication No. 10-1302655 discloses a method for preparing neoagarotetraose and neoagarohexaose, wherein the above-mentioned preparation method is characterized in that agarase derived from Streptomyces coelicolor is reacted with agarose or agar. Further, Korean laid-open patent publication No. 10-1190078 discloses a recombinant expression vector of β -agarase capable of transforming prokaryotes, which comprises a DNA fragment having the base sequence represented by SEQ ID NO.7 (comprising a promoter of trypsin gene (sprT) derived from Streptomyces coelicolor and a signal peptide coding region (coding region)), and a method for producing β -agarase using the same; and a DNA fragment represented by the base sequence of SEQ ID NO.2 (Signal peptide coding region was removed from the. beta. -agarose gene (DagA) derived from Streptomyces coelicolor). Further, Korean laid-open patent publication No. 10-2014-0060045 discloses an enzymatic production method of neoagarobiose or neoagarotetraose using a novel beta-agarase-producing gene. Also, Korean laid-open patent publication No. 10-2009-0044987 discloses a skin whitening composition comprising neoagarotetraose (neoagarotetraose) as an active ingredient. Further, Korean laid-open patent publication No. 10-2013-0085017 discloses a pharmaceutical composition for preventing or treating skin pigmentation diseases, which comprises 3, 6-diether-L-galactose (3, 6-anhydro-L-galactose); a cosmetic composition for skin whitening or moisturizing comprising 3, 6-lacto-L-galactose (3, 6-anhydro-L-galactose); a pharmaceutical composition for preventing or treating inflammatory diseases, which contains 3,6-anhydro-L-galactose (3, 6-anhydro-L-galactose). As described above, in the prior art, in order to prepare a neoagaropectin having a relatively high content of neoagarotetraose (neoagarotetraose) and neoagarohexaose (neoagarohexaose) from agar or agarose, a method of preparing a transformed strain using genetic recombination or isolating and purifying only DagA from β -agarase expressed by a novel strain or a recombinant strain is used. However, to date, there have been few reports on strains capable of producing DagA β -agarase at a commercially applicable level.

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the background of the prior art, and it is an object of the present invention to provide a mutant strain of streptomyces coelicolor which mainly expresses DagA β -agarase having a relatively high activity compared to a parent strain and hardly expresses DagB β -agarase.

Another object of the present invention is to provide a method for efficiently producing DagA β -agarase in a large amount using a mutant strain of Streptomyces coelicolor.

In addition, another object of the present invention is to provide a method for preparing a neoagaro-oligosaccharide having a higher content of neoagarotetraose (neoagarotetraose) or neoagarohexaose (neoagarohexaose) than neoagarobiose (neoagarobiose) from agar or agarose using a mutant strain of streptomyces coelicolor.

Means for solving the problems

The present inventors induced mutation of a wild-type Streptomyces coelicolor A3(2) strain by ultraviolet irradiation, and screened a Streptomyces coelicolor A3(2) _ M22 strain overexpressing beta-agarase for the first time therein, and applied for a patent (Korean laid-open patent publication No. 10-2018. 0019887, 2018.02.27). The present invention relates to the screening and technical features of Streptomyces coelicolor A3(2) _ M22 strain, and the entire contents of the strain are described in Korean laid-open patent publication No. 10-2018-0019881. The primary selected Streptomyces coelicolor A3(2) _ M22 strain was confirmed to express both DagA and DagB β -agarase. Thereafter, the mutation was induced by irradiating ultraviolet rays again to the primarily selected Streptomyces coelicolor A3(2) _ M22 strain, and finally the present invention was completed by selecting the Streptomyces coelicolor A3(2) _ M22-2C43 strain. Among them, the Streptomyces coelicolor A3(2) _ M22-2C43 strain mainly expresses high-activity DagA beta-agarase, and hardly expresses DagB beta-agarase or expresses DagB mutant enzyme having almost no beta-agarase activity.

To achieve the above object, one example of the present invention provides a Streptomyces coelicolor A3(2) _ M22-2C43 strain (accession No.: KCCM 12577P), the beta-agarase (beta-agarase) activity of a culture solution obtained by culturing the strain under the same conditions or the beta-agarase (beta-agarase) activity of a supernatant recovered from the culture solution is at least 1.2 times or more, preferably at least 1.4 times or more, greater than that of a wild-type Streptomyces coelicolor strain, and compared with a wild Streptomyces coelicolor strain or a Streptomyces coelicolor A3(2) _ M22 strain (accession number: KFCC11668P), the strain mainly expresses DagA beta-agarase, and hardly expresses DagB beta-agarase or expresses DagB mutant enzyme with almost no beta-agarase activity. Although the Streptomyces coelicolor A3(2) _ M22-2C43 strain according to one example of the present invention can be obtained by a variety of known mutation methods, it is preferable to mutate the wild-type Streptomyces coelicolor A3(2) parent strain by irradiating it with ultraviolet rays. Specifically, the Streptomyces coelicolor A3(2) _ M22 strain can be obtained by irradiating a wild-type Streptomyces coelicolor A3(2) parent strain with ultraviolet rays to mutate it, and further, the Streptomyces coelicolor A3(2) _ M22-2C43 strain can be obtained by irradiating the Streptomyces coelicolor A3(2) _ M22 strain with ultraviolet rays to mutate it. The Streptomyces coelicolor A3(2) _ M22-2C43 strain can produce beta-agarase with obviously improved activity compared with a wild Streptomyces coelicolor A3(2) parent strain or express the beta-agarase in an obviously improved mode compared with the parent strain. In addition, when the Streptomyces coelicolor A3(2) _ M22-2C43 strain according to one example of the present invention was compared with a wild-type Streptomyces coelicolor strain or Streptomyces coelicolor A3(2) _ M22 (accession No.: KFCC11668P) strain, a DagB gene (refer to SEQ ID NO.2) modified by point mutation (point mutation) in which guanine (G) as the 1420 th DNA base sequence of the normal DagB gene (refer to SEQ ID NO.1) was replaced with cytosine (C) was obtained. In addition, the DagB gene modified by gene mutation contained in Streptomyces coelicolor A3(2) _ M22-2C43 strain (see SEQ ID NO.2) hardly expressed or expressed the DagB mutant enzyme having almost no β -agarase activity when the strain was cultured. Specifically, a wild-type Streptomyces coelicolor strain or a Streptomyces coelicolor A3(2) _ M22 strain (accession No.: KFCC11668P) expresses DagB β -agarase consisting of the amino acid sequence of SEQ ID NO. 5. In contrast, the Streptomyces coelicolor A3(2) _ M22-2C43 strain expresses a DagB mutant enzyme consisting of the amino acid sequence of SEQ ID NO.6 corresponding to the modified DagB gene (cf. SEQ ID NO. 2). In comparison with the normal DagB β -agarase consisting of the amino acid sequence of SEQ ID No.5, the DagB mutant enzyme consisting of the amino acid sequence of SEQ ID No.6 in which the 474 th amino acid guanine (G) was replaced with arginine (R) was confirmed to have almost no β -agarase activity, in particular, almost no activity of decomposing agar or agarose (agarose) to convert to DP2 (neoagarobiose).

Since the Streptomyces coelicolor A3(2) _ M22-2C43 strain according to one example of the present invention has a DagB gene modified by gene mutation, DagA β -agarase is mainly expressed, and DagB β -agarase is hardly expressed or a DagB mutant enzyme having little β -agarase activity is expressed. Therefore, when a culture broth of Streptomyces coelicolor A3(2) _ M22-2C43 strain or a supernatant of the culture broth according to an example of the present invention is used, neoagaropectide having a content of neoagarotetraose (neoagarotetraose) or neoagarohexaose (neoagarohexaose) higher than that of neoagarobiose (neoagarobiose) can be prepared from agar or agarose.

In order to solve the above-mentioned object, one example of the present invention provides a method for producing a β -agarase, comprising: (a) a step of inoculating the Streptomyces coelicolor A3(2) _ M22-2C43 strain in a liquid medium containing Galactose (Galactose) as a carbon source, and culturing the inoculated liquid medium to obtain a culture solution; and (b) a step of obtaining a supernatant by centrifuging the culture medium. In the method for producing β -agarase according to an example of the present invention, the concentration of galactose in the above liquid medium is preferably 0.5% (w/v) to 4% (w/v) in consideration of the β -agarase (β -agarase) activity of the culture solution or the supernatant recovered from the culture solution, and more preferably 1.0% (w/v) to 2.5% (w/v) in consideration of the DagA enzyme activity of the culture solution or the supernatant recovered from the culture solution. In addition, in view of the β -agarase (β -agarase) activity of the culture solution or the supernatant collected from the culture solution, in the method for producing β -agarase according to an example of the present invention, the culture temperature of the Streptomyces coelicolor A3(2) _ M22-2C43 strain is preferably 25 to 35 ℃, more preferably 28 to 32 ℃. In addition, in view of the β -agarase (β -agarase) activity of the culture solution or the supernatant collected from the culture solution, in the method for producing β -agarase according to an example of the present invention, the culture stirring speed of the Streptomyces coelicolor A3(2) _ M22-2C43 strain is preferably 200 to 300rpm, more preferably 210 to 270 rpm. In addition, the culture time of the Streptomyces coelicolor A3(2) _ M22-2C43 strain is preferably 40 to 150 hours, more preferably 48 to 120 hours, taking into account the β -agarase (β -agarase) activity of the supernatant collected from the culture solution.

In addition, another embodiment of the present invention provides a method for producing β -agarase, comprising: (a) a step of inoculating the Streptomyces coelicolor A3(2) _ M22-2C43 strain in a liquid medium containing Galactose (Galactose) as a carbon source, and culturing the inoculated liquid medium to obtain a culture solution; (b) a step of obtaining a supernatant by centrifuging the culture medium; (c) and a step of precipitating β -agarase contained in the supernatant by adding ammonium sulfate to the supernatant. In the method for producing β -agarase according to another example of the present invention, the concentration of galactose in the above liquid medium is preferably 0.5% (w/v) to 4% (w/v) in consideration of the β -agarase activity of the culture solution or the supernatant recovered from the culture solution, and more preferably 1.0% (w/v) to 2.5% (w/v) in consideration of the DagA enzyme activity of the culture solution or the supernatant recovered from the culture solution. In addition, in the method for producing β -agarase according to another example of the present invention, the culture temperature of the Streptomyces coelicolor A3(2) _ M22-2C43 strain is preferably 25 to 35 ℃, more preferably 28 to 32 ℃ in consideration of the β -agarase (β -agarase) activity of the culture solution or the supernatant collected from the culture solution. In addition, in view of the β -agarase (β -agarase) activity of the culture solution or the supernatant collected from the culture solution, in the method for producing β -agarase according to another example of the present invention, the culture stirring speed of the Streptomyces coelicolor A3(2) _ M22-2C43 strain is preferably 200 to 300rpm, more preferably 210 to 270 rpm. In addition, in view of the β -agarase (β -agarase) activity of the supernatant collected from the culture solution, in the method for producing β -agarase according to another example of the present invention, the culture time of the Streptomyces coelicolor A3(2) _ M22-2C43 strain is preferably 40 to 150 hours, and more preferably 48 to 120 hours. In addition, considering the β -agarase (β -agarase) activity of a product purified from the supernatant of the culture solution, in the method for producing β -agarase according to another example of the present invention, it is preferable to add the ammonium sulfate so that the protein saturation concentration of the supernatant reaches 45% to 70%, and considering the activity of DagA enzyme of a product purified from the supernatant of the culture solution, it is preferable to add the ammonium sulfate so that the protein saturation concentration of the supernatant reaches 45% to 55%.

In order to solve the above-mentioned problems, one embodiment of the present invention provides a method for producing novel agaro-oligosaccharides, comprising: (a') preparing a culture solution of Streptomyces coelicolor A3(2) _ M22-2C43 strain or a supernatant of the culture solution; and (b') enzymatically reacting agar (agar) or agarose (agarose) with β -agarase (β -agarase) present in a culture solution of Streptomyces coelicolor A3(2) _ M22-2C43 strain or a supernatant of the culture solution. Preferably, in the method for producing a novel agaro-oligosaccharide according to one embodiment of the present invention, the culture solution is obtained by inoculating and culturing Streptomyces coelicolor A3(2) _ M22-2C43 strain in a liquid medium containing Galactose (Galactose) as a carbon source. In the method for producing a novel agaro-oligosaccharide according to one embodiment of the present invention, the enzyme reaction temperature is preferably 30 to 45 ℃, and more preferably 35 to 42 ℃.

Effects of the invention

If the Streptomyces coelicolor A3(2) _ M22-2C43 strain according to the present invention is used, a very high activity beta-agarase (beta-agarase) can be produced in large quantities. In addition, the Streptomyces coelicolor A3(2) _ M22-2C43 strain according to the present invention hardly expresses DagB β -agarase or a DagB mutant enzyme expressing almost no β -agarase activity, particularly activity of decomposing agar or agarose (agarose) to convert into DP2(neoagarobiose), and thus it is not necessary to additionally isolate and purify DagA enzyme from a culture solution, and neoagarotetraose (neoagarotetraose) or neoagarohexaose (neoagarobiose) can be prepared from agar or agarose by using a culture solution or supernatant of the Streptomyces coelicolor A3(2) _ M22-2C43 strain, compared with neoagarobiose (neoagarobiose).

Drawings

FIG. 1 is a diagram showing the colony morphology (morphology) of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain. The bottom row in FIG. 1 is the result of staining colonies with a staining reagent.

FIG. 2 shows the results of analyzing the DagA enzyme activity of a supernatant sample obtained by culturing Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain, a 50% ASP sample (an enzyme sample obtained by adding ammonium sulfate to the supernatant so that the saturation concentration of protein becomes 50%), and a 70% ASP sample (an enzyme sample obtained by adding ammonium sulfate to the supernatant so that the saturation concentration of protein becomes 70%) by Thin Layer Chromatography (TLC).

FIG. 3 shows the results of analyzing the new agarose composition in the decomposition product by HPLC-ELSD after decomposing 70% ASP sample obtained by culturing Streptomyces coelicolor A3(2) _ M22-2C43 strain by reaction with agarose (agarose).

FIG. 4 shows a part of the results obtained by aligning and comparing the nucleotide sequences of the DagB gene of Streptomyces coelicolor A3(2) wild-type (WT) strain (upper line) with the nucleotide sequences of the DagB gene of Streptomyces coelicolor A3(2) M22-2C43 strain (lower line).

FIG. 5 shows the biological system (system) and the genetic relationship of Streptomyces coelicolor A3(2) _ M22-2C43 strain prepared based on the 16S rRNA base sequence.

FIG. 6 is an enzymatic cleavage map of pUWL201pw vector used in the present invention for cloning the DagB gene.

FIG. 7 shows the β -agarase (β -agarase) activities of the supernatant obtained during the culture of the recombinant strain WT dagB, recombinant strain M22-2C43 dagB and recombinant strain pUWL201pw prepared in the examples of the present invention, on a cultivation day basis.

FIG. 8 is a result of measuring β -agarase activity of a culture broth of Streptomyces coelicolor A3(2) _ M22-2C43 strain according to the kind of carbon source in various media by a reducing sugar quantitation method.

FIG. 9 is a result of measuring β -agarase activity by a reducing sugar quantitation method after culturing Streptomyces coelicolor A3(2) _ M22-2C43 strain in a medium containing a carbon source under various conditions at a temperature condition of 28 ℃ and shaking conditions of 216 rpm.

FIG. 10 is a result of measuring β -agarase activity by a reducing sugar quantitation method after culturing Streptomyces coelicolor A3(2) _ M22-2C43 strain in a medium containing a carbon source under various conditions at a temperature condition of 30 ℃ and shaking conditions of 250 rpm.

Detailed Description

The present invention will be described more specifically with reference to examples. However, the following examples are only for clearly illustrating the technical features of the present invention and are not intended to limit the scope of the present invention.

1. Enzyme activity measuring method

(1) Measurement of the beta-agarase (beta-agarase) Activity of the samples

The β -agarase (β -agarase) activity of the sample was measured by a reducing sugar assay (dinitrosalicylic acid method (DNS method)). Specifically, 490. mu.L of 20mM Tris-HCl solution (pH 7) in which agarose (agarose) was dissolved at a concentration of 0.5% (w/v) was mixed in 10. mu.L of a sample and reacted at 40 ℃ for 15 minutes, and then a DNS reagent (prepared by dissolving 6.5g of dinitrosalicylic acid (dintrosalicylic acid), 325ml of 2M NaOH, and 45ml of glycerol (glycerol) in 1L of distilled water) in the same amount as the reaction solution was added thereto and boiled for 10 minutes, cooled, and the absorbance was measured at 540 nm. The activity with an absorbance of 0.001 at 540nm was defined as β -agarase (β -agarase) activity of 1U (Unit).

(2) Evaluation of DagA enzyme Activity in samples

Streptomyces coelicolor strains produce the enzyme DagA and DagB as beta-agarases (. beta. -agarases). It was reported that the DagA enzyme mainly produces DP4 (neoagarotetraose) and DP6 (neoagarohexaose) by decomposing agar or agarose (agarose), while the DagB enzyme mainly produces DP2(neoagarobiose) by decomposing agar or agarose (agarose). Agarose (agarose) was reacted with an enzyme in a sample to decompose the enzyme, and then the decomposition product was analyzed by Thin Layer Chromatography (TLC), and amounts of DP2(neoagarobiose), DP4 (neoagarotetraose), and DP6 (neoagarohexaose) contained in the decomposition product were compared in a qualitative manner, thereby evaluating the DagA enzyme activity of the sample. Specifically, the activity of β -agarase (. beta. -agarase) of the sample was adjusted to 250U/ml, and 1ml of a 20mM Tris-HCl (pH 7) solution dissolving β -agarase (. beta. -agarase) at a concentration of 0.5% (w/v) was mixed therein, and reacted at 40 ℃ for 16 hours. Thereafter, the reaction solution was boiled for 10 minutes, and the supernatant was recovered by centrifugation. Thereafter, 5. mu.L of the supernatant was dropped onto a TLC silica gel 60 glass plate (TLC silica gel 60 glass plate), and after development was performed 2 times with a developing solvent (a solution in which butanol, ethanol and sterilized distilled water were mixed in a manner of 5:3:2(v/v), respectively), 10% (v/v) sulfuric acid solution (the base solvent was ethanol) was thinly sprayed, and reacted at 110 ℃ for 15 minutes. Thereafter, the shapes of the developed decomposition products were compared in TLC plates. On the other hand, a solution in which DP2(neoagarobiose), DP4 (neoagarotetraose), and DP6 (neoagarohexaose) were mixed at a concentration of 30mg/ml was used as the standard substance solution. 0.5. mu.L of the standard substance solution was dropped onto a TLC silica gel 60 glass plate and developed by the same method.

2. Induction of mutation of actinomycete Streptomyces coelicolor (Streptomyces coelicolor) by Ultraviolet (UV) irradiation and screening of mutant strains overexpressing beta-agarase

(1) Screening of Streptomyces coelicolor A3(2) _ M22 Strain

Streptomyces coelicolor A3(2) wild-type (WT) strain was subjected to stationary culture on an actinomycete basal medium (Minimal medium); hopwood, 1967) on a plate (plate) for 5 days, and 2ml of a 20% (w/v) glycerol solution was plated on the plate, followed by collecting spores for experiments in which mutation was induced by ultraviolet irradiation. Streptomyces coelicolor A3(2) spore (spore) stock (stock) solution (1 μ L) was added to a Petri dish (petri dish), and TSB (tryptone soy broth) was added as a general bacterial nutrient medium, including 17g of tryptone (tryptone), 3g of soy peptone (soytone), 2.5g of glucose, 5g of NaCl, K on the basis of 1L of distilled water₂PO₄2.5g) of medium 10ml, and a thin film was formed by dilution. Thereafter, the culture solution was irradiated with Ultraviolet (UV) light of 30W intensity for 45 minutes at a height of about 30cm in a state where the peripheral illumination was blocked, and then collected and cultured for 8 hours under a temperature condition of 28 ℃, a shaking condition of 180rpm, and a dark condition. The culture broth was spread on MM agar medium on a plate (plate), and then subjected to static culture in an incubator at 28 ℃ for 8 days under dark conditions. Thereafter, surviving colonies (colony) were counted in the cultured plates (plate) and stained with a staining reagent (Congo red). Among them, 1581 colonies having a large clear zone (clear zone) size were screened for the first time. From the primary screened colonies, 1400 colonies (colony) were individually coated with MM liquid medium [ containing 2% (w/v) concentration of agarose (agarose) as carbon source []The cells were plated on glass fiber filter paper (glass filter paper) and incubated at 28 ℃ for 5 days, and then 313 strains having a large number of spores (spoore) were selected twice. The secondarily selected strain was inoculated to RSM3 (containing MgCl based on 1L of distilled water) containing agar oligosaccharide (agaroligosaccharide) at a concentration of 2% (w/v)₂·7H₂O5 g, yeast extract 11g, CaCO₃0.5g) in liquid medium and at a temperature of 28 ℃ andthe culture was carried out for 2.5 days under shaking at 180 rpm. Thereafter, cell debris (cell debris) was removed by centrifuging the culture solution, and the supernatant was recovered. Thereafter, the activity of β -agarase (β -agarase) in the purified supernatant was measured by a reducing sugar quantitation method (DNS method). In addition, Streptomyces coelicolor A3(2) wild-type (WT) strain as a parent strain was also cultured by the same method and conditions, and then the activity of β -agarase (β -agarase) in the purified supernatant was measured. The activity of the wild-type (WT) strain of Streptomyces coelicolor A3(2) was compared with that of the secondarily screened strain, and a mutant strain in which the activity of β -agarase (β -agarase) was the highest was selected as the final strain and named as Streptomyces coelicolor A3(2) _ M22.

(2) Information on the preservation of Streptomyces coelicolor A3(2) _ M22 strain the inventors of the present invention carried out Korean patent preservation of Streptomyces coelicolor A3(2) _ M22 strain finally selected at 17.06.2016 at Korea culture Collection (address: 2. sup. methyl 45. sup. julian Julian 3F, Ministry of Japan) at Korea microorganism culture Collection, named Korea culture Collection, 2, Korea, Seoul, Korea, and the preservation number is KF116CC 3868 11668P.

3. Induction of mutation of Streptomyces coelicolor A3(2) _ M22 strain by ultraviolet irradiation and screening of mutant strain overexpressing DagA beta-agarase

(1) Screening of Streptomyces coelicolor A3(2) _ M22-2C43 Strain

Streptomyces coelicolor A3(2) _ M22 strain (mutant strain of Streptomyces coelicolor A3(2) wild-type (WT) strain) was cultured on an actinomycete complete medium (ISP4 medium) on a plate (plate) for 5 days at rest, and 2ml of a 20% (w/v) glycerol solution was plated on the plate, followed by collection of spores (spore) for experiments in which mutation was induced by ultraviolet irradiation. 1. mu.L of a spore stock (spore stock) solution of Streptomyces coelicolor A3(2) _ M22 strain was added to a Petri dish(s)Petri dish) and TSB (tryptone soy broth; based on 1L of distilled water, the extract comprises 17g of tryptone, 3g of soybean peptone, 2.5g of glucose, 5g of NaCl and K₂PO₄2.5g) 5ml of medium and formed into a thin film by dilution. Then, the culture medium is irradiated with Ultraviolet (UV) rays of 30 to 40W intensity for 24 to 60 minutes at a distance of about 35 to 50cm with ambient illumination blocked, and then the culture medium is collected and cultured for 8 hours under a temperature condition of 28 ℃, a shaking condition of 180rpm, and a dark condition. The culture broth was smeared on MM agar medium and then cultured statically in an incubator at 28 ℃ for 5 days under dark conditions. Thereafter, surviving colonies (colony) were counted in the cultured plates (plate), at which point the mortality rate was 99.2%. After these clear circles (clear zones) were stained with a staining reagent (Lugol's Iodine) and compared in size, strains of different colonies were selected according to morphology (morpholinoy), and cultured on MM agar medium on a plate for 5 days at 28 ℃. Then, the selected strain was inoculated into a liquid medium containing 0.5% (w/v) agarose (containing MgCl. based on 1L of distilled water)₂·7H₂O5 g, yeast extract (yeast extract)11g, CaCO₃0.5g) and cultured with shaking at a temperature of 28 ℃ and a shaking speed of 216rpm for 2.5 days. Thereafter, cell debris (cell debris) was removed by centrifuging the culture solution, and the supernatant was recovered. Thereafter, the supernatant was sterilized and filtered with a 0.45 μm syringe filter (syringe filter) to obtain a purified supernatant. Then, the activity of β -agarase (β -agarase) and the activity of DagA enzyme of the purified supernatant were measured. The mutant strain with the highest beta-agarase (beta-agarase) activity and the highest DagA enzyme activity was selected as the final strain and named Streptomyces coelicolor A3(2) _ M22-2C 43.

(2) Preservation information of Streptomyces coelicolor A3(2) _ M22-2C43 strain

The inventors of the present invention carried out Korean patent preservation of Streptomyces coelicolor A3(2) _ M22-2C43 strain finally selected at 22.09.2017 in Korea at the Korean culture Collection (address: 2 Torrel Seisaku Kogyo, 2 Torrel provincial gate district, Hongji, 45 Breynia mansion, 3F) as an international preservation organization, and the preservation number was KFCC 11742P. In addition, the present inventors requested, on 23.08.2019, the transformation of the Streptomyces coelicolor A3(2) _ M22-2C43 strain (accession No.: KFCC11742P) of the above Korean patent deposit into the International patent deposit under the Budapest Treaty (Budapest treat), which was assigned accession No. KCCM 12577P.

4. Comparison of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain

(1) Comparison of colony morphology (morphology) of the respective strains

FIG. 1 is a diagram showing the colony morphology (morphology) of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain. The lower row of the graph in FIG. 1 shows the result of staining the colony (colony) with a staining reagent.

(2) Culture of Each Strain and preparation of enzyme sample containing beta-agarase

The strains were inoculated in liquid media (containing MgCl. based on 1L of distilled water) containing 0.5% (w/v) agarose, respectively₂·7H₂O5 g, yeast extract 11g, CaCO₃0.5g) was added to 1000ml, and cultured with shaking at 216rpm for 2.5 days at a temperature of 28 ℃. Thereafter, cell debris (cell debris) was removed by centrifuging the culture solution, and the supernatant was recovered. The collected supernatant was used as a sample to measure the activity of β -agarase (β -agarase), and the activity of DagA enzyme was evaluated. Thereafter, the supernatant was sterilized and filtered with a 0.45 μm syringe filter to obtain a purified supernatant. Thereafter, ammonium sulfate was added to the purified supernatant liquid so that the saturated concentrations of the proteins contained in the supernatant liquid reached respectivelyTo 50% and 70%, and precipitating the beta-agarase (beta-agarase) by Ammonium Sulfate Precipitation (ASP), which is one of salting-out methods, followed by centrifugal separation to obtain the purified beta-agarase (beta-agarase) in the form of pellet. According to the contents described in the specification of the Streptomyces coelicolor A3(2) _ M22 strain (Korean laid-open patent publication No. 10-2018-0019881, 2018.02.27), it was indirectly confirmed that when the upper layer containing ammonium sulfate was at a protein saturation concentration of 50%, the DagA enzyme was mainly precipitated, and when the upper layer containing ammonium sulfate was at a protein saturation concentration of 70%, both the DagA enzyme and the DagB enzyme were precipitated. The purified β -agarase (β -agarase) in the form of a pellet was dissolved in 5ml of distilled water, and then the β -agarase (β -agarase) activity was measured and the DagA enzyme activity was evaluated using it as a sample.

(3) Comparison of the beta-agarase (beta-agarase) Activity of enzyme samples obtained from the respective strains

The following table 1 shows the results of measuring β -agarase (β -agarase) activities of supernatant samples obtained by culturing Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain, 50% ASP samples (enzyme samples obtained by adding ammonium sulfate to the supernatant so that the saturation concentration of protein becomes 50%), and 70% ASP samples (enzyme samples obtained by adding ammonium sulfate to the supernatant so that the saturation concentration of protein becomes 70%). The unit of β -agarase (β -agarase) activity is U/ml.

[ Table 1]

As shown in Table 1 above, the highest β -agarase (β -agarase) activity was exhibited in 50% of the ASP samples predicted to be composed mainly of DagA enzyme, which were recovered from the culture broth of Streptomyces coelicolor A3(2) _ M22-2C43 strain.

(4) FIG. 2 shows the results of analyzing the DagA enzyme activity of a supernatant sample obtained by culturing Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) M22 strain, and Streptomyces coelicolor A3(2) M22-2C43 strain, a 50% ASP sample (an enzyme sample obtained by adding ammonium sulfate to the supernatant so that the saturation concentration of protein becomes 50%), and a 70% ASP sample (an enzyme sample obtained by adding ammonium sulfate to the supernatant so that the saturation concentration of protein becomes 70%) by Thin Layer Chromatography (TLC). In FIG. 2, "M" represents a standard substance solution, and lanes "1", "4" and "7" are all enzyme samples obtained from a culture solution of a wild-type (WT) strain of Streptomyces coelicolor A3(2), and lanes "2", "5" and "9" are all samples obtained from a culture solution of a strain of Streptomyces coelicolor A3(2) _ M22, and lanes "3", "6" and "9" are all samples obtained from a culture medium of a strain of Streptomyces coelicolor A3(2) _ M22-2C 43. In addition, lanes "1", "2" and "3" are supernatant samples, lanes "4", "5" and "6" are 70% ASP samples, and lanes "7", "8" and "9" are 50% ASP samples.

As shown in FIG. 2, all enzyme samples obtained from the culture broth of Streptomyces coelicolor A3(2) _ M22-2C43 strain decomposed agarose (agarose) to produce mainly DP4 (neoagarotetraose) and DP6 (neoagarohexaose) regardless of the degree of isolation and purification. In contrast, a supernatant sample obtained from a culture broth of Streptomyces coelicolor A3(2) _ M22 strain and a 70% ASP sample decomposed agarose (agarose) to produce mainly DP2 (neoagarobiose).

FIG. 3 shows the results of analyzing the composition of new agarose in a decomposition product by HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) after decomposing agarose (agarose) by reacting it with a 70% ASP sample obtained by culturing Streptomyces coelicolor A3(2) _ M22-2C43 strain. The agarose (agarose) degradation reaction conditions were the same as those used in the evaluation of the activity of DagA enzyme, and the agarose degradation reaction was repeated 4 times in total under the same conditions. When the composition of the fresh agarose in the decomposition product was analyzed by HPLC-ELSD, NH 2P-504E multimodal column (NH 2P-504E multimodal column) (250 mm. times.4.6 mm) was used as the column, and a mixed solution of acetonitrile (acetonitrile) and water (acetonitrile: water mixing ratio by weight 65:35) was used as the mobile phase. As shown in fig. 3, the content of DP4 (neoagarose) in the agarose (agarose) decomposition product was 5 to 5.5 times that of DP2(neoagarobiose), and the content of DP6 (neoagarobiose) was 3 to 3.5 times that of DP2 (neoagarobiose).

(5) Beta-agarase (beta-agarase) gene information of each strain

The DagA gene and the DagB gene of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain were amplified by PCR reaction, and the DNA base sequences of the amplified PCR products were analyzed. The DagB genes of Streptomyces coelicolor A3(2) wild-type (WT) strain and Streptomyces coelicolor A3(2) _ M22 strain all show the same nucleotide sequence as SEQ ID No. 1. In contrast, the DagB gene of Streptomyces coelicolor A3(2) _ M22-2C43 strain was modified by gene mutation in an alternative manner and represented by the base sequence of SEQ ID NO. 2. FIG. 4 shows a part of the results of comparison by aligning the nucleotide sequence of the DagB gene of Streptomyces coelicolor A3(2) wild-type (WT) strain (upper line) with the nucleotide sequence of the DagB gene of Streptomyces coelicolor A3(2) M22-2C43 strain (lower line). On the other hand, the DagA genes of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain, and Streptomyces coelicolor A3(2) _ M22-2C43 strain all exhibited the same nucleotide sequence as that of SEQ ID NO. 3. Therefore, it is predicted that Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2) _ M22 strain and Streptomyces coelicolor A3(2) _ M22-2C43 strain all express the DagA enzyme having the amino acid sequence of SEQ ID NO. 4. In addition, Streptomyces coelicolor A3(2) wild-type (WT) strain, and Streptomyces coelicolor A3(2) _ M22 strain were predicted to express the normal DagB enzyme having the amino acid sequence of SEQ ID NO. 5. In contrast, it was predicted that the Streptomyces coelicolor A3(2) _ M22-2C43 strain did not express the normal DagB enzyme or could express the mutant DagB enzyme having the amino acid sequence of SEQ ID NO.6 and having almost no β -agarase (β -agarase) activity.

(6) System and genetic relationship of Streptomyces coelicolor A3(2) _ M22-2C43 strain

The 16S rRNA base sequence of Streptomyces coelicolor A3(2) wild-type (WT) strain and the 16S rRNA base sequence of Streptomyces coelicolor A3(2) _ M22-2C43 strain were analyzed by colony (colony) PCR. FIG. 5 shows the biological system and genetic relationship of Streptomyces coelicolor A3(2) _ M22-2C43 strain prepared based on the 16S rRNA base sequence.

(7) Comparison of expression levels according to the DagB Gene

A recombinant vector was prepared by cloning the DagB gene of Streptomyces coelicolor A3(2) wild-type (WT) strain to pUWL201pw vector having the cleavage map of FIG. 6, and a recombinant strain WT DagB was prepared by transforming Streptomyces lividans TK24 strain having no β -agarase (β -agarase) gene using the above recombinant vector. Further, a recombinant vector was prepared by cloning the DagB gene of Streptomyces coelicolor A3(2) _ M22-2C43 strain into pUWL201pw vector, and the recombinant vector was used to transform Streptomyces lividans TK24 strain to prepare recombinant strain M22-2C43 dagB. In addition, a recombinant strain pUWL201pw was prepared by transforming Streptomyces lividans (Streptomyces lividans) TK24 strain with pUWL201pw vector. Thereafter, 3 recombinant strains were cultured, and the culture solution was recovered on the first, second and third days of the culture to obtain a supernatant. Then, the β -agarase (β -agarase) activity of the supernatant sample was measured by a reducing sugar assay (DNS method). FIG. 7 shows β -agarase (β -agarase) activities of supernatant obtained during the culture of the recombinant strain WT dagB, recombinant strain M22-2C43 dagB and recombinant strain pUWL201pw prepared in the examples of the present invention, according to different culture days. As shown in FIG. 7, the DagB gene of Streptomyces coelicolor A3(2) wild-type (WT) strain was expressed at a higher level in the form of normal DagB enzyme having β -agarase (β -agarase) activity, whereas the modified DagB gene of Streptomyces coelicolor A3(2) _ M22-2C43 strain was expressed in the form of DagB mutant enzyme not expressing normal DagB enzyme or hardly expressing β -agarase (β -agarase) activity.

5. Establishing optimal culture conditions for beta-agarase (beta-agarase) of Streptomyces coelicolor A3(2) _ M22-2C43 strain

(1) The type and content of carbon source in the culture medium

Streptomyces coelicolor A3(2) _ M22-2C43 strain was cultured for 4 days on an actinomycete agar medium containing agar at a concentration of 1.5% by weight as a carbon source. Thereafter, in 50ml of RSM3 liquid medium containing one selected from Galactose (Galactose) at a concentration of 1.5% (w/v), succinic acid (succinic acid) at a concentration of 2% (w/v), Glucose (Glucose) at a concentration of 1.5% (w/v), agar (agar) at a concentration of 0.2% (w/v), and agar (agar) at a concentration of 0.5% (w/v) as a carbon source, 3 colonies of strains having a size of 1cm × 1cm were inoculated, and cultured under a temperature condition of 28 ℃ and a shaking condition of 216rpm for 2.5 days. Thereafter, the supernatant was recovered from the culture solution, and β -agarase activity contained in the supernatant was measured.

FIG. 8 shows the results of measuring the β -agarase activity of a culture broth of Streptomyces coelicolor A3(2) _ M22-2C43 strain according to the kind of carbon source in various media by a reducing sugar quantitation method. In FIG. 8, the Y-axis represents the β -agarase activity and the X-axis represents the incubation time. Table 2 below shows the β -agarase activity of the culture broth according to the kind of carbon source in the different media when Streptomyces coelicolor A3(2) _ M22-2C43 strain was cultured for 96 hours.

[ Table 2]

As shown in FIG. 8 and Table 2 above, galactose was used as the carbon source in the medium for optimum production of β -agarase from Streptomyces coelicolor A3(2) _ M22-2C43 strain, and the concentration thereof was confirmed to be 1.5% (w/v).

(2) Culture temperature and culture stirring speed (rpm)

3 colonies of a strain having a size of 1cm × 1cm were inoculated in 50ml of RSM3 liquid medium containing Galactose (Galactose) at a concentration of 1% (w/v) and agar (agar) at a concentration of 0.5% (w/v) as carbon sources, 50ml of RSM3 liquid medium containing Galactose (Galactose) at a concentration of 1.5% (w/v) and agar (agar) at a concentration of 0.5% (w/v) as carbon sources, and 50ml of liquid medium containing agar (agar) at a concentration of 0.5% (w/v) as carbon sources, respectively, and cultured for 2.5 days under a temperature condition of 28 ℃ and a shaking condition of 216rpm or a temperature condition of 30 ℃ and a shaking condition of 250 rpm. Thereafter, the supernatant was recovered from the culture broth, and β -agarase activity contained in the supernatant was measured.

FIG. 9 is a result of measuring β -agarase activity by a reducing sugar quantitation method when Streptomyces coelicolor A3(2) _ M22-2C43 strain is cultured under a temperature condition of 28 ℃ and a shaking condition of 216rpm in a medium containing a carbon source under various conditions. FIG. 10 is a result of measuring β -agarase activity by a reducing sugar quantitation method when Streptomyces coelicolor A3(2) _ M22-2C43 strain is cultured under a temperature condition of 30 ℃ and a shaking condition of 250rpm in a medium containing a carbon source under various conditions. In FIGS. 9 and 10, the Y-axis represents the β -agarase activity and the X-axis represents the incubation time. As shown in FIGS. 9 and 10, when Streptomyces coelicolor A3(2) _ M22-2C43 strain was cultured, the culture conditions for optimum production of β -agarase were confirmed to be a temperature of 30 ℃ and a shaking (stirring speed) of 250 rpm.

As described above, although the present invention has been described by the above-described embodiments, the present invention is not limited thereto. Of course, many modifications may be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the scope of the invention be construed as including all embodiments within the scope of the appended claims.

[ accession number ]

The name of the depository institution: korean Collection of microorganisms

The preservation number is: KFCC11668P

The registration date: 2016.06.17

[ accession number ]

The name of the depository institution: korean Collection of microorganisms

The preservation number is: KFCC11742P

The registration date: 2017.09.22

[ accession number ]

The name of the depository institution: korean Collection of microorganisms

The preservation number is: KCCM12577P

And (3) converting the date: 2019.08.23

Microorganism preservation certificate

2016 (6 months) and 17 days

So that: plum economic and virtuous

The microorganisms deposited by the inventors were accepted as 16 th to 19 th on 6 th and 17 th 2016, and the following microorganism deposit numbers were reported.

The following-

1. Name of microorganism: streptomyces coelicolor A3(2) _ M22

2. Microorganism collection number: KFCC11668P

Korean Collection of microorganisms

Microorganism preservation certificate

9 and 22 months in 2017

So that: dainebiao Limited

The microorganisms deposited by the noble companies were accepted as No. 17-26 on day 22 of 9/2017, and the following microorganism deposit numbers were reported.

The following-

1. Name of microorganism: streptomyces coelicolor A3(2) _ M22-2C43

2. Microorganism collection number: KFCC11742P

Korean Collection of microorganisms

Proof of deposit of microorganisms under the Budapest treaty for patent applications

International template

Proof of original deposit issued by the International depositary agency under the provisions of 7.1

So that: dainebiao Limited

China Mogu road No. 45 (14 (13209) of Central China, south City, Kyogi province, Korea)

¹The date of legal status of the international depository is obtained by applying rule 6.4 (d).

<110> Daindiobio Limited

DyneBio Inc.

<120> Streptomyces coelicolor mutant strain, beta-agarase production method using the same and method for preparing new agaro-oligosaccharide using the same

Mutant strain of Streptomyces coelicolor, method for producing

beta-agarase using the same and method for manufacturing

neoagarooligosaccharide using the same

<130> ZPCT-19-0108

<150> KR 10-2018-0112517

<151> 2018-09-19

<160> 6

<170> KopatentIn 2.0

<210> 1

<211> 2397

<212> DNA

<213> unknown

<220>

<223> DagB Gene derived from Streptomyces coelicolor A3(2)

<400> 1

atgaccgtgc acaagcgcgc ttgcaccact ccgccgccgc gagccagcag gtcgttccgc 60

gtgaggtggc ctgtcctgat agcggccgcc tgcgccgggt tagtcctggc gaccaccagc 120

cctccggccg tcgcggccgg cgctcatgac ctcggcgacg agaccatgct ctacgacttc 180

caggacggcc tggtaccggc cgaggtcggc ccgtaccagg cgaagacgac aatcgtcggt 240

cgcggcgaca agaagctccg ggtcgatttc caggcccgga agaactacta ctcctcgttc 300

tccgtacgcc ccgagcccgt gtggaactgg tcggcggagg agtccgagtc gctcggcatc 360

gcgatggagc tcacgaaccc gagcgaccgc tccgtccagc tcacgatcga tctggagagc 420

tcgaccggcg tcgccacccg cagtgtcaac gtcccggccg gcggcggtgg cacgtactac 480

ttcgacgtcg acagccccgc gctccaccgc gacaccgggc tgcgcgccga tccgtcctgg 540

ctcgcggaca aggacgtcac ctctgcggtc tggatgtggg gctccaagga gacggacacg 600

agccgcatca gccagctgaa cttctacgtc gccggcctgc tgcacgaccg gtcggtcatc 660

gtggacgaca tccgcgtcgt ccgtgacgcg ccggcagacc ccgattacct caagggcctg 720

gtcgacgcct tcggtcagaa caacaaggtc gactacaagg gaaaggtctc caggacgtcg 780

gagattcttc ggcagcgcgc tgccgaagcc aaggaccttc gcaggcatcc ggttccggag 840

gaccggtcca ggtacggcgg ctggctgaac ggtccacgac tcgaggcgac gggcaacttc 900

cgcgtggaga agtaccaggg gcggtggacc ctggtggacc ctgacggcta cctgttcttc 960

tcaaccggca tcgacaacgc ccgcatgttc gactccccaa ccacgacggg ttacgacttc 1020

gaccatgacg cgatccagga gctgccgccc cccagcctga cggccggcgg ccccgaggac 1080

ctcaaccgcg tccagaagtc ggcgctgccg acccgcacga agatgtccga aacccgcgcc 1140

gacctcttca gcaagttgcc caagtaccgc acccgcgcgg gcgagggctt cggttacgcc 1200

cccgacaccc tggccggtcc cgtcgcgcag ggcgagacct acagcttcta caaggcgaac 1260

gtcgcccgga agtaccccgg cagcaactac atggagcggt ggcgggacaa cacggtcgac 1320

cggatgctca gctggggctt cacctccttc ggcaactgga ccgacccgga gatgtacgac 1380

aacgaccgta tcccgtactt cgcccacggc tggatcaagg gcgacttcaa gacggtgagc 1440

accggccagg actactgggg cccgatgccg gacccgttcg accccgcgtt ctccgacgcc 1500

gcagccagaa ccgcgcgagc agtcgccgac gaggtcgcgg acagcccgtt ggcgatcggc 1560

gtattcatgg acaacgaact gagctggggc aacgccggca gtttcagcac ccgttacggc 1620

gtcgtcatcg acaccatgtc acgtgacgcg gcagagagcc ccaccaagtc ggcgttctcc 1680

gacgaactgg aggagaagta cgggaccatc gacgctctca acgccgcgtg gcagacgaca 1740

gttccgtcat gggaggcact ccgtagcggc agtgccgacc tcggctccga cgagaccgcg 1800

aaggagtccg actactccgc gctcatgacg ctctacgcca ctcagtactt caagacggtc 1860

gacgccgagc tcgacaaggt catgccggac catctctacg cgggttcgag gttcgccagc 1920

tggggccgca caccggaggt cgtcgaggca gcgagcaagt acgtcgacat catgagctac 1980

aacgagtacc gcgagggact gcacccgagc gagtgggcgt ttctcgaaga gctcgacaag 2040

cccagcctca tcggtgagtt ccacatggga acgaccacta ccgggcagcc gcatccgggt 2100

ctcgtctcgg cgggaacgca ggccgagcgg gcacggatgt acgccgagta catggaacag 2160

ctcatcgaca acccgtacat ggtgggcggc cactggttcc agtatgccga ctcgcccgtg 2220

actggcagag cactcgacgg ggagaactac aacattggct tcgtctccgt cacggaccgt 2280

ccctacccgg agatcgtcgc cgctgcccgc gacgtgaacc agcgtctcta tgaccgccga 2340

tacggcaacc tggccacggc cgagggacat tacaccggtc ggcgttcagc ggagtag 2397

<210> 2

<211> 2397

<212> DNA

<213> unknown

<220>

<223> DagB Gene derived from Streptomyces coelicolor A3(2) _ M22-2C43

<400> 2