<i>Streptomyces coelicolor </i>mutant strain, method of producing β-agarase by using same, and method of producing neoagaro-oligosaccharides by using same

Abstract

The present invention provides Streptomyces coelicolor strain A3(2)_M22-2C43 obtained by inducing a point mutation in the base sequence of the DagB gene in a wild-type Streptomyces coelicolor strain A3(2) by UV radiation. Since the Streptomyces coelicolor strain A3(2)_M22-2C43 according to the present invention expresses a DagB mutant enzyme expressing little or no DagB beta-agarase or exhibiting little or no beta-agarase activity, there is no need for separate isolation and purification of DagA enzymes from culture fluid, and the culture fluid of the Streptomyces coelicolor strain A3(2)_M22-2C43 or supernatant thereof may be used to produce, from agar or agarose, neoagarose oligosaccharides with a higher content of neoagarotetraose or neoagarohexaose than that of neoagarobiose.

Claims

1. A Streptomyces coelicolor A3(2)_M22-2C43 strain having the accession number KCCM 12577P.

2. A method for producing β-agarase, the method comprising: (a) inoculating and culturing the Streptomyces coelicolor A3(2)_M22-2C43 strain having the accession number KCCM 12577P of claim 1 into a liquid culture medium containing galactose as a carbon source, thereby obtaining a culture fluid; and (b) centrifuging the culture fluid to obtain a supernatant.

3. The method of claim 2, wherein a concentration of galactose in the liquid culture medium is 0.5% (w/v) to 4% (w/v).

4. The method of claim 2, wherein a culture temperature of the Streptomyces coelicolor A3(2)_M22-2C43 strain having the accession number KCCM 12577P is 25 to 35° C. and a culturing agitation speed thereof is 200 to 300 rpm.

5. The method of claim 2, wherein a culturing duration of the Streptomyces coelicolor A3(2)_M22-2C43 strain having the accession number KCCM 12577P is 40 to 150 hr.

6. A method for producing β-agarase, the method comprising: (a) inoculating and culturing the Streptomyces coelicolor A3(2)_M22-2C43 strain having the accession number KCCM 12577P of claim 1 into a liquid culture medium containing galactose as a carbon source, thereby obtaining a culture fluid; (b) centrifuging the culture fluid to obtain a supernatant; and (c) precipitating the β-agarase contained in the supernatant by adding ammonium sulfate to the supernatant.

7. The method of claim 6, wherein the ammonium sulfate is added so that a protein saturation concentration of the supernatant is 45% to 70%.

8. A neoagarooligosaccharide preparation method comprising: (a′) preparing a culture fluid of the Streptomyces coelicolor A3(2)_M22-2C43 strain having the accession number KCCM 12577P of claim 1 or a supernatant of the culture fluid; and (b′) performing enzymatic reaction of agar or agarose with β-agarase present in the culture fluid or the supernatant.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a photograph showing the colony morphology of each of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2)_M22 strain, and Streptomyces coelicolor A3(2) M22-2C43 strain. A picture in a lower row in FIG. 1 shows the result of staining colonies with a dyeing reagent.

(2) FIG. 2 shows analysis results of the DagA enzymic activity of each of supernatant samples obtained through culture of 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 so that the saturation concentration of the protein contained in the supernatant becomes 50%), and a 70% ASP sample (an enzyme sample obtained by adding ammonium sulfate so that the saturation concentration of the protein contained in the supernatant is 70%) using a thin layer chromatography (TLC) method.

(3) FIG. 3 shows an analyzing result of a composition of neo-agarosaccharide in a degradation product using HPLC-ELSD, wherein the degradation product is obtained by reacting agarose with a 70% ASP sample obtained through culture of Streptomyces coelicolor A3(2)_M22-2C43 strain.

(4) FIG. 4 shows some of comparison results of the DagB gene base sequence (upper line; positions 1201-1560 of SEQ ID NO: 1) of the Streptomyces coelicolor A3(2) wild-type (WT) strain and the DagB gene base sequence (lower line; positions 1201-1560 of SEQ ID NO: 2) of the Streptomyces coelicolor A3(2)_M22-2C43 strain as aligned with each other.

(5) FIG. 5 shows the biological lineage and relationship of the Streptomyces coelicolor A3(2)_M22-2C43 strain prepared based on the 16S rRNA base sequence.

(6) FIG. 6 is a cleavage map of the pUWL201pw vector used to clone the DagB gene according to the present disclosure.

(7) FIG. 7 shows the β-agarase activity of each of supernatants obtained from culture of recombinant strain WT dagB, recombinant strain M22-2C43 dagB and recombinant strain pUWL201pw prepared in the examples of the present disclosure based on the culture date.

(8) FIG. 8 shows the result of measuring β-agarase activity of Streptomyces coelicolor A3(2)_M22-2C43 strain culture fluid based on the type of carbon source in the culture medium by a reducing sugar quantitative assay method.

(9) FIG. 9 shows the result of measurement of the β-agarase activity of the culture fluid when Streptomyces coelicolor A3(2)_M22-2C43 strain is cultured in a culture medium containing carbon sources under the temperature condition at 28° C. and the shaking condition at 216 rpm, using a reducing sugar quantitative assay method.

(10) FIG. 10 shows the result of measurement of the β-agarase activity of the culture fluid when Streptomyces coelicolor A3(2)_M22-2C43 strain is cultured in a culture medium containing carbon sources under the temperature conditions at 30° C. and shaking conditions at 250 rpm, using a reducing sugar quantitative assay method.

DETAILED DESCRIPTION OF EMBODIMENT

(11) Hereinafter, the present disclosure will be described in more detail based on Examples. However, the following Examples are intended to clearly illustrate the technical characteristics of the present disclosure, and do not limit the scope of protection of the present disclosure.

(12) 1. Method for Measuring Enzymic Activity

(13) (1) Measurement of β-Agarase Activity of Sample

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

(15) (2) Evaluation of DagA Enzymic Activity of Sample

(16) The Streptomyces coelicolor strain produces DagA and DagB enzymes using β-agarase. DagA enzyme is known to degrade agar or agarose to produce mainly DP4 (neoagarotetraose) and DP6 (neoagarohexaose), while DagB enzyme is reported to produce DP2 (neoagarobiose) mainly by decomposing agar or agarose. DagA enzymic activity of the sample was evaluated by reacting agarose with an enzyme in the sample to decompose the same and then analyzing the decomposition product using a thin layer chromatography (TLC) method, and performing qualitative comparison between amounts of DP2 (neoagarobiose), DP4 (neoagarotetraose), and DP6 (neoagarohexaose) contained in the degradation product. Specifically, the β-agarase activity of the sample was adjusted to 250 U/ml, and 1 ml of a 20 mM Tris-HCl solution (pH 7) ion which the agarose was dissolved at a concentration of 0.5% (w/v) was mixed therewith and the mixture was reacted at 40° C. for 16 hr. Thereafter, the reaction solution was boiled for 10 minutes and then centrifuged to collect the supernatant. Thereafter, 5 μl of the supernatant was added dropwise to a TLC silica gel 60 glass plate, and was developed twice with a developing solvent (a mixed solution of butanol, ethanol, and sterile distilled water at a ratio of 5:3:2 (v/v)). A 10% (v/v) sulfuric acid solution (base solvent being ethanol) was sprayed thinly thereto and then reaction occurred at 110° C. for 15 minutes. Thereafter, patterns of degradation products developed on the TLC plates were compared with each other. In one example, a solution in which each of DP2 (neoagarobiose), DP4 (neoagarotetraose), and DP6 (neoagarohexaose) was mixed therewith at a concentration of 30 mg/ml was used as a standard solution. 0.5 μl of the standard solution was dropped on a TLC silica gel 60 glass plate and was developed using the same method as above.

(17) 2. Mutation Induction of Actinomyces Streptomyces Coelicolor using Ultraviolet (UV) Irradiation and Selection of Mutation Strain Overexpressing β-Agarase

(18) (1) Selection of Streptomyces Coelicolor A3(2)_M22 strain

(19) Streptomyces coelicolor A3(2) wild-type (WT) strain was stationary-cultured for 5 days in actinomyces minimal culture medium (Minimal medium, MM; Hopwood, 1967) on a plate. After dispensing 2 ml of a 20% (w/v) glycerol solution on the plate, spores were collected and used for mutation induction experiments by UV irradiation. 1 μl of Streptomyces coelicolor A3(2) spore stock solution was input into a Petri dish, and 10 ml of a tryptic soy broth (TSB) culture medium (containing 17 g of tryptone, 3 g of soytone, 2.5 g of glucose, 5 g of NaCl, and 2.5 g of K.sub.2HPO.sub.4) based on 1 liter of distilled water) as a general bacterial nutrient culture medium was added thereto and the solution was diluted to form a thin film. Thereafter, after irradiating 30 W ultraviolet (UV) thereto for 45 minutes at a height of about 30 cm while ambient light was blocked, we collected a culture fluid and incubated the same for 8 hr under 28° C. temperature condition, 180 rpm shaking condition and in the dark condition. The culture fluid was plated on a MM agar culture medium on the plate, and then incubated for 8 days in an incubator at 28° C. under the dark condition. Subsequently, viable colonies were counted on the plate, and were stained using a dyeing reagent (Congo red). 1,581 colonies with a large clear zone size were first selected. 1,400 colonies among the first selected colonies were individually dispensed on a glass filter paper coated with a MM liquid culture medium [containing 2% (w/v) concentration of agarose as a carbon source] and then were subjected to stationary culture at 28° C. for 5 days, and then 313 strains with many spore formations were second selected. The second selected strains were inoculated into a RSM3 liquid culture medium (containing 5 g MgCl.sub.2.7H.sub.2O, 11 g yeast extract, and 0.5 g CaCO.sub.3 based on 1 liter of distilled water) containing 2% (w/v) concentration of agarooligosaccharide. Then, incubation thereof was carried out for 2.5 days under a temperature condition of 28° C. and a shaking condition of 180 rpm. Subsequently, the culture fluid was centrifuged such that cell debris was removed therefrom, and then the supernatant was collected. Subsequently, the supernatant was sterilized and filtered with a 0.45 μm syringe filter to collect the purified supernatant. Thereafter, the β-agarase activity of the purified supernatant was measured using the reducing sugar quantitative method (DNS method). Further, the parent strain, that is, the Streptomyces coelicolor A3(2) wild-type (WT) strain was cultured in the same manner and conditions, and then the β-agarase activity of the purified supernatant thereof was measured. We compared the β-agarase activity of the second selected strains with that of the Streptomyces coelicolor A3(2) wild-type (WT) strain. Then, the mutation strain with the highest β-agarase activity was selected as a final strain and was named Streptomyces coelicolor A3(2)_M22.

(20) (2) Deposit Information of Streptomyces Coelicolor A3(2)_M22 strain

(21) The present inventors deposited the final selected Streptomyces coelicolor A3(2)_M22 strain on Jun. 17, 2016 to the Korean Culture Center of Microorganisms, an international depository (Address: 3F, Yurim Building 45, Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, Korea) in a domestic patent application manner. Therefore, an accession number KFCC 11668P was assigned thereto.

(22) 3. Mutation Induction of Streptomyces Coelicolor A3(2)_M22 strain by UV Irradiation and Selection of Mutation Strains Overexpressing DagA β-Agarase

(23) (1) Selection of Streptomyces Coelicolor A3(2)_M22-2C43 Strain

(24) Streptomyces coelicolor A3(2)_M22 strain as a mutation strain of the Streptomyces coelicolor A3(2) wild-type (WT) strain was stationary-cultured for 5 days in an actinomyces complete culture medium (ISP4 medium) on a plate. After dispensing 2 ml of a 20% (w/v) glycerol solution on the plate, spores were collected and used for mutation induction experiments by UV irradiation. 1 μl of Streptomyces coelicolor A3(2)_M22 strain spore stock solution was input into a Petri dish, and 5 ml of a tryptic soy broth (TSB) culture medium (containing 17 g of tryptone, 3 g of soytone, 2.5 g of glucose, 5 g ofNaCl, and 2.5 g of K.sub.2HPO.sub.4) based on 1 liter of distilled water) as a general bacterial nutrient culture medium was added thereto and the solution was diluted to form a thin film. Thereafter, UV rays (UV) of 30 to 40 W intensity are irradiated thereto for 24 to 60 minutes at a distance of about 35 to 50 cm while ambient light was blocked. The culture fluid was collected and incubated for 8 hr under 28° C. temperature condition, 180 rpm shaking condition and dark condition. The culture fluid was plated on a MM agar culture medium, and then was subjected to stationary culture for 5 days in an incubator at 28° C. under dark condition. Thereafter, viable colonies were counted on the plate. In this connection, a mortality percentage thereof was 99.2%. A clear zone thereof was dyed using a dyeing reagent (Lugol's Iodine). After comparing the sizes thereof with each other, strains of colonies having different morphologies were classified from each other and then were stationary cultured at 28° C. for 5 days in MM agar culture medium on a plate. Thereafter, the selected strains were inoculated in a liquid culture medium (containing 5 g MgCl.sub.2.7H.sub.2O, 11 g yeast extract, and 0.5 g CaCO.sub.3 based on 1 liter of distilled water) containing 0.5% (w/v) concentration of agarose. Shaking culture thereof was performed for 2.5 days under a temperature condition of 28° C. and a shaking condition of 216 rpm. Subsequently, the culture fluid was centrifuged to remove cell debris and then the supernatant was collected. Subsequently, the supernatant was sterilized and filtered with a 0.45 μm syringe filter to collect the purified supernatant. Thereafter, the β-agarase activity and DagA enzymic activity of the purified supernatant were measured. A mutation strain having the highest β-agarase activity and DagA enzymic activity was selected as a final strain, and was named Streptomyces coelicolor A3(2)_M22-2C43.

(25) (2) Deposit Information of Streptomyces Coelicolor A3(2)_M22-2C43 Strain

(26) The present inventors deposited the final selected Streptomyces coelicolor A3(2)_M22-2C43 strain on Sep. 22, 2017 at the Korean Culture Center of Microorganisms, an international depository (Address: 3F, Yurim Building, 45 Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, Korea) in a domestic patent application manner and then the accession number KFCC 11742P was allocated thereto. Further, the present inventors applied for conversion of the Streptomyces coelicolor A3(2)_M22-2C43 strain (accession number: KFCC 11742P) deposited in Korea to an international patent deposit based on the Budapest Treaty on Aug. 23, 2019. Thus, an accession number KCCM 12577P was allocated thereto.

(27) 4. Comparison Between Streptomyces Coelicolor A3(2) Wild-Type (WT) Strain, Streptomyces Coelicolor A3(2)_M22 Strain and Streptomyces Coelicolor A3(2)_M22-2C43 Strain

(28) (1) Comparison of Colony Morphologies of Respective Strains

(29) FIG. 1 is a photograph showing the colony morphology of each of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2)_M22 strain, and Streptomyces coelicolor A3(2) M22-2C43 strain. A picture in a lower row in FIG. 1 shows the result of staining colonies with a dyeing reagent.

(30) (2) Culture of Each Strain and Preparation of Enzyme Sample Containing β-Agarase

(31) Each strain was inoculated in 1000 ml of liquid culture medium (containing 5 g of MgCl.sub.2.7H.sub.2O, 11 g of yeast extract, 0.5 g of CaCO.sub.3 based on 1 liter of distilled water) containing 0.5% (w/v) concentration of agarose and was subjected to shaking culture at 28° C. temperature condition and a shaking condition of 216 rpm for 2.5 days. Thereafter, the culture fluid was centrifuged to remove cell debris and the supernatant was collected. Using the collected supernatant as a sample, β-agarase activity thereof was measured, and DagA enzymic activity thereof was evaluated. Thereafter, the supernatant was sterilized and filtered with a 0.45 μm syringe filter to collect the purified supernatant. Thereafter, ammonium sulfate was added to the purified supernatant so that the saturation concentration of the protein contained in the supernatant was 50% and 70%, respectively. The β-agarase enzyme was precipitated via ammonium sulfate precipitation (ASP) as a type of a salting out method, and then the purified 3-agarase enzyme in the form of a pellet was obtained via centrifugation. According to the contents disclosed in the specification of the patent application of Streptomyces coelicolor A3(2)_M22 strain (Korean Patent Application Publication No. 10-2018-0019881, 2018 Feb. 27), it is indirectly identified that when the protein saturation concentration of the ammonium sulfate-added supernatant is 50%, DagA enzyme is mainly precipitated, and when the protein saturation concentration of the ammonium sulfate-added supernatant is 70%, both DagA enzyme and DagB enzyme are precipitated. After dissolving the purified β-agarase enzyme in the form of a pellet in 5 ml of distilled water, the β-agarase activity thereof was measured, and DagA enzymic activity thereof was evaluated.

(32) (3) Comparison of β-Agarase Activities of Enzyme Samples Obtained from Respective Strains

(33) Table 1 below shows a measurement result of the β-agarase activity each of supernatant samples obtained through culture of 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 so that the saturation concentration of the protein contained in the supernatant becomes 50%), and a 70% ASP sample (an enzyme sample obtained by adding ammonium sulfate so that the saturation concentration of the protein contained in the supernatant becomes 70%). A unit of the β-agarase activity is U/ml.

(34) TABLE-US-00001 TABLE 1 Strains S. coelicolor S. coelicolor S. coelicolor Samples A3(2) WT A3(2)_M22 A3(2)_M22-2C43 Supernatant 799 1,163 1,133 50% ASP 2,150 15,875 23,155 70% ASP 20,875 35,119 32,524

(35) As shown in Table 1 above, regarding the 50% ASP sample expected to be composed mainly of DagA enzyme, 50% ASP sample obtained from the culture fluid of the Streptomyces coelicolor A3(2)_M22-2C43 strain exhibited the highest β-agarase activity.

(36) (4) Comparison Between DagA Enzymic Activities of Enzyme Samples Obtained from Respective Strains

(37) FIG. 2 shows analysis results of the DagA enzymic activity of each of supernatant samples obtained through culture of 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 so that the saturation concentration of the protein contained in the supernatant becomes 50%), and a 70% ASP sample (an enzyme sample obtained by adding ammonium sulfate so that the saturation concentration of the protein contained in the supernatant is 70%) using a thin layer chromatography (TLC) method. In FIG. 2, ‘M’ represents the standard solution, all of lanes ‘1’, ‘4’ and ‘7’ denote enzyme samples obtained from the culture fluid of Streptomyces coelicolor A3(2) wild-type (WT) strain, and all of lanes ‘2’, ‘5’ and ‘8’ denote samples obtained from the culture fluid of Streptomyces coelicolor A3(2)_M22 strain. All of lanes ‘3’, ‘6’ and ‘9’ denote samples obtained from the culture fluid of Streptomyces coelicolor A3(2)_M22-2C43 strain. Further, all of lane ‘1’, ‘2’ and ‘3’ are all supernatant samples, all of lanes ‘4’, ‘5’ and ‘6’ are 70% ASP samples, and all of lanes ‘7’, ‘8’ and ‘9’ are 50% ASP samples.

(38) As shown in FIG. 2, all of the enzyme samples obtained from the culture fluid of Streptomyces coelicolor A3(2)_M22-2C43 strain decomposed agarose regardless of the level of separation and purification, thereby producing mainly DP4 (neoagarotetraose) and DP6 (neoagarohexaose). To the contrary, the supernatant sample and the 70% ASP sample as obtained from the culture fluid of Streptomyces coelicolor A3(2)_M22 strain decomposed agarose to produce mainly DP2 (neoagarobiose).

(39) FIG. 3 shows an analyzing result of a composition of neo-agarosaccharide in a degradation product using HPLC-ELSD, wherein the degradation product is obtained by reacting agarose with a 70% ASP sample obtained through culture of Streptomyces coelicolor A3(2)_M22-2C43 strain. The decomposition reaction conditions of agarose are the same as those used in the DagA enzymic activity evaluation. The agarose decomposition reaction was repeated a total of 4 times under the same condition. When analyzing the composition of the neo-agarosaccharide in the decomposition product using HPLC-ELSD, an NH2 P-50 4E multimode column (250 mm×4.6 mm) was used as a column, and a mixed solution of acetonitrile and water (mixing ration of acetonitrile:water being 65:35 based on weight) was used as a mobile phase. As shown in FIG. 3, a content of DP4 (neoagarotetraose) in the decomposition product of agarose is 5 to 5.5 times larger than that of DP2 (neoagarobiose). A content of DP6 (neoagarohexaose) was found to be 3 to 3.5 times larger than that of DP2 (neoagarobiose).

(40) (5) β-Agarase Gene Information of Each Strain

(41) We amplified the DagA gene and DagB gene of each of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2)_M22 strain and Streptomyces coelicolor A3(2)_M22-2C43 strain using a PCR reaction. The DNA base sequence of the amplified PCR product was analyzed. The DagB genes of the Streptomyces coelicolor A3(2) wild-type (WT) strain and the Streptomyces coelicolor A3(2)_M22 strain were found to have the same base sequence represented by SEQ ID NO:1. To the contrary, the DagB gene of the Streptomyces coelicolor A3(2)_M22-2C43 strain was modified via gene mutation as a substitution, and was found to have the base sequence represented by SEQ ID NO:2. FIG. 4 shows some of comparison results of the DagB gene base sequence (upper line) of the Streptomyces coelicolor A3(2) wild-type (WT) strain and the DagB gene base sequence (lower line) of the Streptomyces coelicolor A3(2)_M22-2C43 strain as aligned with each other. In one example, all of 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 were found to have the same base sequence represented by SEQ ID NO:3. Therefore, all of Streptomyces coelicolor A3(2) wild-type (WT) strain, Streptomyces coelicolor A3(2)_M22 strain, and Streptomyces coelicolor A3(2)_M22-2C43 strain are expected to express the DagA enzyme having the amino acid sequence represented by SEQ ID NO:4. Further, Streptomyces coelicolor A3(2) wild-type (WT) strain and Streptomyces coelicolor A3(2)_M22 strain are predicted to express a normal DagB enzyme having the amino acid sequence represented by SEQ ID NO:5. To the contrary, Streptomyces coelicolor A3(2)_M22-2C43 strain is predicted not to express the normal DagB enzyme but to express DagB mutant enzyme with amino acid sequence represented by SEQ ID NO:6 and little β-agarase activity.

(42) (6) Lineage and Relationship of Streptomyces Coelicolor A3(2)_M22-2C43 Strain

(43) The 16S rRNA base sequence of the Streptomyces coelicolor A3(2) wild-type (WT) strain and the 16S rRNA base sequence of the Streptomyces coelicolor A3(2)_M22-2C43 strain were analyzed using colony PCR. FIG. 5 shows the biological lineage and relationship of the Streptomyces coelicolor A3(2)_M22-2C43 strain prepared based on the 16S rRNA base sequence.

(44) (7) Comparison Between Expression Levels by DagB Genes

(45) The DagB gene of the Streptomyces coelicolor A3(2) wild-type (WT) strain was cloned into the pUWL201pw vector with the cleavage map in FIG. 6 to create a recombinant vector thereof Streptomyces lividans TK24 strain which does not have a β-agarase gene was transformed using the recombinant vector to prepare a recombinant strain WT dagB. Further, a recombinant vector was produced by cloning the DagB gene of the Streptomyces coelicolor A3(2)_M22-2C43 strain into the pUWL201pw vector. Streptomyces lividans TK24 strain was transformed using the recombinant vector to prepare a recombinant strain M22-2C43 dagB. Further, a recombinant strain pUWL201pw was prepared by transforming the Streptomyces lividans TK24 strain using the pUWL201pw vector. Thereafter, the three recombinant strains were cultured, and culture fluids thereof were collected on first, second, and third days during the culturing, and the supernatants thereof were obtained. Thereafter, the β-agarase activity of the supernatant sample was measured using the reducing sugar quantitative assay method (DNS method). FIG. 7 shows the β-agarase activity of each of supernatants obtained from culture of recombinant strain WT dagB, recombinant strain M22-2C43 dagB and recombinant strain pUWL201pw prepared in the examples of the present disclosure based on the culture date. As shown in FIG. 7, the DagB gene of the Streptomyces coelicolor A3(2) wild-type (WT) strain was expressed at a high level using a normal DagB enzyme with β-agarase activity. To the contrary, the modified DagB gene of the Streptomyces coelicolor A3(2)_M22-2C43 strain was not expressed using the normal DagB enzyme or was expressed using the DagB mutant enzyme with little β-agarase activity.

(46) 5. Establishment of Optimal Culture Conditions for β-Agarase of Streptomyces Coelicolor A3(2)_M22-2C43 Strain

(47) (1) Type and Content of Carbon Source in Culture Medium

(48) Streptomyces coelicolor A3(2)_M22-2C43 strain was cultured for 4 days in an actinomyces agar culture medium containing agar at a 1.5 wt % concentration as a carbon source. Thereafter, 3 strain colonies, each having a size of 1 cm×1 cm, were inoculate into 50 ml of a RSM3 liquid culture medium containing one selected from 1.5% (w/v) concentration of galactose, 2% (w/v) concentration of succinic acid, 1.5% (w/v) concentration of glucose, agar of 0.2% (w/v) concentration and agar of 0.5% (w/v) concentration as a carbon source, and then were incubated for 2.5 days under a temperature condition of 28° C. and a shaking condition of 216 rpm. Thereafter, the supernatant was collected from the culture fluid and the activity of the β-agarase contained in the supernatant was measured.

(49) FIG. 8 shows the result of measuring β-agarase activity of Streptomyces coelicolor A3(2)_M22-2C43 strain culture fluid based on the type of carbon source in the culture medium by a reducing sugar quantitative assay method. In FIG. 8, the Y-axis represents the β-agarase activity and the X-axis represents the culturing duration. Table 2 below shows the β-agarase activity of the culture fluid based on the type of carbon source in the culture medium when the Streptomyces coelicolor A3(2)_M22-2C43 strain was cultured for 96 hr.

(50) TABLE-US-00002 TABLE 2 2%(w/v) Carbon 1.5%(w/v) 1.5%(w/v) succinic 0.2%(w/v) 0.5%(w/v) source galactose glucose acid agar agar β-agarase 546 137 0 15 72.5 activ- ity(U/ml)

(51) As shown in FIG. 8 and Table 2 above, it is identified that the carbon source in the culture medium for optimal production of β-agarase from the Streptomyces coelicolor A3(2)_M22-2C43 strain is galactose, and a concentration thereof is 1.5% (w/v).

(52) (2) Culture Temperature and Culturing Agitation Speed (rpm) 3 strain colonies, each having 1 cm×1 cm size were inoculated into each of 50 ml of RSM3 liquid culture medium containing 1% (w/v) concentration of galactose and 0.5% (w/v) concentration of agar as a carbon source, 50 ml of RSM3 liquid culture medium containing 1.5% (w/v) concentration of galactose and 0.5% (w/v) concentration of agar as a carbon source, and 50 m1 of RSM3 liquid culture medium containing 0.5% (w/v) concentration of agar as a carbon source. Then, incubation thereof was performed for 2.5 days under a temperature condition of 28° C. and shaking condition of 216 rpm or under a temperature condition of 30° C. and a shaking condition of 250 rpm. Thereafter, a supernatant was collected from a culture fluid and then the activity of β-agarase contained in supernatant was measured.

(53) FIG. 9 shows the result of measurement of the β-agarase activity of the culture fluid when Streptomyces coelicolor A3(2)_M22-2C43 strain is cultured in a culture medium containing carbon sources under the temperature condition at 28° C. and the shaking condition at 216 rpm, using a reducing sugar quantitative assay method. FIG. 10 shows the result of measurement of the β-agarase activity of the culture fluid when Streptomyces coelicolor A3(2)_M22-2C43 strain is cultured in a culture medium containing carbon sources under the temperature conditions at 30° C. and shaking conditions at 250 rpm, using a reducing sugar quantitative assay method. In FIG. 9 and FIG. 10, the Y-axis represents the β-agarase activity and the X-axis represents the culturing duration. As shown in FIG. 9 and FIG. 10, it is identified that the culture conditions for optimal production of β-agarase during culture of the Streptomyces coelicolor A3(2)_M22-2C43 strain are a temperature of 30° C. and shaking (stirring speed) at 250 rpm.

(54) As described above, the present disclosure has been described based on the above examples. However, the present disclosure is not necessarily limited thereto, and various modifications may be implemented within the scope and spirit of the present disclosure. Therefore, the scope of protection of the present disclosure should be construed as including all embodiments belonging to the scope of the claims attached to the present disclosure.

(55) [Accession Number]

(56) Depositary Organization Name: Korean Culture Center of Microorganisms

(57) Accession number: KFCC 11668P

(58) Deposit Date: 2016 Jun. 17

(59) [Accession Number]

(60) Depositary Organization Name: Korean Culture Center of Microorganisms

(61) Accession number: KFCC 11742P

(62) Deposit Date: 2017 Sep. 22

(63) [Accession Number]

(64) Depositary Organization Name: Korean Culture Center of Microorganisms

(65) Accession number: KCCM 12577P

(66) Deposit date: 2019 Aug. 23