JP2020080741A - Agarivorans bacteria, variants thereof, seaweed degrading bacteria and bacteria for degradation of agar, alginic acid, fucoidan, carrageenan, pectin and xylan - Google Patents

Abstract

To provide novel Agarivorans bacteria or variants thereof having degradation activity on agar, alginic acid, fucoidan, carrageenan, pectin and xylan as well as bacteria for seaweed decomposition.SOLUTION: An Agarivorans bacterial strain of the invention has an activity to decompose agar, alginic acid, fucoidan, carrageenan, pectin and xylan. The bacteria for seaweed decomposition comprise Agarivorans bacteria having degradation activity on agar, alginic acid, fucoidan, carrageenan, pectin and xylan.SELECTED DRAWING: None

Description

The present invention relates to a bacterium belonging to the genus Agarivorans, a mutant strain thereof, a bacterium for decomposing seaweed, and a bacterium for decomposing agar, alginic acid, fucoidan, carrageenan, pectin and xylan.

The genus Agarivorans belongs to the family Alteronomonaceaceae within the class Gammaproteobacteria, and is mainly separated from shellfish, seaweed, seawater, soil of tidal flat and the like.

One of the characteristics common to the genus Agariborans is agar resolution. Since agar is one of the major polysaccharides contained in seaweed, especially red algae, it is considered that the genus Agariborans plays a central role in the carbon cycle of the marine environment.

Non-Patent Document 1 describes that the agar-degrading enzyme of Agarivorans albus YKW-34 was purified and characterized.

On the other hand, the ability to decompose alginic acid, which is one of the major seaweed polysaccharides as well as agar, has been reported in the genus Agariborans. Non-Patent Document 2 describes that the alginate-degrading enzyme of Agarivorans albus YKW-34, which is a bacterium belonging to the genus Agariborans, was purified and characterized.

Applied Microbiology and Biotechnology, 78, 265-273, 2008 Enzyme and Microbiological Technology, 41, 828-834, 2007.

However, up to now, no report has been made on a bacterium belonging to the genus Agariborans, which has the activity of decomposing a plurality of polysaccharides other than agar and alginic acid.

The present inventors have found a novel bacterium of the genus Agariborans in marine organisms and completed the present invention. It is an object of the present invention to provide a novel bacterium of the genus Agariborans or a mutant strain thereof having agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity, and a bacterium for degrading seaweed.

In order to achieve the above object, a bacterium of the genus Agarivorans according to the first aspect of the present invention is
It has agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity.

A bacterium for decomposing seaweed according to the second aspect of the present invention,
It consists of agar, alginic acid, fucoidan, carrageenan, pectin and agariborans bacteria having xylan degrading activity.
It is characterized by

The bacterium belonging to the genus Agariborans or a mutant thereof according to the third aspect of the present invention,
Deposited under the deposit number NITE BP-02802.

A bacterium for decomposing seaweed according to the fourth aspect of the present invention,
It consists of the bacterium belonging to the genus Agariborans or the mutant strain thereof deposited under the deposit number NITE BP-02802.

The agar, alginic acid, fucoidan, carrageenan, pectin and xylan-degrading bacteria according to the fifth aspect of the present invention include
It consists of the bacterium belonging to the genus Agariborans or the mutant strain thereof deposited under the deposit number NITE BP-02802.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a bacterium belonging to the genus Agariborans or a mutant thereof having agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity, and a bacterium for degrading seaweed.

It is a figure which shows the result of having measured the agar degrading enzyme activity and alginic acid degrading enzyme activity of an isolate. It is a figure which shows the transmission electron microscope image of the Toyoura001 strain negative staining cell. It is a figure of the phylogenetic tree which shows the phylogenetic relationship between the Toyoura 001 strain|stump|stock and the Alteromomonaceae family bacteria containing the related species, which is deduced from the 16S rRNA gene sequence. The sequence of the Toyoura001 strain is shown in bold. A bootstrap value of over 50% is shown in the node. The 16S rRNA sequence of Pseudomonas aeruginosa DSM 50071T (X06684) was used as an outgroup.

First, the bacterium of the genus Agarivorans according to the present embodiment will be described.

The bacterium belonging to the genus Agarivorans according to the present embodiment is a novel species belonging to the genus Agarivorans, and is a novel microorganism that is systematically close to, but distinctly different from, Agarivorans albus MKT106T. The bacterium of the genus Agariborans can grow using agar, alginic acid, fucoidan, carrageenan, pectin and xylan as nutrient sources. That is, the bacterium of the genus Agariborans according to the present embodiment has agar, alginic acid, pectin and xylan degrading activity.

Next, the bacterium belonging to the genus Agarivorans or its mutant strain deposited under the deposit number NITE BP-02802 according to the present embodiment will be described.

The bacterium belonging to the genus Agariborans deposited under the deposit number NITE BP-02802 is also referred to herein as “Toyoura001”. The bacterium belonging to the genus Agariborance (Toyoura 001) deposited under accession number NITE BP-02802 has the 16S rRNA gene base sequence shown in SEQ ID NO:1.

Toyoura001 was entrusted to the Japan Microbial Depositary Center for Product Evaluation Technology, National Institute for Product Evaluation Technology (Postal code 292-0818, 2-5-8122 Kazusa Kamasa, Kisarazu City, Chiba Prefecture) on November 7, 2018. Deposited at -028022.

In the present specification, a mutant strain of Toyoura001 refers to, for example, 95% or more, preferably 96% or more, 97% or more, 98% or more or 99% or more, more preferably 100% or more of the nucleotide sequence shown in SEQ ID NO:1. It refers to a strain having a 16S rRNA gene nucleotide sequence showing homology. The mutant Toyoura001 has agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity.

The mutant strain of Toyoura001 can be derived from, for example, Toyoura001 by a mutation treatment known to those skilled in the art. Examples of the mutation treatment include irradiation with radiation such as ultraviolet rays and γ rays, contact with mutagenic chemical substances such as methylnitrosourea, N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment, ethylmethane Examples thereof include sulfonic acid (EMS) treatment.

Toyoura001 can be obtained, for example, by dissecting an abalone captured in the sea near Japan, collecting the intestinal contents, applying it to a marine broth agar medium, culturing, and isolating a strain having agar and alginic acid-degrading activity. it can.

Next, the seaweed degrading bacterium according to the present embodiment will be described.

The seaweed degrading bacterium according to the present embodiment is composed of the above-mentioned bacterium belonging to the genus Agariborans or Toyoura001 or a mutant strain thereof. The seaweed-degrading bacterium according to the present embodiment has agar, alginic acid, fucoidan, carrageenan, pectin, and xylan-degrading activity, so that the polysaccharides of seaweed components (e.g., agar, alginic acid, fucoidan, carrageenan, pectin, and xylan) are efficiently used. Decomposes and can decompose seaweed. In the present specification, "seaweed" is a general term for algae of the marine species group, and includes brown algae, red algae and green algae, for example, as brown algae, arame, gagome kelp, etc. As green algae such as Susabinori), Tosakanori and Ogonori, human Exa (Aosa) and the like can be mentioned. In the present specification, “seaweed degradation” is a decomposition of polysaccharides of seaweed components (for example, agar, alginic acid, fucoidan, carrageenan, pectin and xylan) to soften the seaweed and physically decompose the seaweed. It refers to what you do.

Next, the agar, alginic acid, fucoidan, carrageenan, pectin and xylan-degrading bacteria according to the present embodiment will be described.

The agar, alginic acid, fucoidan, carrageenan, pectin and xylan-degrading bacterium according to the present embodiment is composed of the aforementioned bacterium of the genus Agariborans or Toyoura001 or a mutant strain thereof. The agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading bacteria according to the present embodiment have agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity. That is, the bacterium has six decomposition activities of agar decomposition activity, alginic acid decomposition activity, fucoidan decomposition activity, carrageenan decomposition activity, pectin decomposition activity and xylan decomposition activity. The decomposition activity of agar, alginic acid, fucoidan, carrageenan, pectin and xylan is determined by, for example, a medium containing agar, alginic acid, fucoidan, carrageenan, pectin and xylan as the sole carbon sources (for example, Ensor, LA, Stosz, S. K., & Weiner, RM (1999). It can be measured by culturing the bacterium in (in accordance) and comparing the turbidity of the culture solution before and after the culture. Regarding the agar-degrading enzyme activity, for example, the bacterium is suspended in a Marine Broth liquid medium and shake-cultured, and the supernatant after centrifugation is sterilized by a filter to obtain an extracellular enzyme (enzyme solution), After mixing the enzyme solution and a phosphate buffer solution, a substrate solution (phosphate buffer solution containing agar) is added and mixed for reaction, and a dinitrosalicylic acid reagent is added to the reaction solution supernatant after centrifugation and heated. After cooling with ice, the absorbance is measured at 545 nm, a calibration curve is prepared using galactose which is a product of agarose decomposition, and the amount of galactose produced can be measured as the enzyme activity. Regarding the alginic acid-degrading enzyme activity, for example, the bacterium is suspended in a Marine Broth liquid medium and shake-cultured, and the supernatant after centrifugation is sterilized by a filter to obtain an extracellular enzyme (enzyme solution), After mixing the enzyme solution and Tris-HCl buffer, sodium alginate-containing Tris-HCl was added and mixed to react, and the absorbance of the reaction solution supernatant after centrifugation was measured at 235 nm, and the absorbance was 0 per minute. An enzyme activity that increases by 0.01 can be measured as 1 U.

As described above, the bacterium belonging to the genus Agariborans or Toyoura001 or the mutant thereof according to the present embodiment is a novel bacterium belonging to the genus Agarivorans having agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity. It will be possible to use it for highly efficient seaweed degradation technology, which utilizes the polysaccharide degradability that has not been reported in the genus Agariborans. By utilizing the bacterium of the genus Agariborans or Toyoura001 or a mutant thereof according to the present embodiment, for example, it is expected to reduce the amount of seaweed waste and develop a digestible seaweed feed.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

(Example 1)
A novel bacterial strain of the genus Agariborans (Toyoura001) was isolated from the intestinal contents of abalone, and its polysaccharide-degrading activity was examined.

Wild abalone Haliotis discus hannai was captured on the coast of Toyoura-cho, Abuta-gun, Hokkaido. On the day after the capture, the abalone was dissected to collect the intestinal contents and suspended in filter-sterilized artificial seawater (Instant Ocean, Aquarium Systems). The obtained suspension was applied on a 1.5% agar plate of Marine Broth (Difco) and cultured at 15°C for 10 days. After the culture, a plurality of colonies were formed on the agar, but colonies growing while denting the agar were observed. Among the formed colonies, four that dent the agar (strain name: 203 strains, 206 strains, 219 strains, 225 strains), and four that do not dent the agar (strain name: 201 strains, 204 strains, 220 strains) Strains 222 strains), the partial sequence of the 16S rRNA gene was decoded and simply identified. As a result, the ones that dent the agar were all bacteria of the genus Agarivorans, and those that did not dent were the closely related species of Allivirio, Shewanella, and Vibrio bacteria. Presumed to be a species.

The agar-degrading activity and the alginic acid-degrading activity of the above 8 strains (201, 203, 204, 206, 219, 220, 222, 225 strains) were measured. The six strains 201, 203, 204, 206, 219 and 220 and the two strains 222 and 225 were isolated from separate abalone.

A method for measuring agar-degrading enzyme activity and alginate-degrading enzyme activity will be described.
(1) Preparation of enzyme solution The isolated bacterium was suspended in a Marine Broth liquid medium, shake-cultured (15°C, 24h), and centrifuged (2,800rpm, 20 minutes, 4°C), and the supernatant was filtered. Sterilization (DISMIC-25, 0.45 μm) was performed to obtain an extracellular enzyme.

(2) Agar-degrading enzyme activity As a substrate solution for agar-degrading enzyme activity, a 10 mM phosphate buffer solution (pH 6.8) containing 0.25% agar (special grade reagent, Fujifilm Wako Pure Chemical Industries, Ltd.) was used. After mixing the enzyme solution and the phosphate buffer (pH 6.8), the substrate solution was added and mixed, and the mixture was reacted at 30° C. for 30 minutes. The dinitrosalicylic acid reagent was added to the reaction solution supernatant after centrifugation (7,000 rpm, 4°C, 5 minutes), and the mixture was heated at 100°C for 5 minutes. After cooling with ice, the absorbance was measured at 545 nm. A calibration curve was prepared using galactose, which is a product of agarose degradation, and the amount of galactose produced was taken as the enzyme activity.

(3) Alginic acid-degrading enzyme activity The enzyme solution and 0.1 M Tris-HCl buffer (pH 7.5) were mixed, and then 1 M (w/v) sodium alginate-containing 0.1 M Tris-HCl was added and mixed. The mixture was reacted at 30°C for 30 minutes. The absorbance of the reaction solution supernatant after centrifugation (8,000 rpm, 4° C., 5 minutes) was measured at 235 nm. The enzyme activity was defined as 1 U, which is an enzyme activity that increases the absorbance by 0.01 per minute. For the measurement of alginate degrading enzyme activity, Inoue A. et al. , Et al. (2014). Characterization of an Alginate Lyase, FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli. Marine Drugs, 12, 4693-4712. Was referred to.

The results are shown in Figure 1. Of the eight strains, 203, 206, 219, and 225 strains were confirmed to have both agar-degrading enzyme activity and alginate-degrading enzyme activity, and in particular, the "206 strain" had agar-degrading enzyme activity and alginate-degrading enzyme activity. It was shown to be high. Therefore, this "206 strain" was named "Toyoura001 strain" (accession number NITE BP-02802).

(Example 2)
The "Toyoura001 strain" obtained in Example 1 was examined for morphology, physiological properties, chemical properties, genotype characteristics, and the like.

The Toyoura 001 strain grown on the aforementioned 1.5% agar plate of marine broth was isolated, transferred to MB medium, incubated at 15° C. for 24 hours with shaking, and then added to the culture medium until use in the following experiments. It was supplemented with 20% glycerol and stored at -80°C. The comparative Agarivorans albus MKT106T was obtained from the NITE Biological Resource Center (NBRC).

After culturing the Toyoura 001 strain on an MB agar plate at 25° C. for 24 hours, colony morphology and Gram staining were observed. The morphology of cells cultured in MB medium at 25° C. for 24 hours was negatively stained and observed with a transmission electron microscope, and the motility was evaluated by the hanging drop method under an optical microscope. The cells were cultured on MB agar for 24 hours, and the growth temperature range and optimum conditions were determined at 4 to 40°C (4, 10, 15, 20, 25, 30, 35 and 40°C). The pH range and the optimal growth conditions were 20 mM in buffer (MES (pH 5.0 to 6.0), PIPES (pH 6.5 to 7.0), HEPES (pH 7.5 to 8.0), Tricine ( pH 8.5) and CHES (pH 9.0-10.0)) at 25° C. for 24 hours in MB medium. R2A supplemented with 0-8.0% NaCl (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0) The NaCl concentration and optimal growth conditions were examined in (Difco) medium at 25° C. for 48 hours. Growth under anaerobic conditions was tested by incubation on MB agar for 2 weeks at 25°C using AnaeroPack system (Mitsubishi Gas Chemical Co.). For the measurement of catalase activity, see Kim, S. et al. G. Pheng, S.; Lee, Y.; J. , Eom, M.; K. , & Shin, D.; H. (2016). Agarivorans aestuarii sp. nov. , An agar-degrading bacteria isolated from flat. International journal of systematic and evolutionary microbiology, 66(8), 3119-3124. Was referred to. API50CH (bioMerieux) for acid production from carbohydrates, APIZYM (bioMerieux), API20NE (bioMerieux) and API20E (bioMerieux) for various enzyme activities were tested. Regarding the assimilation ability of monosaccharides, disaccharides, and organic acids, the minimum medium (artificial seawater (Park, S., et al. (2014). Agarivorans litoreus sp. nov., a novel) using each of them as a sole carbon source was used. gammaproteobacterium isolated from water and emended description of the genus Agarivorans. Antonie van Leeuwenhoek, 106 (5), and a solution of a solution of vitamins (106), 104(104), and 1041-1047. Was tested. After culturing at 25° C. for 24 hours, the turbidity of the culture solution before and after the culturing was compared to determine the assimilation ability. All API tests were performed at 25° C. according to the manufacturer's instructions, except that the cell suspension was prepared with 3% (w/v) NaCl solution. Sensitivity to antibiotics was determined by determining the sensitivity of each antibiotic (final concentration; μg/ml) (ampicillin (10), carbenicillin (100), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30). , Nalidixic acid (30), neomycin (30), penicillin G (10), streptomycin (10), tetracycline (10) and vancomycin (30)) in MB medium. The culture was incubated at 25°C for 24 hours.

Method of Sasser from 100 mg of cells cultured in MB medium at 25° C. for 24 hours (Sasser, M. (1990). Identification of bacteria by gas chromatographic of cellular fat acids, MIDI Technological. The cellular fatty acid methyl ester was extracted with reference to Table 1 and analyzed by the Sherrlock Microbial Identification System version 6.0 (MIDI Inc.). Further, isoprenoid quinone was purified from 100 mg of cells cultured in MB medium at 25° C. for 24 hours using methanol/chloroform/MilliQ water (10:5:4) and chloroform/MilliQ water (1:1). The obtained purified quinone was subjected to HPLC (Tamaoka, J., Katayama-Fujimura, Y., & Kurashishi, H. (1983). Analysis of bacteriophyla guinea purine humor purine liquor syrup. 31-36.). The polar lipids were extracted with methanol/0.3% NaCl (10:1, v/v) and petroleum ether and the method of Minnikin et al. (Minnikin DE, Collins MD, Goodfellow M. Fatty acid and polar lipid composition of the clef of cation) was extracted. Cellulomonas, Oerskovia and related taxa.J Appl Bacteriol 1979; 47:. 87-95; Collins MD, Jones D.Lipids in the classification and identification of coryneform bacteria containing peptidoglycans based on 2,4-diaminobutyric acid.J Appl Bacteriol 1980; 48:459-470.).

The entire 16S rRNA gene sequence (1,539 bp) (SEQ ID NO: 1) of the Toyoura001 strain was read from the rRNA operon (16S-23S-5S) on the draft genome sequence. A BLAST search was performed to identify adjacent lines of isolates. GENETYX-MAC Ver. 19 (Genetyx) was used to calculate the sequence similarity between the Toyoura001 strain and the standard strain. For phylogenetic analysis, the GenBank/EMBL/DDBJ database was searched for 16S rRNA gene sequences of four standard strains of the genus Agarriborance and strains of the family Alteromonas. MAFFT program (Katoh, K., & Toh, H. (2008). Recent developments in the MAFFT multiple sequence alignment program. Briefing in bioinformatics using 28, 6-2, 9-2, 4). An alignment was generated. MEGA ver. 7.0.21 software (Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: molecular evolutionary analytics analysis 7.0, for bigger datasets, ans. 1870-1874.) using the neighbor-joining method and the Tamura-Nei substitution model (Tamura, K., & Nei, M. (1993). A phylogenetic tree was created based on chimpanzees.Molecular biology and evolution, 10(3), 512-526. Toyoura001 strain and A. albus MKT106T (Yasuike, M., Nakamura, Y., Kai, W., Fujiwara, A., Fukui, Y., Satomi, M., & Sano, M. M. (2013). 106T, an agarolytic marine bacteria. Genome announcements, 1(4), e00367-13. Machine (Yoon, S.H., Ha, S.M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: a taxonomally. united database of 16S rRNA gene sequences and whole-genome assemblies.International journal of systematic and evolutionary microbiology, 67 (5), 1613-1617;. Varghese, N.J., Mukherjee, S., Ivanova, N., Konstantinidis, K. T., Mavromatis, K., Kyrpides, N.C., & Pati, A. (2015). Microbiological species delineation using a used whole genome sequences. Nucleic acids 67-71, 61, 14), 71), 14). I verified it.

The Toyoura 001 strain was cultured at 25° C. for 24 hours, and then white colonies were formed on the MB agar medium. When the culture was continued as it was, the bacteria completely dissolved the agar. The cells were negative for Gram stain, strictly aerobic, highly motile, and formed rod-shaped (width 0.9 to 1.2 μm, length 2.3 to 3.7 μm) single polar flagella. (Figure 2). Colonies were round, slightly convex, white in color, and formed craters around them on MB agar plates. The growth of the Toyoura 001 strain was carried out in the presence of 1.0 to 8.0% NaCl (optimum concentration 5.0%) at pH 5.5 to 10.0 (optimum pH 7.5) and 10°C to 30°C. It was found to occur at the optimum temperature of 25°C. When examined under the same conditions, A. The optimum NaCl concentration of albus MKT106T is 4.0%, and it is known that all other standard strains of the genus Agariborans grow optimally in 2.0 to 3.0% NaCl (Du, ZJ, Lv, GQ, Rooney, AP, Miao, TT, Xu, QQ, & Chen, GJ (2011). Agarivorans gilvus sp. nov. isolated from seaweed. International journal of systematic and evolutionary microbiology, 61(3), 493-496.; Park, S., Park, J.M., J., J., J., Y., T. ).Agarivorans litreus sp. nov. , Y. J., Eom, M.K., & Shin, D. H. (2016) Agarivorans aestuarii sp. nov., an agar-degrading bacterium isolated aflate of sewage sewage. 66(8), 3119-3124.). Therefore, the Toyoura001 strain could be distinguished from other standard strains of agariborans by optimal growth in 5.0% NaCl.

The Toyoura001 strain has alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphorylase and β-galactosidase enzymatic activities, and glycerol, D-xylose, D-galactose, D-glucose, D-fructose and D-. Acid production from mannitol, N-acetylglucosamine, esculin, iron citrate, D-cellobiose, D-maltose, D-lactose, D-melibiose, starch and glycogen was shown. Furthermore, the Toyoura001 strain utilized fumaric acid, malic acid, glucose, galactose, maltose and mannose, but not acetic acid, propionic acid, citric acid, gluconic acid, arabinose and sucrose. Further, the Toyoura001 strain showed sensitivity to ampicillin, carbenicillin, chloramphenicol, erythromycin, nalidixic acid, neomycin, penicillin G, and tetracycline.

The Toyoura001 strain was positive for catalase. Negative for arginine hydrolase, lysine decarboxylase, ornithine decarboxylase, citric acid utilization, H ₂ S production, urease, tryptophan deamination, indole and acetoin production, and protease. All β-galactosidase activities against hydrolysis of esculin and para/ortho-nitrophenyl-β-galactopyranoside were positive. Reduced nitrate to nitrite but not N ₂ . Esterase (C4), esterase/lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl -Β-glucosidase, α-mannosidase and α-fucosidase were negative. Erythol, D-arabinose, L-arabinose, D-ribose, L-xylose, D-adnitol, methyl β-D-xylopyranoside, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, Methyl α-D-mannopyranoside, methyl α-glucopyranoside, amygdalin, arbutin, salicin, D-sucrose, D-trehalose, inulin, D-merezitose, D-raffinose, xylitol, gentiobiose, D-turanose, D-lychtose, D- No acid production was shown from tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconic acid, 2-ketogluconic acid and 5-ketogluconic acid.

The main fatty acids of the Toyoura001 strain are C _16:0 (19.9%), C _16:1 ω7c/C _16:1 ω6c (39.4%) and C _18:1 ω7c/C _18:1 ω6c(16). It was C. 6 _:0 ) and C _12:0 (6.0%). The quinone analysis showed that ubiquinone 8 (Q-8) was the major isoprenoid quinone of the Toyoura001 strain.

Toyoura001 was cultured in 100 mL of a marine broth medium (Difco) at 25° C. for 20 hours while shaking at 80 rpm. Genomic DNA from cultured cells was digested with proteinase K and SDS, extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with isopropanol and ethanol, treated with RNase (Das S, Dash HR. 2014. Basic Molecular Microbiology of). Bacteria p1-34. In Das S, Dash HR (ed), Microbiological Biotechnology-A Laboratory Manual for Bacterial Systems-3-4-78-20.100.7/9. (See the protocol of No. 1). The purity and concentration of the prepared DNA were measured using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and QuantiFluor dsDNA System (Promega). After 500 bp fragmentation of the purified DNA by Covaris M220 (Covaris Inc.), a DNA library was prepared with 500 ng of the fragmented DNA using KAPA HyperPrep Kit (Kapa Biosystems). The integrity of the prepared library was confirmed with a High Sensitivity DNA Kit (Agilent Technologies) using a Bioanalyzer (Agilent Technologies). The paired-end sequence (2×151 bp) of the prepared library was performed with an Illumina MiseSeq sequencer (Illumina). Low quality (Q-score <20) and short length (<128 bases) sequences were analyzed by Sickle ver. 1.33 (Joshi NA, Fass JN. 2011. Sickle: a sliding-window, adapted, quality-based trimming tool for FastQ files (version 1.33). https:/cothin. Used and trimmed and discarded. The rest of the sequence was replaced with SPAdes ver. 3.10.1 (Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Resin VM, Nikolenko SI, Pham S, Prjibelskih Ah, AVS, Prjibelskih Ah, AVS, Prjibelskih Ah, AVs, Prjibelskih Ah, Akh , Pevzner PA. 2012. SPAdes: a new genome assemblly algorithm and it's applications to single-cell sequencing. J Comput Biol 19: 455-477. 0021. It was The obtained Toyoura 001 genome was cloned into Prokka ver. 1.11 (Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30: 2068-2069. doi: 10.1093/bioinformatics/btu153.).

Assembled genome of Toyoura001, 243 contigs _{(maximum: 706,345bp,} N _{50: 186,035bp)} consists, had a G + C content of 44.4% includes 4,731,753Bp. Annotation by Prokka predicted genes for 4,295 protein coding sequences, 1 rRNA operon (16S-23S-5S) and 62 tRNAs. The draft genome contains 5 and 7 genes encoding agarase and arginase, respectively, which are A. It had homology with that of albus MKT106T (homology 77.9-93.3% and 59.3-94.8%, respectively).

Based on the 16S rRNA gene sequence, the Toyoura 001 strains are Agarivorans albus MKT106T, Agarivorans litreus GJSW-6T, Agarivorans aestauari4. Which was below 98.65% of the species segregation criterion (Kim M, Oh HS, Park SC, Chun J. Towerds a taxonomy coherence between severage severage averaging ederiente severage nerve tide identities identities and identities of 16 species). Int J Syst Evol Microbiol 2014;64:346-351.). The phylogenetic relationship between the Toyoura 001 strain and the agariborance strain is that the Toyoura 001 strain is A. albus MKT106T, A. litreus GJSW-6T, A. aestuarii hydD622 and A. It was shown to form an independent branch from gilvus WH0801 (FIG. 3). The closest phylogenetic line is A. The ANI value based on the genomic nucleotide sequence of the Toyoura001 strain for Albus MKT106T is 78.5% (OrthoANI) and 79.1% (gANI) (Yoon, SH, Ha, SM, Kwon, S., . Lim, J., Kim, Y., Seo, H, & Chun, J (2017) .Introducing EzBioCloud:. a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.International journal of systematic and evolutionary microbiology, 67(5), 1613-1617.; Varghese, NJ, Mukherjee, S., Ivanova, N., Konstantinidis, KT, Mavromatis, K., Kyrpides, NC, & Pati, A. (2015). Microbiological species delineation using whole genome sequences. Nucleic acids research, 43(14), 6761-6771.), these values being generally used in the differentiation of species of 94-95%. (Richter, M., & Rossello-Mora, R. (2009). Shifting the genomic gold standard for for the procedural features of the 19th. ). The DNA G+C content of the draft genome of the Toyoura 001 strain was 44.4 mol%, which was equivalent to that of other standard strains of the genus Agariborans.

The following table summarizes the assimilation ability of the polysaccharides of the Toyoura 001 strain. Toyoura 001 strain cultivated in MB medium containing 0.01% agar and 0.02% alginic acid at 25° C. for 24 hours was treated with agar, agarose, alginic acid, fucoidan, k-carrageenan, λ-carrageenan, laminaran, starch, pectin, A medium containing xylan, CM cellulose, inulin, pullulan, and gellan gum as the sole carbon sources (Ensor, LA, Stozz, SK, & Weiner, RM (1999). Expression of multiple complex polysaccharides). -Degrading enzyme systems by marine bacteria strain 2-40.Journal of Industrial Microbiology and Biotechnology, inoculated in a turbidity of 23 (2), 125-126. Regarding agarose and k-carrageenan, the turbidity was visually confirmed because the medium gelled. As a result, bacterial growth was confirmed in agar, agarose, alginic acid, fucoidan, k-carrageenan, λ-carrageenan, starch, pectin, xylan, pullulan, and gellan gum. For agarose and k-carrageenan, liquefaction of the gelled medium was also confirmed. From this, it was shown that the Toyoura001 strain has agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity.

The Toyoura001 strain was proved to be a novel species of the genus Agariborans from homology analysis of the genomic sequence, phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene obtained from the genomic information, morphological observation, physiological properties and the like. Further, it was confirmed that the Toyoura 001 strain could grow when cultured in a medium containing agar, alginic acid, fucoidan, carrageenan, pectin, and xylan as the only carbon sources, and Toyoura 001 strain was confirmed to be agar, alginic acid, fucoidan, carrageenan, pectin And xylan-decomposing activity.

Claims (5)

A bacterium of the genus Agarivorans having agar, alginic acid, fucoidan, carrageenan, pectin and xylan degrading activity. It consists of agar, alginic acid, fucoidan, carrageenan, pectin and agariborans bacteria having xylan degrading activity.
A bacterium for decomposing seaweed, which is characterized in that A bacterium belonging to the genus Agarivorans or a mutant strain thereof deposited under the deposit number NITE BP-02802. It consists of a bacterium belonging to the genus Agariborans or a mutant strain thereof deposited under the deposit number NITE BP-02802.
A bacterium for decomposing seaweed, which is characterized in that It consists of a bacterium belonging to the genus Agariborans or a mutant strain thereof deposited under the deposit number NITE BP-02802.
Bacteria for degrading agar, alginic acid, fucoidan, carrageenan, pectin and xylan, which are characterized in that: