Isolation and Characterization of an Agar-hydrolyzing Marine Bacterium, Pseudoalteromonas sp. H9, from the Coastal Seawater of the West Sea, South Korea

서해안 해수로부터 분리한 한천분해 해양미생물 Pseudoalteromonas sp. H9의 동정 및 특성 연구

지원재¹, 윤영상², 김종희³, 홍순광²*

1국립생물자원관유용자원분석과

2명지대학교생명과학정보학부

3서일대학교식품영양과

Received: March 9, 2015 / Revised: May 11, 2015 / Accepted: May 11, 2015

서 론

한천은홍조류세포벽의주요구성성분으로서

agaropectin

^agarose

^.

3,6-anhydro-

^-

- galactose

^-

-galactose

^unit

^polymer

^,

3,6-anhydro-

^-

- galactose

sulfoxyl, methoxyl, pyruvate

^.

두 종류의 잔기인

3,6-anhydro-

^-

-galactose

^-

- galactose

^-1,3

^,

^-

- galactose

^-1,4

3,6-anhydro-

^-

-galactose

^.

^-

^-agarase

^-

^-agarase

^.

^-agarase

3,6-anhydro-

-galactose

(agarooligosaccharide)

^,

^- agarase

-galactose

(neoagarooligosaccharide)

^{[4]. (}

⁾

^,

^,

^,

Isolation and Characterization of an Agar-hydrolyzing Marine Bacterium, Pseudoalteromonas sp. H9, from the Coastal Seawater of the West Sea, South Korea

Won-Jae Chi

, Young Sang Youn

, Jong-Hee Kim

, and Soon-Kwang Hong

*

1

Biological and Genetic Resource Assessment Division, National Institute of Biological Resource, Incheon 404-170, Republic of Korea

2

Department of Biological Science, Myongji University, Yongin 449-728, Republic of Korea

3

Department of Food and Nutrition, Seoil University, Seoul 131-702, Republic of Korea

An agarolytic marine bacterium (H9) was isolated from the coastal seawater of the West Sea, South Korea. The isolate, H9, was gram-negative and rod-shaped with a smooth surface and polar flagellum. Cells grew at 20 −30

C, between pH 5.0 and 9.0, and in ASW-YP (Artificial Sea Water-Yeast extract, Peptone) media containing 1 −5% (w/v) NaCl. The G+C content was 41.56 mol%. The predominant isoprenoid quinone in strain H9 was ubiquinone-8. The major fatty acids (>10%) were C

ω7c (34.3%), C

(23.72%), and C

ω7c (13.64%). Based on 16S rRNA gene sequencing, and biochemical and chemotaxo- nomic characterization, the strain was designated as Pseudoalteromonas sp. H9 (=KCTC23887). In liquid culture supple- mented with 0.2% agar, the cell density and agarase activity reached a maximum level of OD = 4.32 (48 h) and OD = 3.87 (24 h), respectively. The optimum pH and temperature for the extracellular crude agarases of H9 were 7.0 and 40

C, respec- tively. Thin-layer chromatography analysis of the agarase hydrolysis products revealed that the crude agarases hydrolyze aga- rose into neoagarotetraose and neoagarohexaose. Therefore, the new agar-degrading strain, H9, can be applicable for the production of valuable neoagarooligosaccharides and for the complete degradation of agar in bio-industries.

Keywords: Agarase, marine bacterium, Pseudoalteromonas sp. H9, phylogenetic analysis

*Corresponding author

Tel: +82-31-330-6198, Fax: +82-31-335-8249 E-mail: [email protected]

© 2015, The Korean Society for Microbiology and Biotechnology

기능성식품

^,

^,

^[10].

⁽

⁾

^hydrolase

3,6-anhydro-

-galactose

-galactose

^,

-galactose

. 3,6-anhydro-

-galactose

^,

^[22].

^,

^.

⁽

⁾

고당을생산하기위해서는

^agarase

^,

우선적으로활성이뛰어난

^agarase

^.

본연구그룹은대한민국서해연안의해수로부터

^agarase

,

,

^.

^{, 40}

^C

^agarase

^H9

^,

^H9

^,

^·

^.

재료 및 방법

Agarase 생산 미생물의 분리

해수에서식하는

^agarase

^,

¹⁰

−10

^ASW-YP

(6.1 g Tris base, 12.3 g MgSO

, 0.74 g KCl, 0.13 g (NH

)

HPO

, 17.5 g NaCl, 0.14 g CaCl

, 1% Yeast extract, 0.3% bacto peptone, 1.5% agar; per liter; pH 7.2)

^.

²⁸

^C

²

성장하는모든

^colony

(replica

^{). 28}

^C

²

Lugol's Iodine

(0.05 M Iodine in 0.12 M KI)

^colony

^colony

^.

^ASW-YP

^marine agar (Difco 2216)

²

^colony

^,

^,

^,

³

^.

³

^{16S rRNA}

^.

^H9

^.

균주 H9의 형태적·생리적 특성 분석

균주

^H9

Gram stain kit (BD Biosciences, USA)

용하여염색한후현미경으로관찰하였다

^.

^H9

^flagella

²

1% phosphotungstic acid (PTA)

(JEM1010, JEOL, Japan)

^.

^,

^API 20NE

API ZYM kit (Biomérieux, France)

^.

^,

^NaCl

^2%

²⁸

^C

²

^.

^H9

^{, NaCl}

0 −10% (1%

⁾

^ASW-YP

⁴

^.

^H9

^pH

^, pH 4.0-pH 10.0 (pH 1.0

⁾

⁴

^.

4, 15, 20, 25, 37, 40

C

4

.

H9 균주의 생화학적 특성 분석

균주

^H9

^ASW-YP

²⁸

^C

²

^.

^C18

^-HPLC

^{. HPLC}

^(Younglin SP930D, Korea)

Spherisorb 5

^{m ODS2} (250 mm

4.6 mm, Waters Corp., USA)

^,

⁽

^:

^{= 4:1)}

^1.0 ml/min

^.

methyl ester

^[14]

fatty acid methyl esters (FAME)

Microbial Identification System (MIDI)

^[18]

Gas chromatography (Agilent technologies 6890, USA)

. HP ultra 2

^{(25 m}

0.2 mm phenyl methyl silicone fused silica capillary column)

지방산을분리하고

Sherlock system

였다

^.

¹⁷⁰

^C

⁵

^C

300

C

²

, carrier gas

^H

makeup gas

^N

. Genomic DNA

G+C

-HPLC (C18

^,

^{0.2 M} NH

H

PO

(pH 4.5):acetonitrile = 20:1,

1 ml/min)

^[12].

16S rRNA 유전자 염기서열 해독 및 계통수 제작

균주

^H9

^ASW-YP

²

15,000 rpm

¹⁰

^.

genomic DNA

Genomic DNA extraction

kit (DyneBio, Korea)

^PCR

. 16S rRNA

bacterial universal primer (27F

^1492R)

^[19],

^DNA

은

pGEM-T easy vector (Promega, USA)

^.

^{16S rRNA}

^GenBank

^JF965427

, National Center for Biotechnology Information (NCBI)

BlastN program

GenBank database

^.

EzTaxon database (http://

www.eztaxon.org/)

^{16S rRNA}

^.

Multi alignment

ClustalW program (www.soft82.com/download/windows/clustalW2/)

^5'-

^3'-

^gap

GeneDoc program

^.

(evolutionary distance matrix)

^Kimura’s two-parameter evolutionary model [9]

^, Neighbor-Joining (N-J)

^[17]

^.

(bootstrap value)

^1,000

tree

.

균주 H9의 성장 및 agarase 생산

균주

^H9

^ASW-YP

^0.2%

^agar

첨가된

^ASW-YPA

⁹⁶

(28

C, 180 rpm) 24

^.

600 nm

였고

, 14,000 rpm

¹⁰

agarase

^{. Agarase}

0.2% agarose (AMRESCO LLC., USA)

3,5-dinitrosalicylic acid (DNS)

^[13]

^{540 nm}

^.

^,

20 mM Tris-HCl buffer (pH 7.0)

⁴⁰

^C

^.

균주 H9 배양액으로부터 조효소의 준비

균주

^H9

^ASW-YPA

²⁴

14,000 rpm

¹⁰

^.

ultrafiltration (3 kDa cut- off, PALL, USA)

¹⁰

^.

온도조건에따른

^H9

^agarase

^,

^25-50

^{C (5}

^C

⁾

^agarose

(20 mM Tris-HCl buffer, pH 7.0)

¹⁰

^{, DNS}

^{. pH}

^agarase

^{pH 6.0-} 10.0 (pH 1.0

⁾

⁴⁰

^C

¹⁰

^.

^{20 mM} MOPS buffer (pH 6.0−7.0), 20 mM Tris-HCl buffer (pH 7.0−9.0), 20 mM Glycine-NaOH buffer (pH 9.0−10.0)

^.

Thin layer chromatography 분석

조효소

¹⁰

^l

0.2% agarose

20 mM Tris- HCl buffer (pH 7.0)

⁴⁰

^C

²⁴

응하면서

¹²

, Thin layer

chromatography (TLC)

^[5].

⁽¹⁰

^l)

Silica Gel 60 plate(EMD Merck AG, Darmstadt, Germany)

n-butanol, acetic acid

(2:1:1, v/v/v)

²

^.

^30%(w/v)

¹²⁰

^C

^.

결과 및 고찰

균주 H9의 형태적 특성

충청남도대천 인근에위치하는 호도 해안에 서식하는

agarase

^H9

^.

^H9

Korean Collection for Type Cultures (KCTC)

^KCTC23889

^.

^H9

^marine agar (MA; Difco 2216)

^ASW-YP

^colony

(Fig. 1A),

램염색결과

^Gram

^.

관찰결과

^{, H9}

^1.5 −2.0

^m

^0.6 −0.8

^m

^,

¹

polar flagellum

(Fig. 1B).

균주

^H9

^Na

^Na

^Na

^6%

, 2−3%

^{. H9}

^{pH 4.0}

^{pH 10.0}

^{, pH 5.0} −pH 9.0

는성장할수있고

pH 7.0−pH 8.0

성장이관찰되었다

^.

^H9

¹⁰

^C

⁴⁰

^C

^{, 15}

^C

25−30

C

^.

균주 H9의 생리적 특성

API 20NE kit

^{, nitrate}

reduction, indole production,

-glucose fermentation

urease, arginine dihydrolase,

-galactosidase

. Arabinose,

mannose, potassium gluconate, capric acid, adipic acid,

phenylacetic acid

assimilation

^.

esculin hydrolysis, gelatinase

-glucose, mannitol, N-acetyl-

glucosamine,

-maltose, malic acid, trisodium citrate

의

assimilation

^.

API ZYM kit

^H9

^, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin,

-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase,

-galactosidase,

^-

glucosidase, N-acetyl-

-glucosamidase

, lipase (C14),

-galactosidase,

^- glucuronidase,

-glucosidase,

-mannosidase,

-fucosidase

^.

^H9

^·

Table 1

^.

Fig. 1. Isolation of agarolytic bacterial strain H9. (A) Detection of agarolytic activity on agar plates. The strain was cultured on ASW- YP plates at 28

C for 2 days and the plate was stained with Lugol’s iodine solution. (B) Transmission electron microscopy (TEM) analysis.

Cells cultured on ASW-YP agar plates at 28

C for 2 days were stained with 1% (w/v) phosphotungstic acid and observed with transmission electron microscopy. Bar, 1 μm.

Table 1. Phenotypic and chemotaxonomic characteristics of strain H9 and its related strains.

Characteristics 1 2 3 4 5 6 7 8 9

Pigmentation - - - - - + - - -

Growth at:

4

C - + - - + - + + +

37

C + + - - - - - - -

6% NaCl - + + + + + + + +

Enzyme activity of:

Agarase + - + + - - - - -

Esculin hydrolysis + ND ND - ND ND ND ND +

β

-Galactosidase - ND ND + ND ND ND ND +

Lipase (C14) - + + + ND ND + + -

Fermentation of:

D

-Glucose - + + - + - ND ND -

Assimilation of:

Mannose - - + - - + - - +

N-acetyl-glucosamine + - - - - - - ND -

G+C content (mol%) 41.6 42.7 42.1 42.1 38.9 43.6 40.2 42.1 42.3

Strains: 1, strain H9; 2, Pseudoalteromonas espejiana NCIMB2127

[3, 6]; 3, P. atlantica IAM12927

[1, 6]; 4, P. agarivorans KMM255

[16]; 5, P. carrageenovora ATCC12662

[1, 6]; 6, P. distinct ATCC70051

[7]; 7, P. haloplanktis ATCC14393

[20];

8, P. tetraodonis IAM14160

[8]; 9, P. arctica A37-1-2

[2]. Symbol: +, positive; -, negative; ND, not mentioned.

균주 H9의 계통발생학적 연관관계 규명

균주

^H9

^{16S rRNA}

^NCBI

의

^BlastN

한결과

, Pseudoalteromonas

^.

^H9

^{16S rRNA}

P. espejiana NCIMB2127 (98.98%), P. carrageenovora ATCC12662 (98.78%), P. atlantica IAM12927 (98.64%)

^.

^H9

Pseudoalteromonas

. 16S rRNA

N-J

^.

, 16S rRNA

P. espejiana NCIMB2127, P. carrageenovora ATCC12662, P. atlantica IAM12927, P. agarivorans KMM255

^{(Fig. 2).}

^H9

^·

많은차이점을감안하여균주

^H9

Pseudoalteromonas sp.

H9

^.

Pseudoalteromonas sp. H9의 유전학적 특성 및 세포벽 지방산 조성

균주

^H9

^DNA

^G+C

^41.56%

^.

Pseudoalteromonas

^G+C

³⁸ −44%

^[16].

^H9

quinone-8 (Q-8)

^{, Q-8}

Pseudoalteromonas

^.

^H9

^(>10%)

^C

7c (34.3%),

C

(23.72%), C

7c (13.64%)

^,

Pseudoalteromonas

^[2].

^{, H9}

Pseudoalteromonas

^.

^·

Fig. 2. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequence. A neighbor-joining phylogenetic dendrogram based

on 16S rRNA sequences of Pseudoalteromonas sp. H9

(JN578479) and closely related Pseudoalteromonas species. Numbers at nodes

indicate bootstrap percentages (based on 1,000 resamplings). Bar, 0.01 substitutions per 100 nucleotide positions.

특성들을토대로균주

^H9

Pseudoalteromonas

^.

^H9

Pseudoalteromonas

(Table 1).

H9

N-J phylogenetic tree

monophyletic clade

^.

^H9

Pseudoalteromonas

^.

균주 H9의 성장과 agarase 생산

균주

^H9

^agar

격히 성장하여 배양

²⁴

stationary phase (OD

= 4.22)

^,

⁴⁸

^(OD

= 4.32)

^.

^agar

²⁴

^(OD

= 3.87)

(Fig. 3). H9

^agarase

²⁴

^{, agar}

^1.5

^.

^H9

^agar

의해서성장이촉진될뿐만아니라

^agarase

으로촉진되는것으로예상된다

^.

균주 H9이 생산하는 agarase의 효소적 특성

균주

^H9

^agarase

⁴⁰

^C

^pH 7.0

(Fig. 4). 45

C

^91%

³⁵

^C

⁵⁰

^C

^64%

^.

^H9

^agarase

40−45

C

(Fig. 4A). 40

C

Fig. 3. Cell growth and agarase activities in liquid culture.

Strain H9 was cultured in ASW-YP broth and ASW-YPA (ASW- YP containing 0.2% agar) with vigorous shaking at 28

C for 4 days. Cell density was determined at 600 nm and agarase activ- ity was determined by the DNS method. All data shown are mean values from at least three replicate experiments. , -●- cell growth in ASW-YP; -○-, agarolytic activity in ASW-YP; -■-, cell growth in ASW-YPA; -□-, agarolytic activity in ASW-YPA.

Fig. 4. Biochemical properties of the extracellular agarases.

(A) The effect of temperature. An agarase assay was carried out at pH 7.0 by the DNS method at different temperatures. The max- imum value obtained at 40

C was taken to be 100%. The relative activities are the averages from three independent experiments.

(B) The effect of pH. An agarase assay was carried out at 40

C

at different pH conditions. The maximum value obtained at pH 7.0

was taken to be 100%. -■-, 20 mM MOPS buffer; -●-, 20 mM Tris-

HCl buffer; -

-, 20 mM Glycine-NaOH buffer. All data shown are

mean values from at least three replicate experiments.

였을때

^{, pH 7.0}

^{pH 8.0}

^90%

(Fig. 4B). pH 9.0

^83%

^,

^H9

^agarase

^.

^.

Pseudoalteromonas

^agarase

^,

^-agarase

. Pseudoal- teromonas sp. strain N-1

^-agarase

³⁰

^C

^{pH 7.0}

[21], Pseudoalteromonas sp.

CY24

-agarase AgaB

⁴⁰

^C

^{pH 6.0}

^[11].

Pseudoalteromonas sp. JYBCL 1

^agarase

³⁵

^C

^{pH 7.0}

^[15].

^,

^H9

agarase

agaH92

agarase AgaH92

⁴⁵

^C

^{pH 6.0}

보임을보고하였다

^[5].

AgaH92 agarase

^,

^H9

^agarase

⁽⁴⁰

^C, pH 7.0)

^.

^,

^AgaH92

agarase

연구가필요하다

^.

^AgaH92

^Fe

^agarase

^{, H9}

균주가타균주와는구별되는독특한특성을갖는

^agarase

들을생산할가능성이크다

^.

균주 H9의 agarase를 이용한 한천올리고당의 생산 균주

^H9

²⁴

하여

^agarose

물을

^TLC

^{(Fig. 5).}

^,

^H9

^agarase

(neo)agarotetraose

(neo)agarohexaose

. (Neo)agarooligosaccharide

^.

^.

균주

^H9

^agarase

법에의해서한천으로부터기능성올리고당의생산이가능 하며

^,

^.

요 약

대한민국대천해수로부터

^agarase

^H9

^.

^{16S rRNA}

Pseudoalteromonas espejiana NCIMB2127

(98.98%), Pseudoalteromonas carrageenovora ATCC12662

(98.78%), Pseudoalteromonas atlantica IAM12927

(98.64%), Pseudoalteromonas issachenkonii KMM3549

(98.63%)

^.

^H9

^genomic DNA

^G+C

^41.56%

^quinone- 8

^.

^H9

^C

^7c (34.3%), C

(23.72%), C

7c (13.64%)

^.

^,

^H9

Pseudoalteromonas

Pseudoalteromonas sp. H9

^.

^H9

agarase

40−45

C

pH 7.0−8.0

^{, agarose}

(neo)agarotetraose

(neo)agarohexaose

^.

^H9

^,

Fig. 5. Thin layer chromatography (TLC) analysis of hydroly-

sis product of agarose. The reaction was carried out by using the crude agarases at 40

C for 24 h in 20 mM Tris-HCl buffer (pH 7.0) containing 0.2% agarose, and then the hydrolysis products were separated on a Silica Gel 60 TLC plate. G,

-galactose;

NA6, neoagarohexaose; NA4, neoagarotetraose; AhG, 3,6-anhy-

dro-

-galactose; S1, 12-h reaction sample; S2, 24-h reaction

sample. Major end products, NA4 and NA6, are indicated by

arrow heads.

(neo)agarooligosaccharide

^,

^.

Acknowledgments

This work was supported by a grant from the Next-Generation BioGreen 21 Program (PJ01129301), Rural Development Adminis- tration, Republic of Korea.

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