Isolation and Characterization of Marine Bacterial Strain SH-1 Producing Agar-Degrading Enzymes

한천 분해효소를 생산하는 해양 미생물 SH-1의 분리 및 특성 분석

이재학¹, 이순열²*

1서일대학교식품영양과

2한경대학교생명공학과

Received: November 3, 2014 / Revised: December 4, 2014 / Accepted: December 5, 2014

서 론

최근지상및지하의자원에서해양자원쪽으로관심이이 동되는상황에서해조류또한여러분야에서관심을받고있 다

^.

분은다당류가주를이룬다

^{[5, 15].}

대체에너지개발

^,

다

[2, 3, 6].

지구표면위의생명체가생산하는생리활성물질과그분포 및 종류가 다르다는 면에서도 연구의 대상이 되고 있다

[5, 15].

용한기능성음식및웰빙음식문화가대두되며해조류를이 용하려는연구가진행되고있고최근일부올리고당이의학

적으로효과가있는것으로밝혀지며

^{[14, 17]}

보하는방법에대하여관심이증대되고있다

^.

현재까지다당류를분해하는방법은여러가지가사용이 되고있지만화학적방법의경우에는탄수화물대부분이화 학적성분특히알칼리나산에대하여비교적안정적이므로 유효성분의분리에어려움이있으며비특이적으로분해되는

문제점이있다

^[15].

택적올리고당을획득하기위해서는효소의사용이바람직 하다고볼수있다

^.

[5, 13, 29].

한천은홍조류의세포벽구성성분으로다당류두종류

^,

Isolation and Characterization of Marine Bacterial Strain SH-1 Producing Agar-Degrading Enzymes

Jae-Hag Lee

and Soon-Youl Lee

*

1

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

2

Department of Biotechnology, Hankyong National University, Anseong 456-749, Republic of Korea

A marine bacterial strain producing agar-degrading enzymes was isolated from a mud flat in Jeboo-do (Korea) using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. The isolate, designated as SH-1, was gram-neg- ative, aerobic, and motile with single polar flagellum. 16S rRNA gene sequence similarity analysis showed the isolate SH-1 had the highest homology (96.5%) to marine bacterium Neiella marina J221. Cells could grow at 28-37ºC but not at 42ºC, and the agarase activity of the cell culture supernatant was higher when grown at 28ºC than when grown at 37ºC. Cells could grow when concentrations of 1-5% (w/v) NaCl were added to the growth media with the best growth observed at 3% NaCl, and the agar- degrading enzyme activity of the cell culture supernatant was best when grown at 3% NaCl-containing growth media under the conditions we examined. The crude enzyme prepared from 48-h culture broth of strain SH-1 exhibited an optimum pH and tem- perature for agar-degrading activity at 7.0 and 40ºC, respectively. Zymogram analysis of the crude supernatant and cell extract showed that strain SH-1 produced at least 3 agar-degrading enzymes with molecular weights of 15, 35, and 52 KD. Thinlayer chromatography (TLC) analysis also suggested that HS-1 produces β-agarase to degrade agarose to neoagarooligosaccha- rides.

Keywords: Agar-degrading enzyme, marine bacterium, zymogram, enzyme activity

*Corresponding author

Tel: +82-31-670-5333, Fax: +82-31-670-5333 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

agarose (60-80%)

agaropectin (20-40%)

[10, 20].

적으로많이응용이되었고

^[8]

^{[14, 17],}

로서

^[14],

아가서식품및과자의원료로사용되고있다

^.

^,

[5, 27, 29].

한천분해효소

^(agarase)

최근해양미생물이아닌미생물도생산한다고보고되고있

다

^[27].

는데

^agarose

^-(1

³⁾

agarobiose

-agarase [25]

^-(1

⁴⁾

neoagarooligosaccharide

^-agarase

나눌수있다

^{[13, 25].}

히높은

neoagarooligosaccharide

^-agarase

^{[5, 17].}

^-agarase

고

^,

^-agarase

도활발히진행되고있다

[23, 24, 29].

^-agarase

neoagarotetraose

neoagarohexose

^-agarase

생물의계속적인분리및효소의특성연구는필요하다

^{[5, 17].}

본연구에서는한천분해효소를생산하는세균을서해제 부도의갯벌에서분리하였으며

^,

^SH-1

^.

재료 및 방법

미생물 배양조건 및 시약

개펄에서미생물을분리하고배양하기위하여사용된배지 는

Artificial Sea Water (ASW; MgSO

12.3 g, KCl 0.74 g, (NH

)

HPO

0.13 g, NaCl 17.5 g, CaCl

0.14 g, agar 15 g, Tris base 6.2 g per/1 L, pH 7.2)

^[4]

^Bacto- peptone 10 g, Yeast extract 3 g

^(ASW-YP).

배지 및 효소 활성측정을 위한 시약은 주로

Sigma (MO,

USA)

^PCR

소등은

Takara (Japan)

^.

인천제부도개펄에서다양한해조류를포함하는개펄을 채취하여멸균해수적당량을첨가하여약

¹⁰

^-10

^ASW

²⁸

^C

였다

^{. 4-7}

나움푹파이는세균들을한천분해효소를생산하는균주 로간주하였고필요시

Lugol’s iodine

(25 g/l Iodine, 50 g/l Potassium Iodine)

^.

효소를생산하는것으로확인된후에는주로

^ASW-YP

용하여균주를순수분리하였다

^.

2%

Uranyl acetate

^negative

(JEOL, JEM 1010, USA)

(NICEM, Seoul).

16S rRNA 유전자 염기서열 분석과 계통수(phylogenetic tree) 작성

순수분리한균체로부터

genomic DNA

^16S

rRNA

^PCR

사용하였다

^[19].

^PCR

^bacterial universal primer

^(27F

^{1492R, 9F}

^1512R

518F

^800R)

^,

^DNA

(Daejeon, Korea)

^.

^blastn

^.

^{16S rRNA}

EzTaxon server [7]

^{16S rRNA}

^alignment

ClustalW program [28]

. Phylogenetic tree

^Kimura’s two-parameter evolutionary model [16]

Neighbor-Joining [26]

^.

균주의 세포 성장과 agarase 활성 측정

균주의성장에따른

^agarase

^,

본적인조건으로

0.2% agarose

^ASW-YP

²⁸

^C

¹²

^.

28

C, 37

C, 42

C

^{, NaCl}

^ASW-YP

^NaCl

1%, 3%, 5%

^.

체의 성장은

^{600 nm}

^,

agarase

0.2% agarose

3,5-dinitrosalicylic acid (DNS)

^[21]

^{540 nm}

^.

³

^.

조효소액(crude enzyme) 준비

해수의평균염분농도인

^{3% NaCl}

^ASW-YP

배양액에서

²⁸

^C

²

^.

늄을최종

^75%

^[11].

25 mM Tris-buffer (pH 7.2)

^.

한편배양액으로부터회수된세포펠렛에배양액부피의

25 mM Tris buffer (pH 7.2)

^.

^,

25 mM Tris-buffer (pH 7.2)

^.

pH와 온도에 따른 agarase의 활성 측정

효소활성을위한최적

^pH

^buffer,

20 mM MOPS (pH 6.0-7.0), 20 mM Tris-Cl (pH 7.0-9.0)

^.

²⁰

^C

⁵⁰

^C

³⁰

^pH

^7.0

^.

Zymogram assay 및 박막 크로마토그래피 분석

단백질농도는

Bio-Rad Dc protein assay kit (CA, USA)

^standard

bovine serum albumin (BSA)

^{. SDS-PAGE}

Laemmli [18]

방법을바탕으로

10% polyacrylamide gel

commassie blue

^.

zymographic analysis

^gel

agarose

separating gel

^{[12]. SDS-} PAGE

^gel

2.5% Triton X-100

⁴⁰

^SDS

^{. SDS}

^gel

^agarase

25 mM Tris buffer (pH 7.2)

42

C

^30-60

^Lugol

^commassie

blue

^{. Agarase}

분해산물분석은

agarose 0.2%

50 mM sodium phosphate buffer (pH 7.0)

⁴⁰

^C

^Temujin

[29] silica gel 60 plate (Merck, USA)

n-butanol-acetic acid-DDW (2:1:1)

Thin layer chromatography (TLC)

^.

결과 및 고찰

SH-1 균주의 분리 및 형태학적, 유전학적 특성 분석 제부도개펄에서식하는균주들중한천분해효소를생

산하는해양미생물을분리하고자염분농도가해양의평균 염분농도와같은농도인

^3%(w/v)

artificial sea

water

^ASW

환을보이는균주를선별하여

^SH-1

^{. SH-}

1

^ASW-YP

²⁸

^C

²⁴

면약

^{1-2 mm}

성하고콜로니주위에

^{2-3 mm}

^.

주

^SH-1

¹

을보였으며배양시간이지남에따라점점높은효소활성을 보였다

^{(Fig. 1A).}

SH-1

(transmission electron microscopy)

^.

^0.3-0.7

^m

^1-3

^m

^.

에약

^10-15

^m

(Fig. 1B).

Fig. 1. Agar-degrading activity and transmission electron

micrograph (TEM) of the marine bacterium SH-1. (A) Zymo-

gram plate assay result as a function of cultivation time of the SH-

1. The strain was cultivated on ASW-YP agar plate and Lugol’s

idodine solution was overlaid to detect reducing sugars, degraded

product from agar by agarase. (B) Transmission electron micro-

graph of SH-1. The bacterial cells were removed from ASW-YP

culture plate and examined by transmission electron microscopy

after negative staining with 2% uranyl acetate. Bar, 1 μm.

분리된균주를동정하고자

^{16S rRNA}

^PCR

^.

^blastn

^16S

rRNA

(Fig. 2).

Neiella marina J221

96.5%

. Neiella marina J221

²⁰¹²

(Apostichopus japonicas)

^{, 1}

^[9]

Neiella marina H014

(GenBank: KJ577009.1).

그다음으로높은유사성을보인균군으로는

Pseudoalteromonas

속이약

^92%

^.

보이는

Neiella marina J221

agarase, alginase, cellulase,

amylase

^[9]

서분리한

^SH-1

Neiella marina J221

^SH-1

Neiella

^.

SH-1 균주가 생산하는 한천 분해 효소의 특성 분석

SH-1

분석하고자우선균주의생장조건에따른균주의효소활성 에대하여알아보았다

^{. 28}

^{C, 37}

^C

⁴²

^C

^{, 37}

^C

³⁶

²⁸

^C

³⁷

^C

아졌다

(Fig. 3A).

결과비록생장속도는

³⁷

^C

²⁸

^C

⁵

Fig. 2. Phylogenetic tree analysis of strain SH-1. Tree was constructed from the nucleotide sequence of 16S rRNA gene using the neighbor-joining method [26]. The scale bar indicates a genetic distance of 0.2. The number shown next to each node indicates the per- centage bootstrap value of 1,000 replicates.

Fig. 3. Effect of growth temperature on cell growth and aga-

rase production. (A) Comparison of cell growth in ASW-YP at the

different growth temperature (42

C, ▲; 37

C, ●; 28

C, ■). (B)

Comparison of agarase activity produced by the cells grown at two

different growth temperatures (37

C, ●; 28

C, ■). All data are the

average of three parallel replicates.

(Fig. 3B).

배양액의염분농도에따른효소활성을조사하고자

^NaCl

^{1%, 3%,}

^5%(w/v)

^{. Fig. 4A}

^3%

^.

도에 따른 효소 활성을 알아본 결과

^{Fig. 4B}

NaCl 3%

^{NaCl 1%}

우보다약

^1.6

^.

효소활성을위한최적조건을알아보고자배양액조효소 액을준비하여서로다른

^pH

^.

[13, 14] SH-1

pH 7.0-8.0

활성을보였다

(Fig. 5A).

하여

20, 30, 37, 40, 45,

⁵⁰

^C

^{Fig. 5B}

^30-45

^C

Fig. 4. Effect of salt concentration of the medium on cell growth and agarase production. (A) Cell growth in ASW-YP with the different NaCl concentration at 1, 3 and 5% (w/v) (1%, ▲;

3%, ●; 5%, ■). (B) Comparison of agarase activity produced by the cells grown at the different NaCl concentration (1%, ▲; 3%, ●). All data are the average of three parallel replicates.

Fig. 5. Effect of pH on agarase activity produced by SH-1 strain. (A) Enzyme activity on the different pH (pH 6-7, ▲; pH 7- 9, ●). (B) Enzyme activity on the different temperatures. All data are the average of three parallel replicates.

Fig. 6. Zymogram assay of the agarases produced from SH-

1. Lane 1, crude enzyme extract of cell supernatant; lane 2, crude

enzyme extract of the cell. Size of protein markers are indicated

on the left (in KD).

비

^80%

⁴⁰

^C

^.

배양액과세포질조효소액을한천을포함하는

^PAGE

^zymogram

^{Fig. 6}

^.

^SH-1

³

50-55, 35, 15 KD

^.

^{50-55 KD}

^{35 KD}

^.

^-agarase

^[13, 15]

-agarase

.

^-agarase

^{85 KD}

되고 있다

^{[1, 22].}

agarose

^TLC

neoagarohexose, neoagarotetrose, neoagarobiose

^Rf

^SH-1

^-agarase

^{(Fig. 7).}

요 약

한천을분해하는해양미생물을한천을유일한탄소원으로 하는인공해수한천배지를이용하여제부도개펄에서분

리하였다

^{. SH-1}

한개의극성편모를가지는균이었다

. 16S rRNA

염기서열의유사성분석결과분리된균주는

Neiella marina

J221 [9]

^(96.5%).

28-37

C

⁴²

^C

³⁷

^C

²⁸

^C

^.

^SH-1

1-5% NaCl (w/v)

^3%

^3%

^{. 48}

^pH

^{pH 7.0}

⁴⁰

^C

^.

^zymogram

15, 35, 52 KD

³

^.

^(TLC)

^-agarase

^.

Acknowledgments

The present research has been conducted by the Research Grant of Seoil University in 2013.

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