Production and Characterization of Mannanase from a Bacillus sp. YB-1401 Isolated from Fermented Soybean Paste

된장 분리균 Bacillus sp. YB-1401의 Mannanase 생산성과 효소특성

전호진, 윤기홍*

우송대학교바이오식품과학전공

Received: March 17, 2014 / Revised: April 8, 2014 / Accepted: April 14, 2014

서 론

Mannan

mannose, glucose

^galactose

^,

mannan, glucomannan, galactomannan

galactogluco- mannan

^.

^mannose

^mannose

^glucose

^-1,4

^galactose

^mannose

⁶

^-1,6

^.

β

-1,4-Endo-mannanase (mannanase)

^-mannan

^mannose

-1,4-mannosyl

mannooligosaccharides (MOS)

^,

-1,4-mannosidase

^mannose

^,

mannan

mannobiose

exo-mannanase

Bacteroides fragilis

^[7].

mannose

^galactose

^- galactosidase

^{. Mannan}

요한역할을하는

^mannanase

물에서두루발견되었으며펄프와제지표백공정

^,

^,

첨가용효소로산업적활용성이높다

^[2].

균유래의

mannanases

로활용하기적합한성질을갖는효소를개발하기위해산 성

^,

^,

^[5, 13, 19, 26],

^{[22], Ca}

Clostridium thermocellum [3], Streptomyces thermolilacinus

Thermobifida fusca [9]

mannanases

^.

^,

^.

Production and Characterization of Mannanase from a Bacillus sp. YB-1401 Isolated from Fermented Soybean Paste Ho Jin Joen and Ki-Hong Yoon*

Food Science & Biotechnology Major, Woosong University, Daejeon 300-718, Republic of Korea

A Bacillus strain capable of hydrolyzing locust bean gum was isolated as a producer of extracellular mannanase by way of an enrichment culture in an acidic medium from homemade soybean pastes. The isolate YB-1401 showed a biochemical identity of 61.1% with Brevibacillus laterosporus, while the nucleotide sequence of its 16S rDNA had the highest similarity with that of Bacillus amyloliquefaciens. The mannanase productivity of the Bacillus sp. YB-1401 was drastically increased by mannans.

Particularly, maximum mannanase productivity was reached at approximately 265 U/ml in LB medium supplemented with konjac glucomannan (4.0%). The mannanase was the most active at 55

C and pH 5.5. Mannanase activity was completely maintained after pre-incubation at pH 3.5 to 11.0 for 1 h. The predominant products resulting from the mannanase hydrolysis were mannobiose and mannotriose for LBG, guar gum or mannooligosaccharides. A small amount of mannose was also detected in the hydrolyzates.

Keywords: Bacillus sp. enrichment culture, mannanase, productivity, property

*Corresponding author

Tel: +82-42-630-9742, Fax: +82-42-636-2676 E-mail: [email protected]

© 2014, The Korean Society for Microbiology and Biotechnology

산되는

^mannanase

^{, glycosyl} hydrolase (GH) family 5

²⁶

^{. GH26}

세균성효소가대부분을차지하고

^{, GH5}

는세균유래효소가포함된것으로알려져있다

^.

^GH113

^mannanase

Alicyclobacillus acidocaldarius

^[25].

최근에는

^MOS

[1, 17, 24]

glucomannan

galactomannan

^mannanase

^MOS

glucomannooligosaccharides

[1, 3, 17].

Galactomannan

롯한대부분의아시아국가뿐아니라아프리카지역에서도 고유식품으로 이용되고 있으며

^,

Bacillus

는균주로분리되고그효소의특성이연구되었다

[10, 15,

21].

여이로부터

^mannanase

^Bacillus

^.

재료 및 방법

Mannanase를 생산하는 내산성 균주의 탐색

가정에서제조된된장

²⁵

^pH

^4.5

^LB

³⁷

^C

²

³

³

^{. pH}

^4.5

^LB

(yeast extract, 5 g; tryptone, 10 g; NaCl, 5 g; agar, 15 g; water, 1 liter)

³⁷

^C

²

0.5% locust bean gum (LBG)

LB

congo red

(0.2%)

^LBG

mannanase

^.

분리균의형태적관찰을위해서는그람염색과포자염색을 실시하였으며

^,

API 20E

API 50 CHB kits (Bioméreux

^{, France)}

³⁷

^C

¹

²

^.

^16S

rRNA

색체

^DNA

^,

^{16S rRNA}

^primers

반응

^(PCR)

^PCR

염기서열을결정하였다

^[10].

Mannanase 조효소액 제조

Bacillus

^LB

^4.0%

^LB

^{1% (v/v)}

²⁴

^.

ammonium sulfate (30-75%)

^{, 12,000} rpm

³⁰

^.

10 mM citrate

^{(pH 5.5)}

상등액을취해

mannanase

^.

Mannanase 활성 측정과 반응특성 분석

Mannanase

^{0.5% LBG}

50 mM sodium citrate

^{(pH 5.5)}

⁵⁰

^C

서

¹⁵

^3,5-

dinitrosalicylic acid

^[14]

^,

^{1.0 unit}

¹

^LBG

¹

^mol

의

^mannose

정의하였다

. Mannanase

^pH

^30-65

^C

pH 4.0-8.0

^.

^30-60

^C

mannanase

¹

측정하여결정하였고

^{, pH}

^pH 3.0-12.0

^{50 mM}

⁴

^C

¹

^mannanase

^.

^pH

^citrate

(pH 3.0-6.0), phosphate

(pH 6.0-8.0), Tris

(pH 8.0-9.0), KCl-borate

^{(pH 9.0-} 10.0), carbonate

(pH 10.0-11.0)

phosphate/NaOH

(pH 11.0-12.0)

. Mannanase

의한가수분해산물을분석하기위해서는

^0.5%

^LBG,

guar gum

^2-6

^MOS

^,

20 mM citrate

^{(pH 5.5)}

⁴⁵

^C

⁵

⁹⁵

^C

⁵

chloroform, acetic acid

^{(4.3 : 5 :} 0.7, (v/v))

silica gel-precoated thin layer plate (Merck, Kiesegel, No. 5748)

^.

ethanol 9 ml, p-anisaldehyde 0.5 ml, sulfuric acid 0.5 ml

glacial acetic acid

^{, 120}

^C

¹⁰

^.

활성염색

조효소액을

^SDS-PAGE

polyacrylamide gel

SDS

25% isopropanol

^{50 mM}

phosphate

^{(pH 6.0)}

^15- 30

. Polyacrylamide gel

^{0.2% LBG}

^{1.5% agar}

^gel

⁵⁰

^C

⁴

gel

^{congo red}

polyacrylamide gel

Coomassie brilliant blue

^.

결과 및 고찰

증균 배양을 이용한 mannanase 생산균의 탐색과 분리균 의 특성

된장으로부터

^mannanase

^Bacillus

^pH

^4.5

^LB

²⁵

³

^.

¹

^.

^LB

^{(pH 4.5)}

³⁷

^C

²

²

^,

25

¹

^.

²

^YB-1401

^LBG

mannanase

^.

분리균

^YB-1401

성하는그람양성간균으로확인되어

API 50 CHB

^20E

kit

^{. Table 1}

^YB-1401

^Biomériux

^API

web (https://apiweb.biomerieux.com/jsp/login.jsp)

Brevibacillus laterosporus

^(61.1%)

, Bacillus subtilis

B. amyloliquefaciens

26.9%, B. pumilus

^11%

^.

또한분리균

^YB-1401

^{16S rRNA}

^PCR

^{1,496 bp}

^(GenBank accession no. KJ57222)

^NCBI

^BLAST

16S rDNA database

^.

^B.

amyloliquefaciens FZB42 (NR_075005)

^1,493

¹

^B.

subtilis 168 (NR_102783)

⁴

^.

B. laterosporus

^{16S rDNA}

^(AB112720)

는

^89%

^.

과로는분리균이

^Bacillus

할 수 없다고 하겠다

.

B.

licheniformis

-glucanase [4]

-amylase [11]

, mannanase

B. subtilis

[10, 15], mannanase

^B.

laterosporus

B. amyloliqueaciens

^. YB-1401

^{pH 4.5}

^pH

4.0

^4.5

^LB

¹⁶

^.

YB-1401

^{pH 4.0}

^pH 4.5

¹⁰

^.

B. subtilis

mannanase

[10, 15] B. subtilis

Table 1. Characteristics of the isolate YB-1401.

Sugar utilization Results Characteristics Results

ribose, D-glucose, D-fructose, D-mannose, mannitol, methyl- α-D-gluco- side, arbutin, esculin, salicine, cellobiose, trehalose, glycogen, sucrose +

β-Galactosidase −

Arginine dihydrolase − Lysin decarboxylase − Ornithine decarboxylase −

Citrate utilization −

glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-Xylose, adon- itol, methyl- β-D-xylopyranoside, galactose, sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl- α-D-mannopyranoside, N-acetyl-glucosamin, amygdalin, maltose, lactose, melibiose, inulin, melezitose, raffinose, starch, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-Arabitol, L-Arabitol, Gluconate, 2-keto-gluconate, 5-keto- gluconate

−

H

S production −

Urease −

Tryptophane deaminase −

Tryptophanase −

Acetoin production +

Gelatinase +

Nitrate reduction −

로널리사용되고있는

B. subtilis 168

^{pH 4.5}

배양한결과전혀성장하지못하여분리균

^YB-1401

차이를나타냈다

⁽

^).

부가탄소원에 따른 mannanase 생산성

곰팡이와세균유래의

^mannanase

^mannan

^. B. subtilis BS5 [5], B. subtilis WL-7 [10]

B. subtilis WY34 [6]

, Aspergillus niger BCC4525 [16]

B.

halodurans PPKS-2 [19]

copra meal

mannanase

^.

Bacillus sp. YB-1401

mannanase

0.5% (w/v)

^LB

³⁷

^C

24

mannanase

^.

지에서효소생산성이가장높았으며

^,

^mannan

당류를함유한

^LBG

^{guar gum}

^.

^mannan

palm kernel meal

높지않은것으로나타났다

(Table 2).

달리하였을때그함량이높을수록효소생산량이크게증 가하여

^4%

^.

는곤약을첨가한

^LB

^0.5%

mannanase

mannanase

B. subtilis WL-7

[10]. B. subtilis WY34

^2%

mannanase

^[6].

^LB

는점도가높아지고특히

^2%

^{, 4%}

을첨가한배지는

^agar

태였다

^.

mannanase

는배지의점질도가급격하게저하되고

²⁴

^.

배양시간에따른

mannanase

^(4%)

^LB

³⁶

mannanase

^,

^LB

⁹

^{1 U/ml}

^.

⁹

mannanase

^{10.2 U/ml}

^, 12

155.7 U/ml

¹⁸

이후에는약

265 U/ml

36

시간까지그생산성이유지되었다

^{(Fig. 1).}

^LB

¹²

^,

^LB

Table 2. Effects of additional carbon sources on the mannanase production of Bacillus sp. YB-1401.

Additional carbon sources

(0.5%)

Mannanase productivity

(U/ml)

Additional amount (%)

of konjac glucomannan

Mannanase productivity

(U/ml)

None 0.2 0.3 11.3

Glucose 0.3 0.5 12.1

Xylose 0.7 1.0 16.1

α

^{-Cellulose 0.9 1.5 20.1}

Locust bean gum 8.4 2.0 36.9

Guar gum 4.6 2.5 41.4

Konjac glucomannan 12.2 3.0 113.0

Palm kernel meal 1.3 3.5 128.7

Wheat bran 0.4 4.0 217.8

Fig. 1. Production of mannanase by Bacillus sp. YB-1401 and its growth. The isolate was grown respectively in LB broth sup- plemented with or without konjac glucomannan (4%) for mannan- ase production. Cell growth (closed circles) and mannanase productivity (closed triangles) were observed in the media without additional carbon source. Culture medium containing konjac glu- comannan was used for mannanase production (open circles).

Enzyme activities in the culture filtrates were determined in the

reaction condition of pH 5.5 and 50

C. Each curve represents the

average of three independent experiments.

으로여겨졌다

^.

배양상등액을

ammonium sulfate

효소액을사용하여

^SDS-PAGE

시한결과

^{Fig. 2}

^{38.0 kDa}

도로추정되는

mannanase

는

^SDS-PAGE

^{38-40 kDa}

B. subtilis WY34 [6], B. subtilis BCC41051 [18]

B. subtilis WL-3 [15]

mannanases

^.

Mannanase의 반응특성

조효소액을사용하여반응

^pH

⁵⁰

^C

pH

mannanase

^{pH 5.5}

pH 5.0-6.5

^70%

^{(Fig. 3).}

^pH

^5.5

⁵⁵

^C

^{(Fig. 4).}

^YB-1401

mannanase

^50-70

^C,

pH 5.0-7.0

^Bacillus

mannanases [10, 15, 18, 26]

^.

B. subtilis BS5 [5], B. subtilis G1 [20]

B. halodurans PPKS-2 [19]

mannanases

^.

pH 3-12

^mannanase

pH 3.5-10

¹

Fig. 2. Zymogram (A) and SDS-PAGE (B) of the crude enzyme produced by Bacillus sp. YB-1402. After SDS-PAGE of the con- centrated culture filtrate of isolate YB-1401 grown in LB broth con- taining konjac glucomannan (4%), protein exhibiting mannanase activity, which was indicated with arrow, was analyzed by activity staining with LBG as substrates. Molecular size is shown in kilo- daltons to the right side of the gel.

Fig. 3. pH Optimum and pH stability of the crude enzyme.

Buffers (50 mM) used were as follows: sodium citrate (pH 3-6), sodium phosphate (pH 6-8), Tris (pH 8-9), KCl-borate (pH 9-10), sodium carbonate (pH 10-11), and sodium phosphate/NaOH (pH 11-12). Mannanase activity (closed symbols) was assayed at pH 4-8 using sodium citrate (circles) and sodium phosphate (dia- monds) buffers with constant temperature of 50

C. pH Stability (open symbols) was determined by measuring the residual activ- ities after pre-incubations for 1 h in various pHs at 4

C. Each curve represents the average of three independent experiments.

Fig. 4. Temperature optimum and thermostability of the crude

enzyme. The enzyme reaction was performed at various tempera-

tures (closed symbols) for 15 min in 50 mM sodium citrate buffer

(pH 5.5). Thermostability (open symbols) was determined by mea-

suring the residual activities after pre-incubation for 1 h at different

temperatures. Each curve represents the average of three inde-

pendent experiments.

며

^{, pH 11.0}

^99%

^{(Fig. 3).}

^YB-1401

^mannanase

^pH

^Bacillus

mannanases

^{, pH 4-9}

B. subtilis B23 [26]

B. subtilis G1 [20]

mannanases

^pH

^.

^YB-1401

^mannanase

^{pH 3.0}

¹

^46%

준을유지하여

^{pH 3-10}

Thielavia arenaria XZ7

건에서안정성이낮은것으로나타났다

[13]. Mannanase

열안정성을조사한결과

⁴⁵

^C

¹

^{, 50}

^C

⁵⁰

^C

^{52%, 55}

^C

^15%

^.

^YB-1401

^mannanase

B. subtilis BCC41051 [18], B. subtilis G1 [20]

B. subtilis WL-3 [23]

^Bacillus

mannanases

, B. subtilis BS5 [5]

Sphingomonas

^[27]

mannanases

^.

반응산물

Mannanase

도가

^2-6

^{MOS, LBG}

^{guar gum}

^TLC

^{Fig. 5}

에나타낸바와같이

mannobiose

며이보다중합도가큰

^MOS

mannotriose

mannobiose

mannose

^.

mannotriose

^MOS

B. subtilis WL-3 [23]

Bifidobacterium adolescentis [8]

^.

B. subtilis WY34 [6]

^mannanase

mannotriose

분해하지 못하고

mannotetraose

MOS

^.

LBG

^mannanase

mannotriose

mannobiose

^mannose

되었는데

^{guar gum}

mannotriose

mannobiose

^{. Guar gum}

LBG

galactomannan

^mannose

galactose

, guar gum

galactose

. Mannanase

galactomannan

^mannose

^-1,4

mannose

^galactose

galactose

^{guar gum}

^LBG

^.

^mannanase

^LBG

^guar

gum

^4-10

수있다

^{[12, 23].}

요 약

가정에서제조된된장을산성조건에서계대배양한후균 체외

mannanase

Bacillus sp. YB-1401

^.

Brevibacillus laterosporus

^(61.1%)

^{16S rRNA}

B. amyloliquefaciens

^.

^YB-1401

mannanase

산성은

^mannans

^,

(4%)

^{265 U/ml}

^.

^mannanase

⁵⁵

^C

^{pH 5.5}

pH 3.5-11.0

¹

^.

^LBG

^{guar gum}

mannooligosaccharides

^mannanase

mannobiose

mannotriose

^mannose

^.

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