Enhanced Production of Endo-β-1,4-xylanase from Paenibacillus sp. HX-1 Newly Isolated from Soil Samples at Hambak Mountain in Yongin city, Korea

용인 함박산 토양에서 분리한 Paenibacillus sp. HX-1의 동정과 endo-β-1,4-xylanase 생산 증가를 위한 배지최적화

지원재¹, 김종희², 홍순광¹*

1명지대학교생명과학정보학부

2서일대학교식품영양과

Received : April 9, 2013 / Revised : May 10, 2013 / Accepted : May 11, 2013

서 론

헤미셀룰로오스

(hemicellulose)

^(xylan),

(galactan),

(arabinan)

^,

^{[1, 3].}

^,

^[9].

^D- xylopyranose

^-1,4-

O-acetyl, 4-O-methyl-D-glucuronosyl,

^-L- arabinofuranosyl

(heterogeneous polysaccharide)

^{[10, 25].}

최근자일란은바이오연료나화학원료로사용될수있 는재생가능한바이오메스라는점에서많은관심을모으고

있다

^[1].

-1,4-endoxylanase,

^- xylosidase,

-L-arabinofuranosidase,

-glucuronidase, acetyl xylan esterase, phenolic acid esterase

^[18].

-1,4-xylanases

^-1,4-

xylan

일란분해시작단계에우선적으로작용하는효소이다

^.

^,

-xylosidases

xylooligosaccharides

oligosaccharides

^,

-arabinofuranosidases,

-glucuronidases, acetyl xylan esterases

^[9].

^,

^(xylanase)

^glycosyl hydrolase 10 (GH10)

GH11 family

^{. GH10} family

^pI

^,

(>30 kDa),

^[2].

, GH11 family

^[11]

^pI

^,

^.

Enhanced Production of Endo-β-1,4-xylanase from Paenibacillus sp. HX-1 Newly Isolated from Soil Samples at Ham- bak Mountain in Yongin city, Korea. Chi, Won-Jae

, Jonghee Kim

, and Soon-Kwang Hong

* .

Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Korea,

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

A xylanase-producing bacterium was isolated from a soil sample collected in Yongin city, Korea. The strain was aerobic and gram positive, and grew between pH 5.0 and 11.0, forming a yellow-colored colony. The strain was classified as a novel sub- species bacterium of Paenibacillus barcinonensis by 16S rRNA gene sequence similarity, phylogenetic analysis, phenotypic, and biochemical characteristics, and thus named Paenibacillus sp. HX-1. This strain produced extracellular endo- β-1,4-xyla- nase, and the best xylanolytic activity (205.17 unit/ml) was obtained at 96 h in an optimized TNX medium containing 1% (w/v) bacto tryptone, 1% (w/v) NaCl, and 0.7% (w/v) beechwood xylan at pH 7.0, 37

C and 200 rpm. The endo- β-1,4-xylanase pro- duced by the strain HX-1 yielded xylobiose as the end product from beechwood xylan hydrolysis. The enzyme exhibited opti- mum pH and temperature at pH 7.0 and 45

C, respectively. The remarkable enhancing effect of the TNX medium on xylanase production by HX-1, in spite of its simple formula, may have great advantages for industrial applications of xylanase.

Keywords: Xylanase, endo- β-1,4-xylanase, xylobiose, Paenibacillus sp. HX-1

*Corresponding author

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

© 2013, The Korean Society for Microbiology and Biotechnology

미생물유래의자일라네이즈는다양하게산업적으로응용

된다는점에서최근큰흥미를끌고있다

^[3].

블리칭

(bleaching)

는염소가함유된블리칭용화학약품사용을줄여환경오염 문제를크게줄일수있다

^.

^,

^pH

cellulase-free

^[1].

(clarifying juices)

(agricultural silage)

^.

^,

^,

^(xylose)

용된다

^[3].

질은동물의장내에서음식물의점도를증가시키고

^,

^[24].

^.

^,

이획기적인비용절감공정이될수있기때문이다

^[9].

서

^,

^.

^,

[4, 5, 9].

^,

^HX- 1

^.

^HX-1

^,

균주

^HX-1

배지조성최적화

^,

^.

재료 및 방법

Isolation of xylan-degrading bacteria from soil sample

자일라네이즈

^(xylanase)

^,

¹⁰

^-10

^.

²⁰⁰

^l

0.2% beechwood xylan (Sigma, USA)

Luria-Bertani (LB)

³⁷

^C

2

^{. 1,500}

^{, replica}

^{. 2}

2.0% Congo red

란분해환을형성하는콜로니를선별하였다

^.

(

³⁰⁰

⁾

^replica

^,

³

^.

⁵

^.

⁽

⁴⁰

3

⁾

^{16S rRNA}

^.

^{, 3}

으로밝혀졌기때문에

^,

^HX-1

본연구에사용하였다

^.

고분석용시약은

^Sigma

^(USA)

소및

Taq Polymerase

^Takara

^(JAPAN)

^.

Phylogenetic analysis based on 16S rRNA gene sequence

균주

^HX-1

^DNA

genomic DNA extraction kit (DyneBio, Korea)

. 16S rRNA

bacterial universal primer (27F; 5'-AGAGTTTGATCC TGGCTCAG-3', 1492R; 5'-TACCTTGTTACGACTT-3')

^DNA

polymerase chain reaction (PCR)

^[4].

^DNA

pGEM-T easy vector (Promega, USA)

^{. Applied} Biosystems 3730xl DNA Analyzer

^.

^HX-1

^{16S rRNA}

^NCBI

BLASTN search

^.

(type strain)

^{16S rRNA}

^EzTaxon server [6]

. 16S rRNA

(phylogenetic tree)

PHYLIP suit

^[7]

의

Neighbour-joining (NJ)

^[21]

^. Sequence alignment

^ClustalW

^[23]

을사용하여수행하였고

^{, 5'}

^3'

^gap

^BioEdit

[8]

. Bootstrap

^1,000

. Evolutionary distance matrix

^Kimura two-parameter

^[12]

^.

Chemotaxonomy analysis

균주

^HX-1

respiratory quinone

^Komagata

^-HPLC

^[14],

체의 지방산은 기존의 알려진

^Miller

^[17]

methyl ester

^,

fatty acid methyl

esters (FAME)

Microbial Identification

system (MIDI)

Agilent technologies 6890

GC

HP-1 capillary column (cross-linked methyl siloxane, 30 m

^{0.320 mm}

^0.25

^m)

^-

[22]. DNA G+C content

역상

^-HPLC

^Mesbah

였다

^[15].

Biochemical characteristics

균주

^HX-1

해서

, 0.3% beechwood

LB

^{, 5-55}

^C(5

^C

⁾

^.

^pH

, pH 4.0-12.0(pH 1.0

⁾

^pH

^LB

^{, 37}

^C

^.

^NaCl

0-15%(1%

⁾

^NaCl

^{, 37}

^C

^.

^HX- 1

API 50CH kit (Biomrieux, France)

API Staph kit (Biomrieux, France), API ZYM kit (Biomrieux, France)

^,

배지에

^NaCl

^1%

법에 따라 실험을 수행하였다

^.

^HX-1

0.5 McFarland

^,

37

C

²⁴

^strip

^.

균주

^HX-1

을 탐색하고자 하였다

^.

^bacto tryptone (1%)

beechwood

^(0.3%)

^{NaCl (1%)}

^pH

^7.0

^.

, bacto tryptone

bacto peptone

meat extract

^soytone

^1%

^.

HX-1

³⁷

^C

⁵

^{200 rpm}

였으며

²⁴

^{10 ml}

^.

각각의샘플은

15,000 rpm

¹⁰

, 3,5-dinitrosalicylic acid (DNS)

^[16]

0.2% beechwood

²⁰

^{540 nm}

^.

^(unit/

ml)

^ml

¹

^xylose

⁽

^mole)

^.

배지내최적탄소원을조사하기위해서

^,

(bacto tryptone, 1%)

beechwood

xylose, maltose, glucose, lactose, sucrose, fructose,

carboxylmethylcellulose (CMC)

^0.3%

가되도록첨가하였다

^.

^HX-1

³⁷

^C

⁵

²⁴

^.

, 0.1-1.0% (0.1%

간격

⁾

beechwood

주

^HX-1

^,

된자일라네이즈활성을측정하여최적탄소원농도를탐색 하였다

^.

TNX (Tryptone-NaCl-Xylan)

^.

대한

^{600 nm}

^.

Enzymatic characteristics of secreted xylanases by strain HX-1

균주

^HX-1

500 ml TNX

³

15,000 rpm

¹⁰

하고

^{400 ml}

^.

종농도

^70%

. 15,000 rpm

¹

20 mM Tris- Cl

(pH 8.0) 10 ml

^,

¹²

^.

^pH

^,

pH 6.0-10.0 (pH 1.0

⁾

⁴⁰

^C

¹⁰

^.

^{20 mM} MOPS

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

^{(pH 7.0-} 9.0), 20 mM Glycine-NaOH

(pH 9.0-10.0)

^.

반응의최적온도조건을탐색하기위해서

, 20 mM Tris-Cl

완충액

^{(pH 7.0)}

⁽²⁰

^{C, 30}

^{C, 40}

^C, 45

C, 50

C, 60

C)

¹⁰

^.

^HX-1

^,

(20 mM Tris-Cl

, pH 7.0, 45

C)

beechwood

²⁴

^Chi

^[5]

Silica Gel 60 TLC plate (Merck, USA)

Thin layer chromatography (TLC)

^.

결과 및 고찰

Phylogenetic analysis of newly isolated strain HX-1 based on 16S rRNA gene sequence

경기도용인시처인구에위치한함박산에서채취한토양

샘플로부터분리된자일라네이즈생산균주

^HX-1

기위해

^,

^{16S rRNA}

(Fig. 1A). Bacterial universal primer

^PCR

^,

^HX-1

16S rRNA

Paenibacillus barcinonensis BP-23

, Paenibacillus taichungensis BCRC 17757

, Paenibacillus pabuli JCM 9074

, Paenibacillus amylolyticus NRRL NRS-290

, Paenibacillus tundrae A10b

, Paenibacillus xylanilyticus XIL14

, Paenibacillus tylopili MK2

, Paeni- bacillus illinoisensis NRRL NRS-1356

, Paenibacillus xylanexedens B22a

99.088%, 98.100%, 97.811%, 97.320%, 97.257%, 97.187%, 97.046%, 97.033%, 96.695%

의상동성을보였다

^.

^HX-1

Paenibacillus

(genus)

^(species)

^.

^{1,432 bp}

^{16S rRNA}

^GenBank

JX853820

^.

균주

^HX-1

^{16S rRNA}

phylogenetic tree

. PHYLIP suit program

^{NJ method}

phylogenetic tree

^{, HX-1}

P. barcinonensis [20]

^branch

(bootstrap value = 100%)

(Fig. 1B).

P. taichungensis BCRC 17757

, P. pabuli JCM 9074

, P. xylanilyticus

XIL14

^.

16S rRNA gene sequence

phylogenetic tree

^HX-1

^P.

barcinonensis BP-23

^.

Phenotypic and chemotaxonomic characteristics

균주

^HX-1

찰되었고

^{, LB}

^TNX

²

^.

^HX- 1

^20-45

^C

^35-40

^C

^.

^Na

^ion

요로하지않지만

^1%

^NaCl

하게성장하고

^{, 5%}

^NaCl

었다

^.

^{pH 5.0}

^11.0

^pH

^.

(pH 6.0-8.0)

찰되었고

^,

^{pH 7.0}

였다

^.

균주

^HX-1

^{DNA G+C}

^49.79%

respiratory quinone

^MK-7

^.

, Paenibacillus

^{DNA G+C}

^39- 54%

^,

respiratory quinone

^MK-7

하고있는것으로알려져있는데

^,

^HX-1

와일치함을확인할수있었다

^.

^HX-1

요 지방산은

^anteiso-C

(41.21%), C

(24.02%), C

(7.94%), iso-C

(6.67%), iso-C

(6.70%)

^,

Paenibacillus

^.

^HX-1

^DNA G+C content, respiratory quinone,

Paenibacillus

^.

하지만

^,

^HX-1

^G+C

^(49.79%)

P. barcinonensis BP-23

^G+C

(45.0%)

^.

^{Table 1}

^HX-1

P. barcinonensis BP-23

Fig. 1. Isolation of xylanolytic bacterium from soil sample.

(A) Xylanolytic activity on agar plate. Strain HX-1 was cultured on

LB agar plate containing 0.2% xylan-azure at 37

C for 48 h, and

then 2.0% Congo red solution was overlaid on the plate to detect

xylanolytic activity. (B) Neighbor-Joining phylogenetic tree. The N-

J tree was constructed based on 16S rRNA gene sequences of

strain HX-1 and other Paenibacillus species. Distances was deter-

mined according to the Kimura’s 2-parameter model and boot-

strap values (>50%) based on 1,000 replicates are listed as

percentages at nodes. GenBank accession numbers of 16S rRNA

gene sequences are given in parentheses. Scale bar, 0.01 sub-

stitutions per 100 nucleotides.

는

⁵

^C

nitrate reduction

^- galactosidase, urease, trypsin, N-acetyl-

-glucosaminase

D-arabinose, melezitose, starch, glycogen, gluconate

보이고있다

^.

^HX-1

P. barcinonensis BP-23

16S rRNA gene sequence

^,

^,

^,

^.

⁶

^-

특성에관한비교를

^{Table 1}

^.

Medium optimization for xylanase production from strain HX-1

자일라네이즈생산촉진을위한배지의최적질소원을결 정하기위해

, bacto tryptone, bacto peptone, meat extract, soytone

^HX-1

^.

^bacto peptone, meat extract, soytone

Table 1. Phenotypic characteristics of strain HX-1 and phylogenetically related type strains of genus Paenibacillus.

Characteristic Strain HX-1

P.

taichungensis BCRC 17757

P. babuli JCM 9074

P.

amylolyticus NRRL NRS-290

P.

barcinonensis BP-23

P.

illinoisensis NRRL NRS-1356

P.

xylanilyticus XIL14

Nitrate reduction + − − + − + −

Growth at:

5

C − + + + + − +

45

C + − − − − + +

2% NaCl + + + + + − +

7% NaCl − + + − − + +

pH 4.0 − − − w − + +

pH 10.0 + + + − + + +

pH 11.0 + + + − + − +

pH 12.0 − − − − − − +

Enzyme activity:

β−galactosidase + + + + − + +

Urease + − − + − − −

Trypsin − − − − + − −

α

-Chymotrypsin + + + − + + +

Acid phosphatase + + − + + + +

N-Acetyl- β-glucosaminase − + + + + + −

α-Fucosidase − − − + − − −

Acid production from:

Glycerol w + + − + + +

D-Arabinose − − − − + − −

Inulin − + + − − + +

Melezitose − + + − + − −

Starch + + + + − + +

Glycogen + + + + − + +

Gluconate − − − − + + +

DNA G+C content (mol%) 49.8 46.7 45.0 46.4 45.0 48.1 50.5

Predominant menaquinone MK-7 MK-7 ND MK-7 MK-7 MK-7 ND

Symbols: +, positive; −, negative; w, weak positive.

지의배양액에서는자일라네이즈활성이거의관찰되지않 는반면

, bacto tryptone

^(OD

= 0.198)

(Fig. 2A).

, bacto tryptone

^.

, bacto tryptone

^,

beechwood

, xylose, maltose, glucose, lactose, sucrose, fructose, carboxylmethylcellulose (CMC)

^HX-1

^.

, beechwood

^CMC

^,

(Fig. 2B).

^, Beechwood

^{, CMC}

지보다배양기간동안계속해서최대약

^1.3

^.

beechwood

, beechwood

0.1-1.0%(0.1%

⁾

도로포함하는배지에균주

^HX-1

^,

터 생산된 자일라네이즈 활성을 측정하였다

^{(Fig. 2C).}

Beechwood

^0.7%

^,

^1.6

^.

^1%

bacto tryptone

0.7% beechwood

^.

^HX-1

^TNX

^{, TNX}

^{1% bacto} tryptone, 0.7% beechwood

^{, 1% NaCl}

^, pH 7.0

^.

Fig. 2. Optimization of medium composition for xylanase production by strain HX-1.

(A) Determination of nitrogen source. Strain HX-1 was cultured in liquid medium containing bacto tryptone (●), bacto peptone (◆), meat

extract ( ▲), and soytone (■), respectively. (B) Determination of carbon source. Strain HX-1 was cultured in liquid medium containing

beechwood xylan ( ◆), xylose (■), maltose (▲), glucose (□), lactose (△), sucrose (○), fructose (◇), and carboxylmethylcellulose

(CMC) ( ●), respectively. (C) Determination of optimum concentration of xylan as sole carbon source. Strain HX-1 was cultured in liquid

medium containing various concentrations (0.1-1.0%, increment of 0.1%) of xylan at 37

C for 96 h with vigorous shaking. (D) Cellular

growth and xylanase production in the optimized medium. Strain HX-1 was cultured in optimized medium (TNX) at 37

C with vigorous

shaking. LB medium was used as a control. The cellular growth and xylanase activity was measured at 600 nm and at 540 nm, respec-

tively. All data are the average values of three parallel experiments. ■, cell growth in LB; □, xylanase activity in LB; ●, cell growth in

TNX; ○, xylanase activity in TNX.

Cellular growth and xylanase production of strain HX-1

균주

^HX-1

^TNX

^{, LB}

^1.9

(Fig. 2D). LB

¹

^(OD

= 0.316)

5

^{, TNX}

양

2

(OD

= 0.588)

3

^(OD

= 0.305)

^{. LB}

⁽

27.61 unit/

ml)

^{, TNX}

이배양

²

⁴

^(205.17 unit/ml)

⁵

^{(Fig. 3).}

^HX-1

^7.4

^.

^{, HX-1}

^.

^{, Zheng}

^[27]

Paenbacillus campinasensis BL11

^G1-1

(0.5% NaNO

, 0.3% K

HPO

, 0.03% MgSO

^7H

O, 0.002%

MnSO

, 0.002% FeSO4

^7H

O, 0.002% CaCl

^2H

O, 0.5%

^{; pH 7.0)}

^,

10.5 unit/ml

143.98 unit/ml

였다

^.

^TNX

을갖는다는장점이있으며

^{, HX-1}

산성도월등하게높은것으로판단되었다

.

,

에서최적화된

^TNX

(metal ion

⁾

된다면

^,

^HX-1

자일라네이즈생산을유도할수있을것으로기대된다

^.

균주

^HX-1

는

pH 7.0-8.0

^pH

^,

20 mM Tris-Cl (pH 7.0)

(Fig. 3A).

P. campinasensis G-1 [27]

XynG1-1 (pH 7.5)

Paenibacillus sp. 12-11 [26]

Xyn7c (pH 8.0)

^pH

, P. curdlanolyticus B-6 [19]

Xyl10A (pH 7.0)

P. campinasensis BL11 [13]

XylX (pH 7.0)

^.

^{, 45}

^C

⁵⁰

^C

⁽

^90%)

(Fig. 3B).

P. campinasensis G-1

^XynG1-1 (60

C), Paenibacillus sp. 12-11

Xyln7c (55

C),

P. curdlanolyticus B-6

Xyl10A (60

C)

^.

균주

^HX-1

^TLC

분석한결과

^,

^HX-1

^xylobiose

^endo-

-1,4-xylanase

(Fig. 4). Xylobiose

xylooligosaccharide

^,

, prebiotic

^,

^,

^,

화장품산업등에널리사용될수있다

^[3].

Fig. 3. Enzymatic properties of xylanase produced by strain HX-1.

(A) Effect of pH on xylanase activity. The reaction was carried out at 40

C for 10 min. The values obtained at pH 7.0 using 20 mM Tris-Cl buffer were taken to be 100%. ■, 20 mM MOPS buffer;

●, 20 mM Tris-Cl; ◆, Glycine-NaOH buffer. The relative activities

are the averages from three independent experiments. (B) Effect

of temperature on xylanase activity. The reaction was carried out

at different temperatures in 20 mM Tris-Cl buffer (pH 7.0). The val-

ues obtained at 45

C were taken to be 100%. The relative activities

are the averages from three independent experiments.