Production and Properties of Mannanase and Xylanase by a Bacillus subtilis Isolate

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Bacillus subtilis 분리균의 Mannanase와 Xylanase 생산성과 효소 특성

윤기홍*

우송대학교외식조리영양학부

Received: July 14, 2015 / Revised: August 5, 2015 / Accepted: August 9, 2015

서 론

Hemicellulose는식물의세포벽을구성하는다당류로리

그닌과 cellulose 사이에결합한상태로배열해있고^xylan, mannan, arabinogalactan과^glucan등의다당류의혼합물 이므로한종류의구성잔기로이루어진섬유소와는달리오 탄당^,육탄당과 sugar acid에속하는여러종류의단량체로 구성되어있다. Beechwood(31%), 쌀겨⁽²⁴−^33%),나무껍질 (30%), 목초^(37%), 밀짚^(24%), 옥수수대^(25%), 볏짚⁽²³− 28%)의 hemicellulose 함량이알려져있으나서로차이가있 는결과도보고된바있다[10]. Hemicellulose의주성분다 당류인^xylan과^mannan은식물에따라그종류와구성비율

이차이가크며경질목에는 acetyl-4-O-methylglucuronoxylan이 10−^35%이고 glucomannan은³−^5%수준으로존재하는반면 에연질목에서 galactoglucomannan의함량이¹⁵−^25%정도 로 많으며 arabino-4-O-methylglucuronoxylan의 함량은 15−^25%이다^.또한^mannan다당류는다수의콩과식물종자 의배유와일부비두과류식물의완숙한종자에도존재하며 사료로사용되는대두피(8%), palm kernel meal (30−^35%) 과 copra meal (25−^30%)에는많은양이함유되어있다^.

목재^, 목초^, 농작물 및 농산부산물에 포함되어 있는

hemicellulose는섬유소다음으로가장풍부한재생가능자

원이므로이를소화또는발효가가능한당류로전환함으로 써사료원료의이용효율을높이거나에탄올을비롯한발효 산물의생산에활용하고있다[23]. Xylan과^mannan의분해 에는여러종류의효소가관여하는데이들중^xylan의기본 골격인 β-D-1,4-xylopyranosyl 결합과^mannan다당류의 mannose간의 β-1,4-mannosyl 결합을각각무작위적으로분 해하여중합도가작은올리고당으로전환시키는^xylanase Production and Properties of Mannanase and Xylanase by a Bacillus subtilis Isolate

Ki-Hong Yoon*

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

A bacterial strain capable of hydrolyzing xylan and locust bean gum (LBG) was isolated from the Saemangeum tideland of Korea. Based on the biochemical properties and the 16S rRNA gene sequence, the isolate YB-30 was identified as Bacil- lus subtilis. Xylanase productivity was increased effectively when B. subtilis YB-30 was grown in the presence of wheat bran, while mannanase productivity was increased drastically when grown in the presence of konjac or LBG. Particularly, maximum mannanase and xylanase activities were detected in the culture filtrate of media containing 3.5% konjac and 1%

wheat bran. Both enzyme productivities reached maximum levels in the stationary growth phase. The culture filtrate exhib- ited the highest activity at 60^oC and pH 6.0 for mannanase and at 55^oC and pH 5.5 for xylanase, respectively. Both enzymes were not stable at high temperatures and xylanase was less stable than mannanase. In addition, wheat bran was hydrolyzed to liberate reducing sugar to a greater extent than rice bran by the culture filtrate because the wheat bran con- tained more arabinoxylan than the rice bran. Hence, xylanase and mannanase produced by B. subtilis YB-30 have a potential use as feed additive enzymes.

Keywords: Bacillus subtilis, mannanase, xylanase, characterization

*Corresponding author

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

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[16]와 mannanase [9]는다당류의분해에가장중요한역할 을하는효소이므로산업적유용성이증가되고있다^.곰팡이 와세균으로부터다수의^xylanase와^mannanase가발굴되 었고그유전자에대한특성이규명되었다^.또한이들효소 는산업적으로생산되고있으며^,효소의특성과생산성개선 을위한연구도지속적으로진행되고있다^.

Bacillus속의균은 protease, cellulase, amylase, xylanase와

mannanase 등과같은가수분해효소를분비생산하는특성

을지니고있으며여러종류의균주로부터이들유용효소 가탐색되었다. Mannanase를생산하는균주로 B. subtilis, B. licheniformis, B. amyloliquefaciens, B. halodurans, B.

circulans, B. pumilus를포함한다수의 Bacillus sp.가보고 되었으며[19], xylanase 생산균은 이보다 더다양하여^B.

subtilis, B. licheniformis, B. halodurans, B. circulans, B.

pumilus, B. aerophilus, B. mojavensis, B. alcalophilus, B. cereus, B. amyloliquefaciens, B. trypoxylicola, B. firmus, B. megaterium, B. agaradhaerens, B. lyticus와여러특성 을갖는 Bacillus sp.가알려졌다^[15].또한 endoglucanase, xylanase, pectinase, mannanase를효소복합체로생산하는 B. licheniformis SVD1이보고된바있다^[17].본연구에서 는새만금갯벌로부터^xylanase와^mannanase를생산하는 Bacillus 균주를분리하여효소특성과생산성을조사하였다^.

재료 및 방법

효소 생산균 분리 및 동정

서해안새만금갯벌시료를채취하여^{5% NaCl}이첨가된 nutrient 평판배지에도말한후³⁷^o^C에서²⁴시간동안배양 한 후 형성된 콜로니를 선발하여 0.5% oat spelt xylan (xylan)과 0.5% locust bean gum (LBG)을함유한평판배지 에각각접종하였다^.하룻밤배양한후^{congo red}로염색하 여^xylanase와^mannanase를동시에생산하는균주를선발 하였다^.분리균을 tryptic soy agar에배양하여얻은콜로니 를 McFarland standard 0.5가되도록멸균된 0.45% NaCl 용액에현탁한후 BAC card (Biomereux사^{, France)}에주입 하고이를 Vitek junior 기기에장착하여배양하면서⁶−¹⁵

시간의범위에서판독된생화학적특성을 database와비교

하였다^.분리균의 총 유전체^DNA를 주형으로 하여^16S rRNA의 보존적 지역의 염기서열을 갖는 primers 27F와

1492R로증폭된중합효소연쇄반응^(PCR)산물을정제하여

16S rRNA 염기서열을분석하였다^. 효소 활성 측정

Mannanase 활성은^LBG를, xylanase 활성은^{oat spelt}

xylan을기질로하여각각효소반응후에유리된환원당을

3,5-dinitrosalicylic acid (DNS) 방법으로 다음과같이정량 함으로써측정하였다^.증류수에현탁시킨 1% (w/v) LBG 용 액또는^xylan용액^{0.5 ml}와 200 mM sodium phosphate 완충액^{(pH 6.0}또는 pH 5.5) 0.25 ml를효소용액^{0.25 ml}와 혼합하여적정온도에서¹⁵분동안반응시켰다^{. DNS}용액³ ml를첨가하여반응을정지시키고끓는물에서⁵분동안방

치하여 발색시킨 후^{540 nm}에서 흡광도를 측정하였다^.

Mannose 또는^xylose를표준물질로사용하여동일조건에서 발색시켜조사한흡광도와비교함으로써유리된환원당의양 을결정하였다^.효소활성도^{1.0 unit}는위의조건에서¹분동 안^LBG또는^xylan으로부터¹μ^mol의^mannose또는^xylose 에상응하는환원당을생성하는효소의양으로정의하였다^.

활성염색

조효소액을^SDS-PAGE한 후 polyacrylamide gel에서 SDS를 제거하기위해 25% isopropanol를포함한^{50 mM} sodium phosphate buffer (pH 6.0)로세척한후동일완충액 에¹⁵−³⁰분간방치하였다. Polyacrylamide gel을꺼내어동 일한 완충용액에 0.2% oat spelt xylan 또는^{0.2% LBG}와 1.5% agar를녹여제조한^gel을중층하여비닐로봉입한후 40^oC에서¹시간³⁰분동안반응시킨후중층한^gel을^congo red로염색하여분해지역을관찰하였다^.

조효소액 제조와 반응특성 분석

분리균 B. subtilis YB-30을효소생산에적합한배지에서 약¹⁸시간 배양한 후 원심분리하여 얻은 배양상등액을 ammonium sulfate (30−^70%)로처리하여침전된단백질을 10 mM sodium phosphate 완충액^{(pH 6.0)}에현탁하고투석 한후조효소액으로사용하였다^.효소활성에미치는반응 온도와^pH의영향을조사하기위하여³⁰−⁶⁵^o^C와^{pH 4.0}− 8.0의범위에서각각^xylanase와 mannanase 활성을측정하 였다^.열안정성을조사하기 위하여조효소액을서로다른 온도에서⁶⁰분간방치한후그잔존활성을측정하였다^.

농산부산물의 가수분해

농산부산물인 쌀기울^(1%)또는 밀기울^(1%)과^{50 mM} sodium phosphate 완충액^{(pH 6.0)}를포함하는반응액에효

소량을달리 첨가하여최종 부피가^{0.5 ml}이되도록하고

45^oC에서반응하면서일정시간마다반응액에존재하는환 원당을조사하였다^.

결과 및 고찰

Mannanase와 xylanase의 생산균의 분리와 특성 새만금 갯벌 시료로부터 분리된 균주 중에서^{oat spelt}

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xylan (0.5%)이나 LBG (0.5%)가첨가된^LB평판배지에서

xylan과^LBG을동시에분해하며분해환이큰균주를선발

하고이를^YB-30으로명명하였다^.분리균^YB-30은그람양

성간균으로확인되어^Bacillus속균주로추정되었으므로^,

Vitek의^{BAC card}를사용하여²⁹개항목의생화학적특성 을 조사하였다^.그 결과^YB-30는²⁰종류의 탄수화물 중 sucrose, glucose, arabinose, xylose, mannitol, salicin, inulin, ribose, maltose, trehalose, palatinose, sorbitol, N- acetyl-D-glucosamine을탄소원으로이용하였고, potassium thiocyanate, mandelic acid, polyamido-hyqrostreptin와

같은선택성이높은성장저해제나^{7% NaCl}이첨가된배지

에서포도당을발효하였으며 B. subtilis와유사도가^99%로 나타났다^.또한 분리균의^{16S rRNA}를^PCR로 증폭한 후

1,516 bp 크기의염기서열을결정하였으며이를미국^NCBI

의^BLAST검색방법을사용하여세균들의상응하는염기서

열과비교한결과 B. subtilis CE1 (Genbank accession No.

JQ435698)와 B. subtilis 168 (CP010052)를비롯한여러종 의 B. subtilis와 그 서열이 일치하여 분리균^YB-30은^B.

subtilis에속하는것으로판단되었다. B. subtilis CE1는작 물의뿌리병원성균에항균활성을보이는균으로습지대에 서분리되었다^[3].

한편 B. subtilis YB-30 균주가갯벌에서분리되었으므로 B. subtilis 168과내염성을비교하였다^{. NaCl}의농도가¹− 10% 범위에서달리 첨가된^LB평판배지와액상배지에서 16시간배양하여 성장정도를 비교한결과 액상배지에서 NaCl이^7%이상첨가된배지에서는두균주가모두성장이 약간은저해되었으나^{10% NaCl}을첨가한배지도성장이일 어났다^.평판배지에서는^NaCl의농도가높아질수록콜로니 크기가작아지면서^{10% NaCl}첨가배지는비정상적인형태 를보였으나두균주간에차이가거의없었다. 이로보아분 리균은갯벌에서분리되었지만내염성과는관련이없음을 알수있었다^.

부가탄소원에 따른 효소 생산성

Mannanase와^xyalanase는배지에포함된탄수화물종류

와함량에의해 생산성에큰영향을 받으며^{, mannan}이나

xylan이함유된배지에서그분해효소생산이증가되는것

으로보고된바가있다^{[8, 15].}따라서^YB-30의효소생산성 에미치는탄수화물의영향을조사하기위해^LB액체배지 를기본배지로하여단당류와이당류는^1%,고분자성탄소 원은 0.5% (w/v)가되도록각각첨가하여³⁷^o^C에서진탕배 양하였다^.배양시간은^LB배지를기준으로하여효소생산 성이더이상증가하지않는것으로나타난¹⁵시간으로결 정하고배양상등액에존재하는효소활성을측정하였다^.

분리균^YB-30의^xylanase의생산성은부가탄소원에의

해큰영향을받지않는데비해 mannanase 생산성은영향

을크게받는것으로나타났다(Table 1). 밀기울이첨가된배 지에서^xylanase생산성이^4.6배 정도 증가하였으나^{, oat} spelt xylan에의해서는²배정도수준으로증가하여다른탄

소원에비해 크게^xylanase생산성을증가시키지 않았다^.

Mannanase의생산성은 konjac, LBG와^{guar gum}등과같

은^mannan다당류가첨가된배지에서크게증가하였으며^,

특히^konjac에의한생산성증가는가장높아^LBG를첨가 하였을때보다³배수준에해당하였다^.한편단당류나이당 류가^xylanase와 mannanase 생산성에미치는영향은약간 의 차이를 나타내었으며, glucose, mannose, maltose, sucrose를함유한배지에서는 mannanase 생산성은변화가

거의 없었으나^xylanase생산성은²배 이상 증가하였고

lactose가첨가된배지에서는^xylanase생산성이변화하지 않았으나 mannanase 생산성이 약⁵배정도 증가하였다^. Xylose와 galactose 첨가배지에서는두효소의생산성이모 두^2.5배정도수준으로증가되었다^.

Xylanase와^mannanase의생산성을증가시키는효과가높 은밀기울과^konjac을각각^0.1−^5%범위로첨가된배지에서 효소생산성을조사하였다^.밀기울의첨가량이많아질수록

xylanase의생산성이증가하여^4%에서는생산성이최대에

도달하였다(Table 2). 밀기울성분중어떤성분이효소생 산성을증가시키는지확인하기위해밀기울을증류수에현 Table 1. Effects of additional carbon sources on the mannanase and xylanase production of B. subtilis YB-30.

Additional carbon sources

Mannanase productivity (U/ml)

Xylanase productivity (U/ml)

None 0.5 0.5

Glucose 0.5 1.1

Mannose 0.5 1.4

Lactose 2.6 0.5

Maltose 0.6 1.4

Sucrose 0.6 1.6

Mannitol 0.5 0.4

Fructose 0.5 0.9

Xylose 1.3 1.2

Galactose 1.3 1.3

α-Cellulose 3.1 0.5

Locust bean gum 44.0 1.2

Guar gum 11.3 0.8

Konjac 129.4 1.8

Oat spelt xylan 1.3 1.0

Rice straw 2.7 1.9

Wheat bran 2.9 2.3

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탁한후원심분리하여불용성물질과수용성물질로분리 하고이를밀기울^4%기준으로^LB배지에각각첨가한배

지에서^xylanase생산성을조사한결과수용성물질이함유

된배지^{(4.5 U/ml)}에서보다불용성물질을함유한배지에서

효소생산성이높았다(17.7 U/ml). 불용성과수용성으로구

분하지않고밀기울이그대로첨가된배지에서는^xylanase

생산성이^{20.9 U/ml}로나타났는데이는불용성물질과수용

성물질이따로첨가된배지에서각각생산된효소활성의 합계와유사한수준이었으며^,이들결과로보아밀기울의불

용성성분이^xylanase의생산을유도하는주된역할을하는

것으로추측된다^.

한편^mannanase는소량의^konjac이첨가된배지에서도 생산성이급격하게증가하였으며^{, 3}−^3.5%를첨가하였을때 mannanase 생산성이최대에이르렀다^{. Xylanase}와^mannanase 의생산성을동시에증가시키기위해서 3.5% konjac과동시

에밀기울의첨가량을달리하여제조한배지에서효소생산 성을조사한결과밀기울첨가량이^1%일때두효소의생산 성은모두최대로나타났으나^{, 2%}이상에서는두효소의생 산성이모두감소하였고특히 mannanase 생산성이크게감 소하였다^.

Konjac (3.5%)과밀기울^(1%)을동시에첨가한배지에서배 양시간에따라효소생산성의변화를조사하였다^.배양초기

에는첨가된^konjac에의해배양액의점도가너무높아배

양액을채취하기에적합하지않았으며점성이감소된¹²시 간을배양한후부터배양액의효소활성과생균수를조사하 였다^.그결과^{Table 4}에나타낸바와같이배양시간이¹⁸시

간이후에는생균수가더이상증가하지않았으며^{, xylanase}

는¹⁸시간에최대생산성을보이고이후에는약간씩감소하 였으나^mannanase는¹⁸시간이후에도생산성이약한수준 으로증가하였다^.그러므로효소생산성은정지기에이르렀 을때최대로 생산되었음을알 수있는데, B. halodurans PPKS도^YB-30과유사하게균의성장과연계하여^xylanase 의생산성이증가하고정지기에는최대에도달하는데비해^[13]

B. amyloliquefaciens의^mannanase는생육이정지기에도 달하였을때부터생산되기시작하여정지기기간중계속증 가하는것으로보고되었다^[21].

또한^mannanase와^xylanase를모두생산하는 B. halodurans PPKS02는옥수수피^,쌀기울^,밀기울을^1%첨가한배지에서

xylanase 생산성이증가하며특히옥수수피를첨가한배지

에서^{85 U/ml}의가장높은생산성을보였고^[13],탈지된코 코아야자박을첨가한배지에서는 mannanase 생산성이크

게증가하는것으로나타나^{[18] YB-30}의효소와같이서로

다른물질에의해효소생산성이증대되었다. B. pumilus는 B. subtilis YB-30과같이밀기울이나^xylan에의해생산성 이 증가하였고[12], Bacillus sp.와 B. amyloliqufaciens는 xylan이 첨가된 배지에서^xylanase생산성이^{28 U/ml}와 10.5 U/ml로각각보고되었다^{[2, 15].}또한^xylose를첨가하 였을 때도 분리균과 같이 Bacillus sp.와 Microbacterium sp.의^xylanase생산성도증가하는현상을보인바있다^.한 Table 2. Effects of additional amounts of wheat bran or konjac

on mannanase or xylanase production.

Added amount (%) of wheat

bran

Xylanase productivity

(U/ml)

Added amount (%) of konjac

Mannanase productivity

(U/ml)

None 0.6 None 0.7

0.1 2.3 0.1 44.9

0.3 4.0 0.3 80.5

0.5 5.7 0.5 121.9

1.0 10.2 1.0 164.5

1.5 12.8 1.5 180.4

2.0 14.1 2.0 183.8

2.5 16.2 2.5 203.5

3.0 20.2 3.0 236.9

3.5 20.5 3.5 236.0

4.0 22.5 4.0 232.2

4.5 22.5 4.5 212.2

5.0 22.2 5.0 185.1

Table 3. Effects of mixture of konjac and wheat bran on mannanase and xylanase production.

Amount of wheat bran (%) added with 3.5% konjac

(U/ml)

0 19.4 234.0

1 22.0 291.9

2 18.2 164.1

3 16.5 162.6

4 18.8 156.6

Table 4. Growth and enzyme production of YB-30 according to incubation time.

Culture time (h)

(U/ml)

Cell growth (cfu/ml)

12 15.0 121.46 4.6× 10⁸

15 18.3 198.90 3.2× 10⁹

18 21.5 213.40 5.7× 10⁹

21 18.8 220.13 5.6× 10⁹

24 17.3 222.80 5.7× 10⁹

(5)

편목화밭에서분리된 B. mojavensis는밀기울보다귀리기 울에서생산성이높으며^{, 2%}일때최대생산성을보였다^[1].

Mannanase 생산성은 B. subtilis BS5 경우에는^konjac 분말을첨가한배지에서⁹시간만에최대생산성에도달하며 약 1,241 U/ml의생산성을보여^[6]분리균^YB-30보다생산 성이높았으며^,된장에서분리된 B. amyloliquefaciens YB- 1402는분리균^YB-30과유사하게^LBG보다^konjac을첨가

한배지에서 mannanase 생산성이높은 것으로 알려졌다

[21].

효소 특성

배양상등액에존재하는^mannanase와^xylanase를확인하

기위해서^SDS-PAGE를수행한후활성염색을실시한결과

크기가약^{26 kDa}와^{33 kDa}로추정되는위치에^xylanase와

mannanase 활성을보이는단일한밴드가각각관찰되었다

(Fig. 1). YB-30의^xylanase는²²−^{23 kDa}인 B. firmus [4]

와 B. halodurans PPKS-2 [18]의 xylanases 보다는크며^, mannanase는대부분이^{38 kDa}인^Bacillus유래의 mannanases [6]에비해 작고 B. circulans [11]의 mannanase (31 kDa) 보다는큰것으로확인되었다^.그리고활성염색된단백질이 한군데에서만확인된것으로보아분리균^YB-30은^xylanase

와^mannanase를각각한종류씩생산하는것으로추정되

며, mannanase의활성이매우미약하게나타난것은^SDS-

PAGE 과정중실활후활성염색과정중에활성의회복율

이낮은때문으로여겨진다^.

배양상등액을 ammonium sulfate로처리하여제조한조 효소액을사용하여반응^pH와온도가효소활성에미치는영 향을조사한결과^mannanase는⁶⁰^o^C와 pH 6.0, xylanase 는⁵⁵^o^C와^{pH 5.5}에서각각최대활성을보였다(Fig. 2). B.

halodurans PPKS-2의 mannanase [18]와 xylanase [13]는 모두^{pH 11}과⁷⁰^o^C에서최대활성을보여분리균의효소와

Fig. 1. SDS-PAGE and enzyme zymograms of the culture filtrate of B. subtilis YB-30. After SDS-PAGE of the filtrate of B.

subtilis YB-30 cultures grown in LB broth supplemented with konjac (3.5%) and wheat bran (1%) for mannanase and xylanase production, proteins exhibiting mannanase (lane M) or xylanase (lane X) activity were analyzed by activity staining with LBG or oat spelt xylan as substrates, and indicated by arrows, respectively. Lane P represents gel stained by Coomassie blue. Molecular size of standard proteins (lane S) is shown in kilodaltons to the right side of the gel.

Fig. 2. Effects of reaction temperature and pH on the enzyme activities. Temperature profiles (triangles) were obtained by measuring the mannanase activity (A) with a fixed pH 6.0 and xylanase activity (B) with a fixed pH 5.5 at different temperatures. The pH profiles (circles) were obtained by measuring the enzyme activities at various pH’s and at a constant temperature of 50^oC. Buffers (50 mM) used were as follows: sodium citrate (pH 3-6; -●-), and sodium phosphate (pH 6-8; -○-).

(6)

크게달랐으며, B. circulans CGMCC 1416의^mannanase는 pH 7.6와⁵⁸ôC [11], B. subtilis WL-3의 mannanase [22]는 pH 6.0과⁵⁵ô^C에서 각각 최대활성을 보여^YB-30의 mannanase와유사하였다^{. YB-30}의효소보다는낮은온도 와낮은^pH에서최대활성을갖는^mannanase도 B. subtilis BS5로부터확인되었다[6]. B. subtilis B10의 xylanase (pH 6.0, 50ôC) [5]는분리균의효소와유사한조건에서최대활성 을보이는반면에 B. amyloliquefaciens XR44A의^xylanase [2]는^{pH 7-9}와⁷⁰ôC, Bacillus sp. xylanase [15]는^{pH 8.0} 과⁶⁰ô^C에서각각최대활성을갖는것으로알려졌다^.

열안정성을조사하기위해서조효소액을여러온도에서 1시간방치한후에잔존활성을측정한결과⁴⁰^o^C에서방치

되었을때는^mannanase의활성이유지되었으나이보다높

은온도에서는급격하게실활되었고^{, xylanase}는³⁰ô^C이하 에서는안정하였으며⁴⁰ô^C에서는약^20%이상실활되었다 (Fig. 3). B. mojavensis AG137 xylanase는^45-55ô^C에서 1시간방치하였을때잔존활성이⁶⁸−^{50% [1]}으로분리균의 효소보다약간높았으며^{, 65}ô^C에서¹시간방치하여도실활 되지않는 Bacillus sp.의 xylanase [15]와⁷⁰ô^C에서³시간 방치후에도안정한 B. halodurans PPKS-2의^mannanase 와^xylanase에비해^YB-30에의해생산되는효소는열안정 성이낮았다^.

밀기울과 쌀기울의 분해능

밀기울과쌀기울은가격이저렴하여사료로많이사용되 고있으며[20] 영양성분으로단백질, 지방, 전분과함께비전 분성다당류를포함하고있다^.비전분성다당류의함량은각 각⁴⁵−^50%와¹⁸−^23%로밀기울에많이존재하며^xylan과

mannan은비전분성다당류에속한다. B. subtilis YB-30의 효소에의한밀기울과쌀기울의분해능을조사하기위해조 효소액을사용하여밀기울과쌀기울을가수분해하였다^.조

효소액에존재하는탄수화물가수분해효소로는^xylanase

(77.4 U/ml), mannanase (1,571.5 U/ml)와 함께^CMCase (1.1 U/ml) 및 amylase (107.5 U/ml)의활성이관찰되었다^. 밀기울^(1%)이나쌀기울^(1%)을포함한반응액에조효소액첨 가량을^(0.01−^{0.04 ml)}다르게하여반응하였을때효소사용 량과반응시간에따라환원당의양이증가하였으며쌀기울 보다밀기울을기질로하였을때많은환원당이생성되었다

(Fig. 4). 또한쌀기울은반응시간이진행됨에따라효소량에

따른생성된환원당량의차이가줄어드는경향이있지만^,이 Fig. 3. Thermostability of the crude enzyme. Thermostability

was determined by measuring the residual activities of mannanase (-○-) and xylanase (-●-) after pre-incubation at different temperatures for 1 h.

Fig. 4. Amounts of the reducing sugar liberated from rice bran (A) and wheat bran (B) by crude enzyme of B. subtilis YB-30.

For enzymatic reaction, reaction mixtures consisting of rice bran (1%) or wheat bran (1%) used as substrate and 50 mM sodium phosphate buffer (pH 6.0) were supplemented with 10 µl (-●-), 20µl (-○-) and 40 µl (-▲-) of crude enzyme, followed by incubating at 45^oC for 15 min. Amounts of the liberated reducing sugar were determined by DNS method.

(7)

에비해밀기울은⁶⁰분간반응후에도사용된조효소량에따 라생성되는환원당량의차이가크게유지되었다^.이러한현 상으로밀기울에존재하는 arabinoxylan의함량이^32%수 준으로많은반면에쌀기울에는^10%미만의 arabinoxylan 이포함되어있으므로^{[7, 14]}조소효액의^xylanase에의해생 성된환원당량의차이가일어난때문이라할수있다^.

한편밀기울에포함된전분의함량은쌀기울보다는적거 나유사하므로쌀기울보다밀기울로부터환원당이더많이 생성되는것이조효소액에존재하는^amylase의작용이라고 할수없으며더구나이들전분은분해가어려운저항성전 분으로알려져있다^.밀기울과쌀기울의^mannan함량은^1%

이하로매우소량존재하므로조효소액의^mannanase는환 원당생성에크게관여하지못하였을것으로판단된다^.그러 므로 B. subtilis YB-30으로부터생산된^xylanase는밀기울

과같이^xylan의함량이높은곡물이나그부산물의이용효

율을높이며^,동시에^mannanase는^mannan의함유량이높 은 copra meal, palm kernel meal과대두피의이용효율을 높일수있을것으로판단되므로 B. subtilis YB-30은여러 종류의곡물을혼합한가축사료에발효생균제나효소생산 균으로사용되기적합할것으로보인다^.

요 약

새만금갯벌로부터^xylan과 locust bean gum (LBG)을분

해하는미생물을분리하여그생화학적특성과^{16S rRNA}

서열을조사한결과 Bacillus subtilis로확인되었다^.분리균 YB-30은^konjac이나^LBG가존재한상태에서배양할경우

mannanase의생산성이급격하게증가되며^,밀기울이존재

하에서는^xylanase의생산성이증가되였다. Konjac (3.5%) 과밀기울^(1%)이동시에첨가된배지에서균의성장이정지 기에진입하였을때^mannanase와^xylanase의생산성이최 고수준에도달하였다^.배양상등액의^mannanase와^xylanase 는⁶⁰^o^C와 pH 6.0, 55^oC와^{pH 5.5}에서각각최대활성을보 였다^.고온에서는두효소모두안정하지는않았으며^xylanase

가^mannanase보다안정성이낮았다^.밀기울은쌀기울보다

많은양의 arabinoxylan을함유하고있으므로배양상등액으

로분해하였을때밀기울로부터생성되는환원당의양이더 많은것으로확인되었으며^,이로보아 B. subtilis YB-30에 의해생산되는^xylanase와^mannanase는사료첨가용효소 로활용가능성이높다고여겨진다^.

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