Production and Characterization of β-galactosidase from Bacillus licheniformis Isolated from Doenjang

된장에서 분리된 Bacillus licheniformis의 β-galactosidase 생산성과 효소특성

진현경, 윤기홍*

우송대학교바이오식품과학전공

Received: October 15, 2014 / Revised: November 25, 2014 / Accepted: November 25, 2014

서 론

β-galactosidase는미생물을비롯하여동식물에두루존 재하며 ^lactose를 ^glucose와 ^galactose로분해하는효소이고 식품산업에 활용성이 높다^. 우유^, 유제품과 유장으로부터 lactose를제거하거나^[2] 장내유용미생물의성장을증진시키 는갈락토올리고당^[26]을비롯한 lactulose [6], lactosucrose [16]의생산에사용되며또한대장균유래의β-galactosidase 가작용하지못하는재조합숙주균에서표지효소로이용되 고있다^[27].

효소의생산균주에따라서β-galactosidase의 ^lactose 가 수분해능과당전이효율을비롯하여반응특성이다르므로 산업적활용에적합한효소특성과생산성을확보하기위해 내열성이나 저온성 균주를 비롯한 여러 균주로부터 β^-

galactosidase가연구되었다[22]. Bacillus stearothermophilus [8], B. circulans [26]와 B. coagulans [1]는 ³종이상의 β^- galactosidase isozymes를생산하는데^, 온천에서분리된 ^B.

coagulans RCS3의 isozyme III은다른 ^isozymes와는달리

균체외로분비되며 ^lactose 분해능이우수한것으로확인되

었다^. 또한 B. circulans의효소는 ^isozymes간에면역학적으

로동일하나 ^lactose 분해능과당전이에의한갈락토올리고

당 생산능에는 차이가 큰 것으로 보고되었다^. 한편 ^B.

licheniformis의 β-galactosidase는 당 전이효율은 낮으나 lactose 분해에적합한효소를생산하는것으로알려졌다^[12].

Bacillus속균주중 B. megaterium [23], B. licheniformis [12], B. stearothermophilus [5, 8]와 B. coagulans [1]로부 터β-galactosidase 효소와유전자의특성이보고되었으며^, 북극에서분리된 Alkalilactibacillus ikkense로부터저온에 서활성이높은 β-galactosidase의유전자도클로닝되었다 [22]. β-galactosidase의활용성을증대시키기위해서단백질

의구조를기반으로하여 ^lactose 가수분해능이향상된변이체

가개발되었으며^[5], 담체에고정화된 B. stearothermophilus와 B. circulans 유래의효소가당전이반응과 ^lactose 분해반응 Production and Characterization of β-galactosidase from Bacillus licheniformis Isolated from Doenjang

Hyun Kyung Jin and Ki-Hong Yoon*

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

A bacterial strain was isolated from homemade doenjang (Korean fermented soybean paste) as a producer of the extracellular β-galactosidase, capable of hydrolyzing lactose to liberate galactose and glucose residues. The isolate YB-1414 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. The pro- duction of β-galactosidase by B. licheniformis YB-1414 reached maximum levels of 6.2 U/ml in culture medium containing wheat bran (1%) and yeast extract (2.5%) as carbon and nitrogen sources, respectively. Particularly, the insoluble fraction was more effective for β-galactosidase production than the soluble extract of wheat bran. The enzyme exhibited maximum activity for hydrolysis of para-nitrophenyl-β-D-galactopyranoside (pNP-βGal) under reaction conditions of pH 6.0 and 55-60^oC. Its hydrolyzing activity for pNP-βGal was drastically decreased by the addition of low concentrations of galactose, but only slightly decreased by glucose, with 85% of maximal activity in the presence of 400 mM glucose.

Keywords: Bacillus licheniformis, β-galactosidase, 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

에사용되었다^{[3, 7].} β-galactosidase의생산성을증가시키 기 위해서는 B. stearothermophilus의 효소 유전자를 ^B.

subtilis [9] 또는 B. amyloliquefaciens [10]에서발현시켰다^. 또한 B. subtilis [13]와 B. licheniformis [18]의β-galactosidase 생산성에영향을미치는배지성분에대한연구도수행되었다^. 최근들어청국장의기능성에관심이높아지면서생청국 장이나건조된청국장의분말또는환의형태로유유와혼 합하여건강식으로이용되고있다^. β-galactosidase 활성이 높은청국장은우유와함께섭취할경우유당불내증을방 지하는효과가예상되므로β-galactosidase를생산하는미생 물을청국장발효균으로활용하기위해청국장으로부터 ^B.

licheniformis가분리된바있으며^[28], 본연구에서는된장 으로부터β-galactosidase를생산하는 ^Bacillus속균주중효 소생산성이우수한미생물을분리하여반응특성을조사하 였다^.

재료 및 방법

β-galactosidase 생산균의 탐색

Spizizen 최소배지^[25]의 탄소원인 포도당과 ^sodium citrate 및질소원인 ammonium sulfate 대신에대두분 ^(1%) 이첨가된변형배지^(K2HPO₄, 7 g; KH₂PO₄, 1.5 g; MgSO₄· 7H₂O 0.5 g; MnCl₂· 4H₂O, 0.04 g; CaCl₂· 2H₂O, 0.003 g;

FeSO₄·7H₂O, 0.0025 g; (NH₄)₆Mo₇O₂₄·4H₂O, 0.002 g; water, 1 L)에가정에서수집된된장을접종하여 ^37oC에서약 ³일간 진탕배양하고동일한배지에 ^3-4일간격으로 ³회계대배 양을실시하였다^. 최종적으로계대배양액을동일한성분의 평판배지에도말하고 ^37oC에서 ⁴일간배양한후형성된콜 로니중에서서로다른모양을보이는콜로니를채취하였다^. 이들분리균을 ^LB 액체배지에서 ²⁴시간동안진탕배양한 후 배양상등액을 회수하여 1 mM para-nitrophenyl-β^-D- galactopyranoside (pNP-βGal; Sigma, USA)을 포함한 20 mM sodium phosphate 완충액^{(pH 6.0)}에 첨가하고

45^oC에서 ²시간동안방치한후반응액의색깔을관찰함으

로써β-galactosidase 활성을갖는균을탐색하였다^. 분리균주의 동정

그람염색과포자염색을통해분리균의형태를조사하였으 며^, 균체현탁액을 ^{API 20E}와 API 50CHB (Biomereux사^, France) kits에제조사의지침을따라접종하고 ^37oC에서배 양하면서 ¹일과 ²일째각각관찰하여탄수화물이용능과생 화학적특성을판별하였다^. 분리균의총염색체 ^DNA를주 형으로하고^, 세균의 ^{16S rRNA} 유전자의보존적지역의염 기서열 5'-AGAGTTTGATCCTGGCTCAG-3' (E. coli 16S rRNA 유전자염기서열 8-27), 5'-GGTTACCTTGTTACGACTT-

3' (E. coli 16S rRNA 유전자염기서열 1492-1510)을 ^primer로

사용하여중합효소연쇄반응^(PCR)을실시한후증폭된 ^PCR

산물을정제하여분리균의 ^{16S rDNA} 염기서열을분석하였다^. β-galactosidase 조효소액 제조

분리균 B. licheniformis YB-1414를 ^LB 배지에서 ¹⁶시간

동안배양한배양액을 ^{1% (v/v)}가되도록동일성분의본배

양액에접종하고 ^37oC에서 ³⁶시간동안진탕배양하였다^. 원 심분리하여얻은배양상등액을 ammonium sulfate (25-75%) 로처리하고침전된단백질을 10 mM sodium phosphate 완충

액^{(pH 6.0)}에현탁하여동일완충액으로투석한후조효소

액으로사용하였다^.

β-galactosidase 반응특성 분석

β-galactosidase 활성을결정하기위해서는 ^{1 mM pNP-} β^Gal과 50 mM sodium phosphate 완충액^{(pH 6.0)}을포함 한반응액에효소를첨가하여 ^50oC에서 ¹⁰분간반응시킨후 반응액의 ²배부피의 ^{1 M Na}2CO₃ 용액을첨가하여반응을 종결시키고 ^{405 nm}에서 흡광도를 측정하였다^{. para-} nitrophenol (pNP)을표준물질로하여얻은검량곡선을사 용하여효소반응에의해생성된 ^pNP의양을결정하였다^. 효소의활성도 ^{1 unit}는 ¹분동안 ¹ μ^mol의 ^pNP를유리시 키는효소량으로정의하였다^. 효소활성에미치는반응온도 와 ^pH의영향을조사하기위하여 ^30-70oC와 pH 5.0-8.0의 범위에서 각각 β-galactosidase 활성을 측정하였다^. β^-

galactosidase의열안정성을조사하기위하여조효소액을

서로다른온도에서일정시간방치한후잔존활성을측정하 였다^. β-galactosidase에의한최종가수분해산물을조사하 기위해 lactose (1%)와과량의조효소액을포함한반응액을 40^oC에서 ⁵시간반응시킨후반응액을열처리하여단백질을 제거하고상등액을취해 chloroform, acetic acid와증류수 (4.3 : ⁵ : 0.7, (v/v)) 혼합용액을 전개용액으로하여 ^silica gel-precoated thin layer plate (Merck, Gemany)에서박층 크로마토그래피를수행하였다^. 전개된물질을발색시키기위 해서는 9 ml ethanol, 0.5 ml p-anisaldehyde, 0.5 ml sulfuric acid와 glacial acetic acid 몇방울을혼합한발색제용액을 뿌린후^{, 120oC}에서 ¹⁰분간방치하였다^. 당이효소활성에

미치는영향을분석하기위하여기질로 ^pNP-β^Gal을포함한

반응액에종류와농도가다르게당을첨가한상태에서반응 을수행한후β-galactosidase 활성을측정하였다^.

결과 및 고찰

β-galactosidase 생산균의 분리와 특성

된장으로부터고분자물질의가수분해효소를생산하는 ^B.

subtilis, B. licheniformis 및 B. amyloliquefaciens가분리 된바있으며^{[11, 29],} 여러종류의 ^Bacillus속및이와관련 된균이발효장류에다수존재하는것으로보고되었다^[19].

된장을직접복합평판배지에도말하여균주를분리할경우 우점균이나한정된균종만빠르게성장할수있으므로^[17]

탄소원과질소원대신대두분을첨가한최소배지를사용하 여계대배양과분리과정을실시하고평판배지에서형성된 콜로니의모양이서로다른균을분리하였다^. 가정에서제조 된된장 ⁶⁰점시료로부터균을분리한결과된장시료당약 1-4개의균주가분리되었다^. 총 ¹⁵⁰개분리균을 ^LB 액체배 지에배양하여얻은배양상등액의β-galactosidase 활성을조 사하였으며^, 그결과 ⁵개분리균주를제외하고는대부분이 β-galactosidase 활성이관찰되지않거나미약한것으로나 타났다^. 따라서효소생산성이우수한 ⁵개분리균의배양상 등액내효소활성을비교하여최종적으로β-galactosidase 활 성이높은분리균 YB-1414를확보하였다.

분리균 ^YB-1414는포자를형성하는그람양성간균이며^,

API 50CHB와 ^{20E kit}를사용하여조사한생화학적특성을 Biomeriux의 API web (https://apiweb.biomerieux.com/

jsp/login.jsp)에서다른균주와비교한결과 B. licheniformis

와유사도가 ^99.9%로가장높게 나타났고당이용성도 ^B.

licheniformis와동일하였다^. 분리균은 arginine dihydrolase, gelatinase와 ^oxidase 활성을 보였고^{, nitrate} 환원능과 citrate 이용능이있으며 ^acetoin을생성하였다^. 또한 ^lysine decarboxylase, ornithine decarboxylase, urease와 tryptophan deaminase의활성은없고^{, indole}과황화수소를생성하지못 하였다^. 분리균의 ^{16S rRNA} 유전자를 ^PCR로 증폭한 후 1,430 bp 크기의염기서열을결정하였으며(Genbank accession No. KM660627) 이를미국 ^NCBI의 ^BLAST 검색방법을사 용하여세균들의상응하는염기서열과비교하였다^. 그결과^, B. licheniformis DSM 13 (NR_118996)과 BCRC 11702 (NR_116025)의 ^{16S rDNA} 서열과가장상동성이높았으며^, 2개의염기를제외하고는동일하였다^. 이상의형태적^, 생화 학적특성및 ^{16S rDNA} 서열의결과로볼때분리균 ^YB- 1414는 B. licheniformis인 것으로 판단된다^. 분리균 ^YB-

1414의고분자 물질의 분해능을조사하기위해 ^{1% skim}

milk, 0.2% potato starch, 0.5% xylan과 0.5% carboxymethyl

cellulose를각각첨가한평판배지에서하룻밤배양하여분

해환을관찰하였으며그결과분리균은 ^xylan을분해하지못 하였고나머지물질을모두분해하는것으로나타났다^. 한편 청국장에서도β-galactosidase를생산하는 B. licheniformis 가분리된바있는데^[28], 분리균 ^YB-1414는청국장에서분 리된β-galactosidase 생산균과는달리 ^acetoin을생성하고^, citrate, rhamnose, sorbitol, melibiose, inuline과 β-gentiobiose 를탄소원으로이용하는차이점을보였다^.

B. licheniformis YB-1414의 β-galactosidase 생산성 β-galactosidase는균체내효소로존재하는경우가많으며 B. stearothermophilus [4, 8], B. megaterium ATCC 14581 [23], B. subtilis KL88 [20]에서균체내효소로보고 된바있다^. 또한청국장에서 분리된 B. licheniformis YB- 1105도배양상등액보다균체내에β-galactosidase 활성이

높은 것으로 알려졌다^[28]. 한편 면양유에서 분리된 ^B.

subtilis [13]와온천에서분리된 Bacillus sp. [21]는균체외 로β-galactosidase를분비생산하며, B. coagulans RCS3의 경우 5개의β-galactosidase isozymes 중한개가배양상등 액에서관찰된바있다^[1]. 분리균 B. licheniformis YB-1414 도배양상등액에서β-galactosidase가높은활성으로관찰되 므로균의성장과효소생산성과의관계를조사하기위해 ^LB 액체배지에서진탕배양하면서일정시간마다배양액을채취

하여 ^{600 nm}에서 흡광도와 배양상등액에 존재하는 β^-

galactosidase의활성을측정하였다^.

B. licheniformis YB-1414는배양시간이 ¹²시간되었을때 최대성장도에이르렀고이때부터 β-galactosidase 생산이시 작되어정지기를지나사멸기에도달한후까지효소생산성 이지속적으로증가하였으며 ³⁰시간이되었을때 ^{2.8 U/ml} 로최대에이르렀고이후에도배양상등액에존재하는효소 활성이감소하지않고그대로유지되었다^{(Fig. 1).} 균체외 β^- galactosidase를생산하는 Bacillus sp.의경우도효소생산 성이정지기이후에지속적으로증가되는현상을보여분리 균과유사하였으며^, 이러한현상은균체내에서합성된효소 가균체외로확산되는데걸리는시간의차이때문인것으로

Fig. 1. Growth and β-galactosidase production of B. licheni- formis YB-1414. B. licheniformis YB-1414 was grown at 37^oC in LB medium. The cell growth (-●-) was determined by measuring absorbance of the cell culture at wavelength of 600 nm. β-galac- tosidase activities (-○-) were determined with the culture filtrate.

The curve for β-galactosidase production represents the average of three independent experiments within standard errors of 1.5%

between them.

추정된 바 있다^[21]. 또한 B. stearothermophilus의 β^- galactosidase 유전자를함유한 B. subtilis 재조합균주는균 체내β-galactosidase를생산하므로효소생산성이균의성 장과연계된형태로정지기에이를때까지지속적으로증가 하여최대생산성이약 ^{6.3 U/ml}로나타났다^[9].

β-galactosidase 생산성은배지내탄소원과질소원성분에 따라영향을받는다고알려져있으므로 potassium phosphate (0.01%)와 magnesium sulfate (0.1%)를기본성분으로하고

탄소원과질소원을달리하여분리균 ^YB-1414의효소생산

성을조사하였다^. 질소원으로 peptone (0.5%)을함유하는배

지에서로다른탄소원을 ^0.5%가되도록각각첨가한상태

에서분리균을접종하여 ^37oC에서 ³⁶시간동안진탕배양한 후배양상등액의효소활성을측정한결과 ^{Table 1}에보인 바와같이밀기울을비롯하여쌀기울, locust bean gum 또

는 ^{guar gum}을첨가한배지에서효소생산성이증가되었으

며 glucose와 lactose를비롯한단당류나이당류가첨가된배 지에서는탄소원을첨가하지않은배지보다효소생산성이

감소하였거나큰변화가없는것으로나타났다^. 이는쉽게 대사되는 ^glucose와 ^lactose보다귀리가루^, 밀기울과쌀기울 이함유된배지에서 B. subtilis의β-galactosidase 생산성이증 가되거나^, 왕겨를첨가한고체배지에서 B. licheniformis의β^- galactosidase 생산성이증가한현상과유사하지만^{[13, 18],} B. megaterium [23]과 B. stearothermophilus [9] 및 ^B.

stearothermophilus의β-galactosidase 유전자가 함유된재 조합 B. amyloliquefaciens [10]에서효소생산이 ^lactose에 의해유도된다는결과와는달랐다^. 또한밀기울을첨가한배 지에서효소생산성이가장높았으므로밀기울의농도를달 리한배지에서동일조건으로배양하였을때밀기울첨가량 이 ^1%가될때까지는효소생산성이증가되었으나 ^1.5% 이 상의밀기울이첨가되었을때는효소생산성이약간감소하 는현상을보였다(Table 1).

탄소원으로밀기울첨가량을 ^1%로고정하고유기질소원

(0.5%)의종류를달리한배지에서효소생산성을조사하였

을때 yeast extract 또는 ^tryptone이첨가된배지에서효소

Table 1. Effects of additional carbon sources on the β-galactosidase production from B. licheniformis YB-1414 Additional carbon sources

(0.5%)

β−galactosidase activity

(U/ml) Amount (%) of wheat bran β-galactosidase activity (U/ml)

None 0.114± 0.004 None 0.201± 0.005

Glucose 0.143± 0.002 0.3 0.692± 0.002

Sucrose 0.000± 0.000 0.5 0.878± 0.008

Lactose 0.191± 0.003 0.7 1.152± 0.006

Galactose 0.114± 0.005 1.0 1.781± 0.008

Arabinose 0.000± 0.000 1.5 1.453± 0.009

Melibiose 0.000± 0.000 2.0 1.294± 0.009

Raffinose 0.000± 0.000 2.5 1.231± 0.006

Locust bean gum 0.321± 0.003 3.0 1.252± 0.005

Guar gum 0.192± 0.002

Wheat bran 0.762± 0.008

Rice bran 0.534± 0.008

Table 2. Effects of additional nitrogen sources on the β-galactosidase production from B. licheniformis YB-1414 Additional nitrogen sources

(0.5%)

β-galactosidase activity

(U/ml) Amount (%) of yeast extract β-galactosidase activity (U/ml)

None 0.083± 0.005 0.0 0.042± 0.003

Corn steep powder 0.003± 0.002 0.5 1.485± 0.009

Soytone 0.851± 0.001 0.8 2.385± 0.010

Yeast extract 1.312± 0.004 1.0 3.092± 0.010

Tryptone 1.153± 0.009 1.5 4.793± 0.017

Peptone 0.884± 0.009 2.0 6.152± 0.001

Casein hydrolyzate 0.742± 0.005 2.5 6.205± 0.014

3.0 5.523± 0.011

생산성이높았으며, soytone, peptone 또는 ^casein 가수분해 물이첨가된배지에서는서로유사한수준의효소생산성을 보였고특이하게도 corn steep powder가함유된배지에서는

효소가거의생산되지않았다(Table 2). 효소생산성을가장

크게 증가시킨 yeast extract의 첨가량을 달리하였을 때

yeast extract의첨가량이많아질수록β-galactosidase 생산

성이증가하여 ^2.5%에서최대생산성을보이고 ^3.0%에서는

약간감소하였다. B. subtilis도분리균 ^YB-1414와유사하게 yeast extract를질소원으로사용하였을때효소생산성이가 장높았으나^, 분리균과는달리 ^peptone이 ^tryptone 보다는 효소생산능을더증가시키는것으로보고되었다^[13].

한편밀기울의불용성물질과수용성물질중어느것이 효소생산성을증가시키는지확인하기위해서밀기울 ^1%를 증류수에현탁하고원심분리후불용성의침전물과수용성 의상등액부분으로나누고 yeast extract (2.5%)와기본성 분을각각첨가하여제조된배지에서효소생산성을비교하

였다^{. Table 3}에보인바와같이밀기울의불용성물질이첨

가된배지에서효소생산성은밀기울이그대로첨가되었을 때보다약간낮은것으로나타났지만수용성물질이첨가된 배지에서보다더높았다^. 또한밀기울의수용성물질이함 유된 배지에서의 효소 생산성은밀기울이 첨가되지않고 yeast extract만첨가된배지에서보다 ²배이상높은것으 로나타났다^. 이로보아β-galactosidase의생산성을증가시 키는물질이밀기울의불용성물질과수용성물질에모두있 으며불용성물질에좀더많이존재하는것으로판단된다^. 한편 yeast extract를첨가하지않고밀기울만을첨가한배 지에서는효소생산성이매우낮았는데이는질소원이거의 없어균의성장이낮아진때문으로여겨진다^.

B. licheniformis의 β-galactosidase의 반응특성 배양상등액을 ammonium sulfate (25-75%)로처리하여제 조된조효소액을사용하여반응조건에따른β-galactosidase 활성을측정하였다^. 그결과 ^{pH 6.0}과 ^55-60oC에서최대활 성을나타냈으며, pH 5.5-6.5 범위에서는최대활성의 ^90%

이상의활성을보였다^{(Fig. 2).} 분리균효소의최적반응온

도는 ^50oC에서최대 효소활성을 보이는 B. licheniformis

DSM 13 [12]과 B. licheniformis YB-1105 [28]의 효소에 비해 높았으며, B. coagulans L4 [14]와 B. megaterium 2- 37-4-1 [15]의효소와는유사하였고^, 최적반응온도가 ^65oC 와 ^70oC로보고된 B. coagulans RCS3 [2], B. subtilis [13]

와 B. stearothermophilus [4]의효소보다는낮았다^. 한편북

극에서 분리된 A. ikkense가 생산하는저온활성 효소는

20-30^oC에서최대 활성을보이는것으로보고되었다^[22].

또한 B. licheniformis DSM 13, B. coagulans [1]와 ^B.

stearothermophilus [4] 유래효소의최적 ^pH는 ^6.0-7.0 범위 로알려져분리균의효소와유사하였으며, B. megaterium 2-37-4-1과 A. ikkense의효소 및 B. stearothermophilus의 변이체효소^[5]는 ^{pH 7.5-8}의범위에서최대활성을보이는 것으로보고되었다^.

30-60^oC 범위의온도에서조효소액을 ³⁰분과 ⁶⁰분동안 Table 3. β-galactosidase production from B. licheniformis YB-1414 according to soluble and insoluble fraction of wheat bran

Additional sources for

β-galactosidase activity (U/ml)

Carbon (1%) Nitrogen (2.5%)

Total wheat bran None 0.075± 0.006

None Yeast extract 1.384± 0.011

Total wheat bran Yeast extract 6.223± 0.018

Soluble extract of wheat bran Yeast extract 3.205± 0.012

Insoluble fraction of wheat bran Yeast extract 5.447± 0.019

Fig. 2. Effects of reaction temperature and pH on the β-galac- tosidase activity. Temperature profile (triangles) was obtained by measuring the β-galactosidase activities at different temperatures and pH 6.0. The reactions were done at 50^oC and various pHs for determining the pH profile (circles). Buffers used were as follows:

sodium citrate (-●-), sodium phosphate (-○-). Each curve rep- resents the average of three independent experiments within stan- dard errors of 2% between them.

각각열처리한후잔존활성을조사한결과 ^{Fig. 3}에보인바 와같이 ^40oC 이하에서는 ⁶⁰분동안방치하여도안정하였 으나^{, 45oC} 이상의 온도에서는 ¹시간 방치하였을 때 β^- galactosidase는서서히실활되기시작하였으며 ^60oC에서는 30분방치하였을때도잔존활성이거의관찰되지않는것으 로보아 ^60oC 이상에서는매우불안정한것으로판단된다^. 그러므로분리균의β-galactosidase는 ^55oC에서반감기가 ⁹⁴ 시간인 Bacillus sp.의효소^{[21], 65oC}에서반감기가 ²시간인 B. coagulans RCS3의 효소와 반감기가 ⁵⁰시간인 ^B.

stearothermophilus ATCC 8005의효소에비해열안정성이 낮으며^{[2, 4], 55oC}에서 ³⁰분만에 ^70%가실활된 B. coagulans L4의 효소와 ^45oC에서 ¹시간만에 ^70%가 실활된 ^B.

licheniformis YB-1105의균체외효소보다는열안정성이높 았다^{[14, 28].}

pNP-β^Gal의가수분해능이있는β-galactosidase 중에서 실제 ^lactose를거의분해하지못하는경우도있으므로^[24]

분리균이생산하는 β-galactosidase의 ^lactose 가수분해산물 을 ^TLC로 분석하였다^. 그 결과 ^{Fig. 4}에 보인 바와 같이 lactose가완전히분해되지는않았지만^, 분해산물로 ^galactose

와 ^glucose의이동도가동일한반응산물이생성된것으로보

아 ^lactose를분해하는것을알수있다^. 실제β-galactosidase 는 ^lactose의분해산물인 galactose 또는 ^glucose에의해가 수분해활성이저해를받는것으로알려져있는데^, 반응산

물에상당량의 ^lactose가분해되지못한상태인것으로보아

YB-1414의β-galactosidase도 ^lactose 가수분해과정에서반 응산물에의해크게저해를받은것으로여겨진다^.

β-galactosidase의 가수분해 활성에 미치는 당의 영향 β-galactosidase, β-glucosidase, β-xylosidase와 α^- galactosidase 등을포함하는여러종류의 glycosidase는가 수분해반응시반응산물에의해효소활성이저해되는것으 로알려져있다^. 따라서 ^YB-1414의β-galactosidase 활성에 미치는당의영향을분석하기위해 ^{1 mM pNP-}β^Gal을기질 로하고 glucose, galactose, xylose, mannose의첨가농도를 달리하여가수분해활성을측정하였다^. 그결과 ^galactose에 의한가수분해활성의저해도가가장큰것으로나타났으며^,

20 mM 이상이존재할경우효소활성이 ^50% 이상저해되었

다(Fig. 5). B. coagulans RCS3 [2], B. stearothermophilus [9], B. licheniformis DSM 13 [12]과 B. licheniformis YB- 1105 [28]의효소도 ^galactose에의해활성이강력하게저해 를받는것으로알려져있으나, B. subtilis KL88의효소는 galactose가 ^20% 존재할때도 ^50% 이상의β-galactosidase 활성을유지하는것으로보고되었다^[16].

Glucose는 ^galactose에비해분리균의효소활성을약하게 저해하였으며^{, 400 mM}의존재하에서도약 ^85%의활성을 보였다^{. Glucose}와유사한 ^mannose도효소활성을약하게저 해하였으며^{, 400 mM} 존재하에서약 ^91% 이상의활성을보 였다^. β-galactosidase에의한 ^lactose 분해산물로 ^glucose와 galactose가동시에생성되는데 ^glucose 보다 ^galactose에의 한효소활성저해도가큰것은 ^galactose는효소의활성부위

에경쟁적저해제로작용하는반면에 ^glucose는비경쟁적저

해제로작용하기때문인것으로알려졌다^[16]. 그러므로대

부분의β-galactosidase는분리균의효소와유사하게 ^glucose Fig. 3. Thermostability of the β-galactosidase in culrue filtrate.

Thermostability was determined by measuring the residual activi- ties of β-galactosidase after pre-incubations for 30 min (-●-) and 1 h (-○-) at the different temperatures. Each curve represents the average of three independent experiments within standard errors of 2% between them.

Fig. 4. Thin-layer chromatogram of hydrolysis product of lactose. Reactions were done using lactose as a substrate by culture filtrate (lanes 1 and 2) at 40^oC for 5 h . Reaction products were analyzed from reaction mixtures before (lane 1) and after reaction (lane 2). Authentic sugar abbreviations are as follows:

Gal, galactose; Glc, glucose.

에의한활성의저해도가 ^galactose에비해훨씬낮으나^[20, 28], B. licheniformis DSM 13의효소경우는 ^glucose에의 해서도효소활성이크게저해되는것으로보고되었다^[12].

한편 ^xylose에의해서는효소활성이저해를받지않았을뿐

아니라^{, xylose} 농도가 ^{300 mM} 이하에서는β-galactosidase 활성이미약하게증가되는현상을보였는데이와같은현상 은 B. licheniformis YB-1105에서생산된효소에서도보고 된바있다^.

요 약

가정에서 제조된 된장으로부터 ^lactose를 ^glucose와 galactose로가수분해하는균체외β-galactosidase의생산균 이분리되었다^. 분리균 ^YB-1414는형태적특성^, 생화학적성 질 및 ^{16S rRNA} 유전자 염기서열에 근거하여 ^Bacillus licheniformis로 확인되었다^. 탄소원과 질소원으로 밀기울 (1%)과 yeast extract (2.5%)를사용하였을때 B. licheniformis YB-1414의β-galactosidase 생산성이최대 ^{6.2 U/ml}에이르 렀다^. 특히 밀기울의 불용성 성분이 수용성 성분보다 β^- galactosidase 생산성을더증가시키는것으로확인되었다^. β-galactosidase의 para-nitrophenyl-β-D-galactopyranoside 가수분해활성은 ^{pH 6.0}과 ^55-60oC에서가장높았으며^, 낮 은농도의 ^galactose에의해서도크게저해를받았다^. 그러 나 ^glucose에의해서는β-galactosidase의가수분해활성이

약하게저해를받으며 400 mM glucose가존재하여도최대

활성의 ^85%에해당하는가수분해활성을보였다^.

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