The Korean Journal of Microbiology (2011) Vol. 47, No. 3, pp. 225-230 Copyright c 2011, The Microbiological Society of Korea Microbacterium sp. 분리균의 Hemicellulases 생산성과효소특성 윤기홍 * 우송대학교식품생물과학과 Production and Properties of Hemicellulases by an Isolate of Microbacterium sp. Ki-Hong Yoon Department of Food Science & Biotechnology, Woosong University, Daejeon 300-718, Republic of Korea (Received September 14, 2011 / Accepted September 20, 2011) A bacterium producing the extracellular mannanase and xylanase was isolated from Korean farm soil by successive subcultures in a minimal medium supplemented with palm kernel meal (PKM) and rice bran. The isolate YB-1106 showed 98% similarity with Microbacterium arabinogalactanolyticum on the basis of 16S rrna gene sequences. The additional carbohydrates including locust bean gum (LBG) and PKM increased the mannanase of the YB-1106, while the xylanase of the isolate was increased by wheat bran, oat spelt xylan, rice bran and xylose. Particularly, maximum mannanase and xylanase activities were obtained in the culture filtrate of tryptic soy broth supplemented with 1% LBG or 2% wheat bran, respectively. Both enzyme activities were produced at stationary growth phase. The mannanase of culture supernatant was the most active at 50 C and ph 6.0, while xylanase of culture supernatant was the most active at 55 C and ph 6.5. The predominant products resulting from the mannanase or xylanase hydrolysis were oligosaccharides for LBG or xylan, respectively. Keywords: Microbacterium sp., mannanase,, property, xylanase Hemicellulose는고등육상식물의세포벽을구성하는다당류로리그닌과 cellulose사이에결합하여배열해있으며 xylan 과 mannan이주요구성물질이다. 식물성바이오매스자원인 hemicellulose는목재에서 cellulose 다음으로풍부한성분일뿐아니라곡물에도다량함유된비전분성다당류로이를효소적으로분해하여발효가능한당으로전환하거나 (1), 사료영양효율을개선시키는연구가활발히진행되고있다 (6). Xylan의기본골격인 β-d-1,4-xylopyranosyl 결합을무작위로가수분해하는 β-1,4-xylanase (xylanase) 와 mannan 다당류의기본구성당인 mannose간의 β-1,4-mannosyl 결합을무작위적으로분해하는 β-1,4-mannanase (mannanase) 는 hemicellulose 분해에가장중요한역할을하는효소로알려져있으며 (2) 그유용성이높아지고있다. 세균과곰팡이에서다수의 xylanases 와 mannanases효소와그유전자에대한특성이규명되었는데, mannanase는대부분이 glycosyl hydrolase (GH) family 5와 * For correspondence. E-mail: ykh@wsu.ac.kr; Tel.: +82-42-630-9742; Fax: +82-42-636-2676 26에속하며 GH26에는주로세균성효소가대부분을차지하고, GH5의효소로는곰팡이유래또는세균유래효소가포함되어있으며, xylanase는대부분의효소가 GH 10과 11에속해있다. Microbacterium속의미생물로 xylan 분해균 (10), chitosanase 생산균 (15), α-glucosidase 생산균 (14), cellulase 생산균 (12) 등이분리된바있지만 mannan 분해균에대한보고는없으며, 또한 xylanase와 mannanse에대한연구도거의이루어진바없다. 따라서본연구에서는농후배양을통해분리한 hemicelluloses에대한분해능을갖는 Microbacterium속분리균의 xylanase와 mannanase 생산성과그특성을조사하였다. 재료및방법 Hemicellulases 생산균의탐색국내밭토양시료를채취하여 0.25% 의밀기울과 palm kernel meal (PKM) 이첨가된 SMM 배지 [0.2% (NH 4) 2SO 4, 1.4%
226 Ki-Hong Yoon K 2HPO 4, 0.6% KH 2PO 4, 0.1% trisodium citrate H 2O, 0.02% MgSO 4 7H 2O] 에접종하여 30 C에서약 4일간진탕배양하고동일한배지에 3-4일간격으로 3회계대배양을실시하였다. 계대배양액을 0.25% oat spelt xylan과 0.25% locust bean gum (LBG) 가탄소원으로첨가된 SMM 평판배지에도말하여 30 C에서콜로니가형성될때까지배양하였다. 분리균의 16S rrna 유전자염기서열분석분리균의 16S rrna 유전자염기서열을분석하기위해서분리균의총염색체 DNA를주형으로하고, 세균의 16S rrna 유전자의보존적지역의염기서열 5 -AGAGTTTGATCCTGG CTCAG-3 (E. coli 16S rrna 유전자염기서열 8-27), 5 -GG TTACCTTGTTACGACTT-3 (E. coli 16S rrna 유전자염기서열 1492-1510) 을프라이머로사용하여중합효소연쇄반응 (PCR) 을실시하였다. PCR 반응액의조성은주형 DNA (20 ng), 10 mm Tris (ph 8.3), 50 mm KCl, 1.5 mm MgCl 2, 0.2 mm dntp, 50 pmol primers와 2.5 U Pfu polymerase로구성하여 94 C에서 30초, 55 C에서 30초, 72 C에서 50초간반응을 30회반복하여 16S rrna를코드하는 DNA 단편을증폭하였다. 증폭된 PCR 산물을정제하여 ABI PRISM BigDye terminator cycle sequencing kit와 373A automatic DNA sequencer (Perkin Elmer Co., USA) 를사용하여염기서열을결정하였다. 효소활성측정 Mannanase 활성은 LBG를, xylanase 활성은 oat spelt xylan을기질로하여각각효소반응후에유리된환원당을 3,5-dinitrosalicylic acid (DNS) 방법 (7) 으로다음과같이정량함으로써측정하였다. 증류수에현탁시킨 1% (w/v) LBG 용액또는 xylan 용액 0.5 ml와 200 mm sodium phosphate (ph 6.5/6.0) 0.25 ml를효소용액 0.25 ml와혼합하여적정온도에서 15분동안반응시켰다. DNS 용액 3 ml를첨가하여반응을정지시키고끓는물에서 5분동안방치하여발색시킨후 540 nm에서흡광도를측정하였다. Mannose 또는 xylose 를표준시료로사용하여동일조건에서발색시켜조사한흡광도와비교함으로써유리된환원당의양을결정하였다. 효소활성도 1.0 unit는위의조건에서 1분동안 LBG 또는 xylan 으로부터 1 μmol의 mannose 또는 xylose에상응하는환원당을생성하는효소의양으로정의하였다. 활성염색조효소액을 SDS-PAGE한후 polyacrylamide gel에서 SDS 를제거하기위해 25% isopropanol를포함한 50 mm sodium phosphate buffer (ph 6.0) 로세척한후동일완충액에 15-30 분간방치하였다. Polyacrylamide gel을꺼내어동일한완충용액에 0.2% xylan 또는 0.2% konjac과 1.5% agar를녹여제조한 gel을중층하여비닐로봉입한후 40 C에서일정시간반응시킨후중층한 gel을 congo red로염색하여분해된부분을관찰하였다. 반응산물분석농산부산물을기질로하여과량의효소를첨가하고 40 C에서반응액의 ph는최적으로조절하여 4시간반응을수행하였다. 반응후에반응액을 3분동안 95 C에서열처리한후원심분리하여단백질침전물을제거하고상등액을적정량취해 chloroform, acetic acid와증류수 [4.3:5:0.7, (v/v)] 혼합용액을전개용액으로하여 silica gel-precoated thin layer plate (Merck Kiesegel, No. 5748) 에서박층크로마토그래피를수행하였다. 전개된물질을발색시키기위해서는 9 ml ethanol, 0.5 ml p-anisaldehyde, 0.5 ml sulfuric acid와 glacial acetate 몇방울을혼합한발색제용액을뿌린후, 120 C에서 10분간방치하였다. 결과및고찰농후배양을통한 hemicellulases 생산균의탐색작물재배토양의토착미생물중에서 xylanase나 mannanase 를생산함으로써 hemicellulose를분해하는미생물을탐색하기위해서 xylan 성분을포함하고있는밀기울과 mannan 성분을포함하고있는 PKM을최소배지인 SMM에유일탄소원으로첨가하여작물재배토양을첨가한후 30 C에서진탕배양하고일정기간마다동일배지에계대배양함으로써 hemicellulose의분해능을갖는미생물을농후배양하였다. 농후배양액을 xylan과 LBG를유일탄소원으로첨가한평판배지에도말하여성장한콜로니를선발한후고분자물질을분해할수있는지분석하기위해 0.5% oat spelt xylan, 1% skim milk, 0.2% potato starch, 1% tributyrin과 0.5% carboxymethyl cellulose (CMC) 를각각첨가한 LB 평판배지에서 48 시간배양하여분해환을조사한결과, hemicelluloses 성분인 xylan과 LBG에대한분해능이있는분리균 YB-1106은전분의분해능은높았으나, CMC, 단백질및 tributyrin은분해하지못하는것으로확인되었다. 그람양성무포자단간균으로확인된분리균 YB-1106으로부터 1,399 bp 크기의 16S rrna 유전자서열을결정하여 (GenBank accession No. JN664949) 미국 NCBI의 BLAST 검색방법을사용하여기존에등록된세균들의상응하는염기서열과비교한결과, Microbacterium arabinogalactanolyticum DSM 8611과 98% 상동성을보였으며 (GenBank accession No. NR_044932), M. paraoxydans CF36 (NR_025548), M. foliorum (NR_025368) 과 M. esteraromaticum DSM 8609 (NR_026468) 와 97% 수준의상동성을보였다. 따라서분리균 YB-1106은 Microbacterium 속에속하는균으로판단되었다. 부가탄소원에따른효소생산성 Xylanase와 mannanase는 hemicellulose를분해하는주요효소이므로분리균 YB-1106이생산하는 xylanase와 mannanase 의활성을조사하였다. 미생물유래의다당류분해효소는배양액중의탄수화물성분에의해그생산성이영향을받는경우가많으며, Microbacterium 속균주에서도 colloidal chitosan
Microbacterium sp. 분리균의 hemicellulases 227 Table 1. Effects of additional carbon sources on the mannanase and xylanase production of Microbacterium sp. YB-1106 Additional carbon sources Cell growth (OD 600) Mannanase Xylanase None 5.9 0.12 0.17 Glucose 6.8 0.15 0.34 Glycerol 7.1 0.12 0.18 Lactose 8.5 0.14 0.18 Maltose 4.0 0.12 0.43 Sucrose 5.4 0.13 0.19 Xylose 5.4 0.12 0.99 α-cellulose ND 0.12 0.46 Locust bean gum ND 1.32 0.30 Oat spelt xylan ND 0.12 1.36 Palm kernel meal ND 0.45 0.27 Rice bran ND 0.14 1.16 Rice straw ND 0.14 0.39 Wheat bran ND 0.18 1.51 ND, not determined. 이 chitosanase 생산성을증가시키거나 (15) CMC가 cellulase 의생산성을증가시킨다는사실이보고되었다 (12). 따라서분리균 YB-1106의 hemicellulases 생산성에탄수화물이미치는영향을조사하기위해 LB 액체배지를기본배지로하여부가탄소원을 0.5% (w/v) 가되도록각각첨가하여 30 C에서 24 시간배양한후배양상등액에존재하는 xylanase와 mannanase 활성을조사한결과, Table 1에보인바와같이 xylan뿐아니라농산부산물인밀기울과미강이첨가된배지에서 xylanase 의생산성이크게증가하였으며대부분의단당류나이당류는효소생산에크게영향을미치지않았으나, xylose가첨가된배지에서도 xylanase 생산성이크게증가하였다. 한편 mannanse의생산성은 LBG를첨가한배지에서크게증가하였으며 PKM이첨가된배지에서도효소생산량이약간증가하였다. LBG를첨가한배지에서 mannanase의생산성이증가된경우는 Bacillus 균주등에서다수보고된바있으며 (4), PKM에의한 mannanase 생산성이증가되는현상은 Streptomyces galbus에서보고되었다 (3). 또한 xylan이나밀기울에의해 Bacillus pumilus에서 xylanase의생산성이증가되었고 (8) xylose에의해서 B. subtilis AMX-4의 xylanse 생산성이크게증가하는것으로밝혀졌다 (19). 또한배지에유일탄소원으로 lactose를첨가하였을때 Microbcterium sp. 의 cellulase 생산성도크게증가한다고보고되었다 (12). LBG가 PKM보다분리균 YB-1106의 mannanase 생산성을더크게증가시키므로 LBG의첨가량을달리한 LB 배지에서분리균을배양한후배양상등액에존재하는 mannanase의활성을조사한결과 1.0% 첨가할때까지는첨가량에따라효소생산량이서서히증가하였으나, 1.5% 이상첨가된배지에서는효소생산성이급감하여배양상등액에서거의효소활성 Table 2. Effects of various amounts of LBG or wheat bran on mannanase or xylanase production of Microbacterium sp. YB-1106 Additional amount (%) of LBG Mannanase Additional amount (%) of wheat bran Xylanase None 0.12 None 0.17 0.1 1.01 0.1 0.87 0.3 1.05 0.3 1.30 0.5 1.11 0.5 1.49 0.7 1.22 0.7 2.84 1.0 1.75 1.0 3.34 1.5 ND 1.5 3.97 2.0 ND 2.0 5.76 ND, not determined. 이관찰되지않았다. 또한 xylanase의생산성을가장많이증가시킨밀기울의첨가량을달리하여배양한후효소생산성을분석한결과 2% 첨가할때까지지속적으로증가하는현상을보였다 (Table 2). 한편 LB, tryptic soy broth, nutrient broth에밀기울 (2%) 또는 LBG (1%) 을각각첨가하여분리균의효소생산성을조사한결과다른배지에서보다 TSB 배지에서두효소의생산성이모두높았다 ( 결과미제시 ). 그러므로 LBG (1%) 를첨가한 TSB 배지에서배양시간에따른 mannanase 생산성을조사한결과 LBG를첨가하지않았을때는균의성장이정지기후반에이르기까지효소생산이거의되지않았으나, LBG를첨가한배지에서는정지기중반에효소생산이급격하게증가하는것이확인되었는데이는배양중초기에 YB-1106에의해낮은양으로생산된 mannanase가배양액의 LBG를분해함으로써그분해산물이배양후기에 mannanase의생산유도하는데작용한것으로추정된다 (Fig. 1A). 밀기울 (2%) 을첨가한 TSB 배지에서는 xylanase 생산성이정지기후반에급격하게증가하였는데이는 mannanase 생산성이증가한배양시간보다는 4-6시간더늦은시간이다 (Fig. 1B). 이와같이정지기에이르렀을때 hemicellulases의생산성이급격하게증가하는현상은균의성장과연계하여효소생산성이증가하는 Bacillus속균주와는다른양상인데 (4), 배양시간이 10일이되었을때 cellulase 생산성이최대에이르는 Microbacterium sp. 에비해서는분리균의 hemicellulases 생산성이빠른편이라판단된다 (12). 한편각각의배지에서분리균의배양상등액을농축한후활성염색을실시한결과밀기울을첨가한배지에서는약 38 kda에해당하는 xylan 분해활성단백질이관찰되었고, LBG 를첨가한배지에서는 50 kda 이상크기의 konjac 분해활성단백질이 3개확인되었다 (Fig. 2). 분리균의 xylan 분해활성단백질의분자량은 B. subtilis AMX-4의 xylanase (23 kda) (17) 보다는크지만 Cellulosimicrobium sp. HY-12의 xylanase (39 kda) (9) 와비슷한수준이다. Konjac은 mannanase와 cellulase에의해모두분해되므로활성염색에서 konjac 분해단백질로밝혀진 3개활성단백질이모두 mannanase라고할
228 Ki-Hong Yoon (A) 2.5 100 (B) 5 100 Mannanase 2.0 1.5 1.0 0.5 0.0 10 1 0.1 Cell growth (OD 600 ) Xylanase 4 3 2 1 0 10 1 0.1 Cell growth (OD 600 ) 0.01 0 5 10 15 20 25 30 35 40 0.01 0 5 10 15 20 25 30 35 40 Culture time (h) Culture time (h) Fig. 1. Production of mannanase (A) or xylanase (B) by Microbacterium sp. YB-1106 and its growths. The isolate was grown respectively in TSB broth supplemented with or without LBG (1%) for mannanase production and with or without wheat bran (2%) for xylanase production. Cell growths (squares) were observed in the media without additional carbon sources (closed symbols), culture media containing additional carbon sources including LBG and wheat bran were used for enzyme production (open symbols). Enzyme activities (circles) in the culture filtrates were determined in the optimal reaction conditions. Each curve represents the average of three independent experiments 수는없으나, 분리균 YB-1106은 CMC 분해활성이없는것으로확인되었으므로활성염색에서나타난 konjac 분해는 mannanase 활성에의한것으로추정된다. 따라서분리균이생산하는 mannanases는다수의세균이생산하는 35-40 kda 크기의 mannanases와는달리 Rhodothermus marimus가생산하는 mannanases와같이크기가큰효소로추정된다 (11). 효소반응특성배양상등액을 ammonium sulfate로처리한후투석하여제조한조효소액을사용하여반응 ph와온도가효소활성에미치는영향을조사하였다 (Fig. 3). 그결과 xylanase의최적반응온도는 55 C, ph는 6.5로확인되었으며, sodium phosphate 완충용액보다는 Tris 완충용액에서활성이높았고 mannanase 는 50 C와 ph 6.0에서최대활성을보였다. 조효소액을여러온도에서각각 1시간방치한후에잔존활성을측정한결과 xylanase와 mannanase는 40 C에서는 90% 이상의활성을유지하였으나 50 C 이상에서는급격하게실활되어 10% 미만의활성만보였다 ( 결과미제시 ). 한편 Microbacterium sp. OU01 이생산하는 2 종류의 chitosanases는 ph 6.2와 50 C, ph 6.6 과 60 C에서각각최대활성을보이며 (16), M. laevaniformans 의 levanbiohydrolase는 ph 6.0과 30 C에서최대활성을갖는것으로보고되었는데 (13) 이로보아 Microbacterium속균주가분비생산하는고분자물질분해효소의반응은주로 ph 6.0-6.5 범위에서활성이높은것으로추정된다. 분리균의효소에의한농산부산물의분해산물을조사하기위해조효소액을사용하여미강, 밀기울, PKM 및 xylan과 LBG을분해한후생성된분해산물을조사하였다. 그결과 Fig. 4에보인바와같이 mannanase 조효소액에의한 LBG의주요분해산물로 mannotriose와 mannobiose 사이의이동도를보이는물질이생성되었으며, PKM, 미강과밀기울도 mannanase 에분해되어 mannotriose 보다이동도가큰분해산물이관찰되었다. Xylanase 조효소액에의한 xylan 분해산물중에서는 xylobiose와 xylose 사이의이동도를보이는물질이주요반응산물로확인되었으며, 미강과밀기울분해물질에서는 xylotriose 와이동도가유사한물질이주요산물로생성되었다. 그러나 PKM은 xylanase에의해서관찰가능한수준으로분해산물이생성되지않았다. 그리고분리균이생산하는 mannanase 또는 xylanase에의한 LBG 또는 xylan의분해산물중 mannose 혹 (A) (B) Fig. 2. Zymograms of the mannanase and xylanase produced by Microbacterium sp. YB-1106. After SDS-PAGE of the concentrated filtrates of Microbacterium sp. cultures grown in TSB broth containing wheat bran for xylanase production (A) or PKM for mannanase production (B), respectively, proteins exhibiting mannanase or xylanase activity were analyzed by activity staining with oat spelt xylan or konjac as substrates. Molecular size is shown in kilodaltons to the right side of the gel.
Microbacterium sp. 분리균의 hemicellulases 229 (A) Temperature (B) ( C) ( o 30 40 50 60 Temperature( ( C) o C) 30 40 50 60 70 Relative mannanase activity (%) 100 80 60 40 20 Relative xylanase activity (%) 100 80 60 40 20 0 4 5 6 7 8 9 10 ph 0 4 5 6 7 8 9 10 ph Fig. 3. Effects of reaction temperature and ph on the enzyme activities. Temperature profiles (close symbols) were obtained by measuring the mannanase activity with a fixed ph 6.0 and xylanase activity with a fixed ph 6.5 at different temperatures. The ph profiles (open symbols) were obtained by measuring the enzyme activities at various ph s and at a constant temperature of 50 C. Buffers (50 mm) used were as follows: sodium citrate (ph 3-6; - -), sodium phosphate (ph 6-8; - -), and Tris (ph 8-9; - -). Each curve represents the average of three independent experiments. 은 xylose는탐지되지않았는데, 이는 xylan으로부터분해산물로 xylose를비롯한자일로올리고당을생성하는 Bacilllus licheniformis의 xylanase (5), LBG로부터 mannose를비롯한만노올리고당을분해산물로생성하는 B. subtilis WL-3의 mannanase (18) 와는다른특성으로보인다. 적요유일탄소원으로 palm kernel meal (PKM) 과밀기울을함 유한최소배지에서농후배양하여작물재배토양으로부터 xylan과 locust bean gum (LBG) 에대한분해활성이있는균을분리하였다. 분리균 YB-1106의 16S rrna 유전자염기서열을조사한결과 Microbacterium arabinogalactanolyticum와 98% 유사도를보였다. 분리균의 xylanase는밀기울, oat spelt xylan, 미강및 xylose와같은부가탄소원에의해생산성이증가된반면에 mannanase는 LBG와 PKM에의해생산성이증가되었다. 특히 Xylanase는밀기울 2% 를첨가한배지, mannanase는 1% LBG를첨가한배지에서각각생산성이가 (A) (B) Fig. 4. Thin-layer chromatograms of hydrolysis products of various substances with the concentrated culture filtrates showing mannanase activity (A) and xylanase activity (B). The reaction mixtures containing the culture filtrates and substrates in 40 mm sodium phosphate buffer (ph 6.0) were incubated for 4 h at 40 C. (A) Lanes: 1 and 2, PKM hydrolysates before and after reaction; 3 and 4, LBG hydrolysates before and after reaction; 6 and 7, wheat bran hydrolysates before and after reaction; 8 and 9, rice bran hydrolysates before and after reaction; 5, authentic standards including mannose (M1), mannobiose (M2) and mannotriose (M3). (B) Lanes: 1 and 2, rice bran hydrolysates after and before reaction; 3 and 4, wheat bran hydrolysates after and before reaction; 8 and 9, oat spelt xylan hydrolysates after and before reaction; 10 and 11, PKM hydrolysates after and before reaction. Authentic standards: lanes 5, xylose (X1); 6, xylobiose (X2); 7, xylotriose (X3) and xylotetraose (X4).
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