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1 The Krean Jurnal f Micrbilgy, Vl. 45, N. 3, September 2009, p Cpyright 2009, The Micrbilgical Sciety f Krea ³ l Bacillus lichenifrmis WL-12 Cellulase p Á½ Á»y* w t w Bacillus lichenifrmis WL-12 carbxymethyl cellulase (cellulase) w w ³ ³ q l DEAE-Sepharse Q-Sepharse f j m v mw cellulase w. z y 163 U/mg, SDS-PAGE w d 49.5 kda ùkû. ph C y, SDS (5 mm) w cellulase y w š Cu 2+ (5 mm) w. cellulase CMC, knjac, barley β-glucan lichenan ƒ ww ù xylan, lcust bean gum p-nitrphenyl-β-glucpyranside ww w. Cellligsaccharides WL-12 cellulase ww cellbise celltriseƒ cellbise w ƒ j celltrise, celltetrase cellpentase ww ù cellbise ww w y. Key wrds ý B. lichenifrmis, cellulase, E. cli, prperties, purificatin End-β-1,4-glucanase (carbxymethyl cellulase : cellulase) ex-β-1,4-glucanase, β-glucsidase cellulase z Á s y,, ƒ ƒœ w z. Cellulase ³, q š glycsyl hydrlase (GH) family w ƒ j. Cellulase Trichderma reesei q š ù, q z ƒ w» w w p ³ cellulase z w š. k œ q z ƒ ph w j ƒ ph w ³ cellulase w š (4, 9), cellulase w Saccharmyces cerevisiae Zymmnas mbilis y z j š cellulase z ƒ û» w Bacillus cagulans cellulase q cellulase w y v w cellulase z w (17). Bacillus lichenifrmis B. subtilis z š, w ³ ƒ w GRAS ³ B. lichenifrmis DSM3» (21). B. lichenifrmis š ƒ w z ³ ƒ w k w z w p š *T whm crrespndence shuld be addressed. Tel: , Fax: ykh@lin.wsng.ac.kr. B. lichenifrmis ƒ z ü α-amylaseƒ, y e prtease (18), cellulase-chitsanase (8). xylanase (5), β-1,3-1,4-glucanase (15, 20), cellulase (2, 3, 14), mannanase (7) w z p ù ƒ. w chitsanase w MB-2ƒ (6), 14A rp w z (1) w. wr ü ƒ š B. lichenifrmis WL-12 cellulase B. lichenifrmis ATCC z w» y (23), x ¾ p w šƒ ³ cellulase xw z wš z p mw. ³ v B. lichenifrmis WL-12 cellulase xw» w v ³ pet23a(+) E. cli BL21(F - - mpt hads B (r B m B- ), gal dcm(de3)) ƒƒ w, ampicillin (100 µg/ml) ƒw LB (yeast extract, 5 g; tryptne, 10 g; NaCl, 5 g; water, 1 L) w. DNA Cellulase sw» w WL-12 cellulase w w w v x wš» w w 12CelF (5 -GGAAAACATATGTCATATATG AAACGTTC-3 : NdeI sites; underlined) 12CelR (5 -CGTC AAACTCGAGTTATTTAGGTTCAGTG-3 : XhI sites; underlined) 257

2 258 Jng Duk Park et al. Kr. J. Micrbil primers w pfu DNA plymerase PCR w. PCR 95 C 3 w z 95 C 30, 62 C 40, 72 C 3 30z wš 72 C 10 ew ww. Cellulase Cellulase w w E. cli BL21(DE3)/pPES3 ampicillin ƒ LB w 37 C k w 600 nm Ÿ ƒ 0.7 IPTG ƒ 0.5 mm ƒw z 25 C 5 k w. w z w ³ 50 mm Tris-HCl buffer (ph 8.0) xkwš q ³ q w z 15,000 g, 4 C 20 w ³ q w. ³ q ammnium sulfate ƒw 30~70% (w/v) zw e 50 mm Tris-HCl buffer (ph 8.0) xkw w n w z, DEAE- Sepharse f j m v ww. NaCl (0~0.8 M) w y z ultrafiltratin w z 50 mm Tris-HCl buffer (ph 8.0) n wš Q-Sepharse f j m v ww. w z w» w NaCl (0~0.6 M) zw z y z ultrafiltratin wš z w. Cellulase y d Cellulase y CMC» w z z y 3,5-dinitrsalicylic acid (DNS) w d w. xk k 1.0% (w/v) CMC 0.5 ml, 200 mm sdium citrate buffer (ph 5.5) 0.25 ml z 0.25 ml yww 55 C 15 g. DNS 3ml ƒw jš ò 5 ew k z 540 nm Ÿ d wš glucse t w w g w Ÿ w y w. z y 1.0 unit w 1 CMC l 1 µml glucse w y w z w. p-nitrphenyl-β-glycsides wy w» w» 1 mm w 10 k z 2 v 1M Na 2 CO 3 ƒw jš 405 nm Ÿ d w. z y 1 1µml p- nitrphenl j z 1unit w. 5748) d j m v ww. j» w 9 ml ethanl, 0.5 ml p-anisaldehyde, 0.5 ml sulfuric acid glacial acetate yww z, 120 C 10 ew. š Cellulase x B. lichenifrmis WL-12 cellulase ³ x j» w signal peptide sww cellulase ƒ s 12CelF 12CelR primers w PCR ww. Primers pet23a(+) cellulase j w» w NdeI XhI w. sw cellulase NdeI XhI w z wš w z pet23a(+) w w v ppes3 w (Fig. 1). ppes3 cellulase» w PCR ƒ ù y w. w v ppes3 E. cli BL21(DE3) w x y LB w 37 C k w IPTG w. x cellulase y» w IPTG z 25 C û 5 w. ³ z wš q q w z w ³ q w cellulase y w v» ³ q 5.28 U/ml z y ù, ³ q 1% w w z y. w ³ cellulase ³ ü w Cellligsaccharides» w z wwš 95 C 3 w z w e wš w n-prpanl, nitrmethane [7:1:2, (v/v)] yw w silica gel-precated thin layer plate (Merck Kiesegel, N. Fig. 1. Structure f recmbinant plasmid ppes3 cntaining the cellulase gene. Black bar indicates the cellulase gene. The arrws indicate the directin f transcriptin f genes. Abbreviatins are as fllws: T7, T7 RNA plymerase prmter; CelS, cellulase gene; Amp, ampicillin resistance gene; Ori-f1, f1 rigin; Ori, replicatin rigin f plasmid pbr322.

3 Vl. 45, N. 3 B. lichenifrmis cellulase p 259 y, ³ cellulase ƒ signal peptide swwš wš ³. ³ cellulase w ³ z ƒ x z ƒ ³ ƒ š ƒ. B. lichenifrmis ü α-amylase T7 prmter w ³ x z ƒ ³ ³ ƒ α-amylase signal peptide w š(19), B. lichenifrmis end-β-1,3-1,4-glucanase ³ 40% ³ š š (15). w puc19 j WL-12 cellulase x y k ³ cellulase ƒ ³ š( ), B. subtilis x y WL-12 cellulase 7U/ml w (23). E. cli BL21(DE3)/pPES3 cellulaseƒ WL-12 cellulase p» IPTG ƒw z 25 C û signal peptideƒ z w. B. subtilis mannanase T7 prmter w BL21(DE3) IPTG ƒw 37 C x w signal peptide sww ³ ü mannanse w ù, signal peptide sww mannanase w y (11). wr B. subtilis 1N 2 cellulases T7 prmter w BL21(DE3) x w» 0.52 U/ml 0.22 U/ml y w (13) ³ WL-12 celllulase {. Cellulase BL21(DE3)/pPES3 l cellulase SDS-PAGE y w 3 y bandsƒ y ( ). WL-12 cellulaseƒ w y w cellulse binding dmain (CBD) š ³ cellulase C- w CBDƒ w y cellulase y w y bandsƒ. w x ³ cellulases ³ xw š š(3, 9), WL-12 cellulase B. subtilis x g 2 y bandsƒ (23). B. lichenifrmis B cellulase ³ xw w prtelysis x (3). Cellulase w» w ³ ³ q ammnium sulfate (30%~70%) zw DEAE-Sepharse f j m v w resin w š cellulase resin w NaCl cellluase. SDS-PAGE y w resin w z Fig. 2. SDS-PAGE f the cellulase purified frm recmbinant E. cli. Lane 1, the mlecular weight markers; 2, the purified enzyme. Mlecular size is shwn in kildaltns t the left side f the gel. w z w resin w cellulase j»ƒ y. j»ƒ j cellulase w» w NaCl w z cellulase y z ultrafiltratin wš Q-Sepharse f j m v w. Q-Sepharse w NaCl w z y z w SDS-PAGE w Fig kda cellulaseƒ y ³ B. lichenifrmis NBL420 cellulase-chitsanase w (8).» cellulase 56.8 kda w j»ƒ 7 kda signal peptide ù C- ƒ. ³ B cellulase 42 kda l d 15 kda š (3). B. lichenifrmis B cellulase y bandsƒ ù, 35 kda w w cellulase y ƒ š (2) kda B. lichenifrmis cellulaseù(15) Bacillus cellulases (<42 kda) w ³ WL-12 cellulase j. wr BL21(DE3) x B. subtilis 1N(13) cellulase 54 kda y B. stearthermphilus N. 236 cellulase 95 kda š (10).

4 260 Jng Duk Park et al. Kr. J. Micrbil Fig. 4. Thermstability f the cellulase. The residual relative activity was determined by measuring after preincubatin fr 3 h at varius temperatures with a fixed ph 6.5. Fig. 3. Effects f reactin temperature and ph n the cellulase. Temperature prfile ( þ ) was btained by measuring the cellulase activities at different temperatures and ph 5.5. The reactins was dne at 55 C and varius phs fr determining the ph prfile (dtted line). Buffers used were as fllws: sdium citrate ( ù ), sdium phsphate ( ø ), Tris ( ). Cellulase p ³ w WL-12 cellulase y e ph w w. WL-12 cellulase 55 C ph 5.5 y (Fig. 3). B. lichenifrmis B kda cellulase ³ 42 kda cellulase 65 C ph 6.0 y WL-12 cellulase š (2, 3). Bacillus cellulases ƒ 50~70 C, ph 5.0~7.0 y z ƒ WL-12 cellulase Bacillus cellulase p q. w w» w w z 3 ew y d w 50 C 3 ¾ w ù 55 C 1 e z z y y 3 e z y w (Fig. 4). WL-12 cellulase B B. amylliquefaciens DL-3 z w û r (3, 12), NBL420 ù Bacillus sp. CH43 HR68 z w (8, 16). CMC» w WL-12 cellulase w z y 163 U/mg y ³ B cellulase y (60.7 U/mg) š B kda cellulase y (183 U/mg) w, DL-3 cellulase y (6,070 U/mg) û. ù yw cellulase y e w w» w 5mM ƒw z w. Table 1 SDS w z y w, yw z y w ƒ j ùkû. Cu 2+ B. lichenifrmis B cellulase WL- 12 cellulase y ƒ gš, WL-12 cellulase y w e Mn 2+ cellulase y e w w š (3, 10, 12, 16). w EDTA Bacillus z WL-12 cellulase y ww, DL-3 cellulase y ww š B. subtilis AH18 cellulase y ww (12, 22).» p z w CMC w ƒ Table 1. Effects f metal ins and ther reagents n the cellulase activity Effectr (5 mm) Relative activity (%) Nne KCl NiCl MgCl MnCl CaCl CuCl FeCl FeCl EDTA 94.0 SDS 0.0

5 Vl. 45, N. 3 B. lichenifrmis cellulase p 261 Table 2. Substrate specificity f the purified cellulase Substrates Relative activity (%) Carbxymethylcellulse 100 Knjac 176 Barley β-glucan 292 Lichenan 83 Lcust bean gum Oat spelt xylan p-nitrphenyl-β-celbiside 19 p-nitrphenyl-β-glucside p-nitrphenyl-β-xylside p-nitrphenyl-β-galactside, nt detected. w y w. z 0.5% w x w, cellulase glucseƒ β-1,4 w CMC ƒ w β-1,3-1,4 w barley β-glucan w ƒ w 2 lichenan w ƒ w CMC w 83% (Table 2). Lichenan barley β-glucan glucseƒ β-1,3-1,4 w wš β-1,3 w β-1,4 w ƒ lichenan barley β-glucan lichenan w β-1,3 w. Lcust bean gum ù at spelt xylan cellulase w ƒ wƒ ù WL-12 cellulase mannanase xylanase y y. Knjac glucmannan cellulase glucse» w wwš mannanase mannse» w ww. WL- Fig. 5. Thin-layer chrmatgram f hydrlysis prducts f β-1,4- linked cellligsaccharides. The reactin mixtures cntaining the purified cellulase and cellligsacchrides in 50 mm sdium citrate buffer (ph 5.5) were incubated at 50 C. Reactin prducts were analyzed frm reactin mixtures befre (lanes C2B, C3B, C4B, and C5B) and after reactin (C2A, C3A, C4A, and C5A). C2 t C5 represent fr cellbise, celltrise, celltetrase, and cellpentase, and G fr glucse. 12 cellulaseƒ lcust bean gum ww w knjac w cellulase y w. WL-12 cellulase CMC β-glucan w Bacillus sp. CH43 HR68 cellulases w» p (16). ù B DL-3 cellulase β-glucan ww CMC wy û (3, 12), DL-3 B. stearthermphilus N. 236 WL-12 cellulase xylan ww š (22). wr p-nitrphenyl-βglycsides» w w w p-nitrphenyl-βcellbiside ww β-xylsidase β-galactsidase y β-glucsidase y. cellulases ƒ p-nitrphenyl-β-cellbiside ww p-nitrphenyl-βglucside ww w (3, 9, 12, 22). Cellulase w ƒ w w» w» glucse w ƒ cellbise, celltrise, celltetrase š cellpentase w z w z ƒ w TLC w. cellbise x w ù w ƒ 3 š w ùkû celltrise w ƒ ƒ û (Fig. 5). Cellpentase, celltetrase celltrise ƒ w B cellulase w glucse š cellbise celltriseƒ, B cellbiseƒ celltrise ùkû (3). p B cellulase celltrise ww w WL-12 cellulase ww. š x 1. Berensmeier, S., S.A. Singh, J. Meens, and K. Buchhlz Clning f the pela gene frm Bacillus lichenifrmis 14A and bichemical characterizatin f recmbinant, thermstable, highalkaline pectate lyase. Appl. Micrbil. Bitechnl. 64, Bischff, K.M., A.P. Rney, X.L. Li, S. Liu, and S.R. Hughes Purificatin and characterizatin f a family 5 endglucanase frm a mderately thermphilic strain f Bacillus lichenifrmis. Bitechnl. Lett. 28, Bischff, K.M., S. Liu, and S.R. Hughes Clning and characterizatin f a recmbinant family 5 endglucanase frm Bacillus lichenifrmis strain B Prc. Bichem. 42, Cavac-Paul, A Mechanism f cellulase actin in textile prcesses. Carbhydr. Plym. 37, Damian, V.B., R. Ward, E. Gmes, H.F. Alves-Prad, and R. Da Silva Purificatin and characterizatin f tw xylanases frm alkalphilic and thermphilic Bacillus lichenifrmis Appl. Bichem. Bitechnl , Ekwati, C., P. Hariyadi, A.B. Witart, J.K. Hwang, and M.T. Suhartn Bichemical characteristics f chitsanase frm the Indnesian Bacillus lichenifrmis MB-2. Ml. Bitechnl. 33, Feng, Y.Y., Z.M. He, L.F. Sng, S.L. Ong, J.Y. Hu, Z.G. Zhang, and W.J. Ng Kinetics f ß-mannanase fermentatin by Bacillus lichenifrmis. Bitechnl. Lett. 25, Hng, I.P., H.K. Jang, S.Y. Lee, and S.G. Chi Clning and

6 262 Jng Duk Park et al. Kr. J. Micrbil characterizatin f a bifunctinal cellulase-chtsanase gene frm Bacillus lichenifrmis NBL420. J. Micrbil. Bitechnl. 13, Jung, K.H., Y.C. Chun, J.C. Lee, J.H. Kim, and K.H. Yn Clning and expressin f a Bacillus sp cellulase gene. Bitechnl. Lett. 18, Kim, S.H., S.G. Ch, and Y.J. Chi Purificatin and characterizatin f carbxymethyl cellulase frm Bacillus stearthermphilus N J. Micrbil. Bitechnl. 7, Kweun, M.A., J.Y. Shn, and K.H. Yn High-level expressin f a Bacillus subtilis mannanase gene in Escherichia cli. Kr. J. Micrbil. Bitechnl. 32, Lee, U.J., B.K. Kim, B.H. Lee, K.I. J, N.K. Lee, C.H. Chung, Y.C. Lee, and J.W. Lee Purificatin and characterizatin f cellulase prduced by Bacillus amylliquefaciens DL-3 untilizing rece hull. Biresur. Technl. 99, Li, W., X. Huan, Y. Zhu, Q. Ma, and Y. Chen Simultaneus clning and expressin f tw cellulase genes frm Bacillus subtilis newly islated frm glden takin (Budrcas taxiclr Bedfrdi). Bichem. Biphys. Res. Cmmun. 383, Liu, Y., J. Zhabg, Q. Liu, C. Zhang, and Q. Ma Mlecular clning f nvel cellulase genes cel9a and cel12a frm Bacillus lichenifrmis GXN151 and synergism f their encded plypeptides. Curr. Micrbil. 49, Llberas, J., J.A. Perez-Pns, and E. Querl Mlecular clning, expressin and nucletide sequence f the end-β-1,3-1,4-d-glucanase gene frm Bacillus lichenifrmis. Predictive structural analyses f the encded plypeptide. Eur. J. Bichem. 197, Mawadza, C., R. Hatti-Kaul, R. Zvauya, and B. Mattiassn Purificatin and characterizatin f cellulases prduced by tw Bacillus strains. J. Bitechnl. 83, Ou, M.S., N. Mhammed, L.O. Ingram, and K.T. Shanmugam Bacillus cagulans requires less cellulases fr simultaneus saccharificatin and fermentatin f cellulse t prducts than mesphilic micrbial bicatalysts. Appl. Bichem. Bitechnl. 155, Sareen, R., U.T. Brnscheuer, and P. Mishra Clning, functinal expressin and characterizatin f an alkaline prtease frm Bacillus lichenifrmis. Bitechnl. Lett. 27, Shahhseini, M., A.A. Ziaee, and N. Ghaemi Expressin and secretin f an α-amylase gene frm a native strain f Bacillus lichenifrmis in Escherichia cli by T7 prmter and putative signal peptide f the gene. J. Appl. Micrbil. 95, Teng, D., J.H. Wang, Y. Fan, Y.L. Yang, Z.G. Tian, J. Lu, G.P. Yang, and F. Zhang Clning f β-1,3-1,4-glucanase gene frm Bacillus lichenifrmis EGW039 (CGMCC 0635) and its expressin in Escherichia cli BL21 (DE3). Appl. Micrbil. Bitechnl. 72, Veith, B., C. Herzberg, S. Steckel, J. Feesche, K.H. Maurer, P. Ehrenreich, S. Baumer, A. Henne, H. Liesegang, R. Merkl, A. Ehrenreich, and G. Gttschalk The cmplete genme sequence f Bacillus lichenifrmis DSM13, an rganism with great industrial ptential. J. Ml. Micrbil. Bitechnl. 7, W, S.M., H.K. Jung, and S.D. Kim Clning and characterizatin f a cellulase gene frm a plant grwth prmting rhizbacterium, Bacillus subtilis AH18 against phytphthra blight disease in red-pepper. Kr. J. Micrbil. Bitechnl. 34, Yn, K.H Clning and expressin f a Bacillus lichenifrmis cellulase gene. Kr. J. Micrbil. 42, (Received August 10, 2009/Accepted September 1, 2009) ABSTRACT : Prperties f a Bacillus lichenifrmis Cellulase Prduced by Recmbinant Escherichia cli Jng Duk Park, Yen A Kim, and Ki-Hng Yn* (Department f Fd Science and Bitechnlgy, Wsng University, Daejen , Republic f Krea) Carbxymethyl celluase (cellulase) was purified frm cell-free extract f the recmbinant Escherichia cli carrying a Bacillus lichenifrmis WL-12 cellulase gene by DEAE-Sepharse and phenyl-sepharse clumn chrmatgraphy with specific activity f 163 U/mg prtein. The mlecular mass f the purified enzyme was estimated t be apprximately 49.5 kda by sdium ddecyl sulfate-plyacrylamide gel electrphresis. The enzyme had a ph ptimum at 5.5 and a temperature ptimum at 55 C. The activity f the enzyme was cmpletely inhibited by SDS (5 mm), and slightly enhanced by Cu 2+ (5 mm). The cellulase was active n CMC, knjac, barely glucan and lichenan, while it did nt exhibit activity twards xylan, lcust bean gum, and p-nitrphenyl-β-glucpyranside. The predminant prducts resulting frm the cellulase hydrlysis were cellbise and celltrise fr cellligsaccharides including celltrise, celltetrase and cellpentase. The enzyme culd hydrlyze cellligsaccharides larger than cellbise.