The Krean Jurnal f Micrbilgy, Vl. 45, N. 3, September 2009, p. 257-262 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 5.5 55 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: 82-42-630-9742, Fax: 82-42-636-2676 E-mail: 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 14580 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
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.
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-41361 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. 2 49.5 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- 41361 cellulase 42 kda l d 15 kda š (3). B. lichenifrmis B-41361 cellulase y bandsƒ ù, 35 kda w w cellulase y ƒ š (2). 27.4 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).
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-41361 35 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-41351 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-41351 cellulase y (60.7 U/mg) š B-41351 35 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-41361 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 100.0 KCl 101.3 NiCl 2 106.4 MgCl 2 97.7 MnCl 2 98.4 CaCl 2 97.0 CuCl 2 119.5 FeCl 2 98.0 FeCl 3 95.8 EDTA 94.0 SDS 0.0
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-41351 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-41351 cellulase w glucse š cellbise celltriseƒ, B-41351 cellbiseƒ celltrise ùkû (3). p B- 41351 cellulase celltrise ww w WL-12 cellulase ww. š x 1. Berensmeier, S., S.A. Singh, J. Meens, and K. Buchhlz. 2004. Clning f the pela gene frm Bacillus lichenifrmis 14A and bichemical characterizatin f recmbinant, thermstable, highalkaline pectate lyase. Appl. Micrbil. Bitechnl. 64, 560-567. 2. Bischff, K.M., A.P. Rney, X.L. Li, S. Liu, and S.R. Hughes. 2006. Purificatin and characterizatin f a family 5 endglucanase frm a mderately thermphilic strain f Bacillus lichenifrmis. Bitechnl. Lett. 28, 1761-1765. 3. Bischff, K.M., S. Liu, and S.R. Hughes. 2007. Clning and characterizatin f a recmbinant family 5 endglucanase frm Bacillus lichenifrmis strain B-41361. Prc. Bichem. 42, 1150-1154. 4. Cavac-Paul, A. 1998. Mechanism f cellulase actin in textile prcesses. Carbhydr. Plym. 37, 273-277. 5. Damian, V.B., R. Ward, E. Gmes, H.F. Alves-Prad, and R. Da Silva. 2006. Purificatin and characterizatin f tw xylanases frm alkalphilic and thermphilic Bacillus lichenifrmis 77-2. Appl. Bichem. Bitechnl. 129-132, 289-302. 6. Ekwati, C., P. Hariyadi, A.B. Witart, J.K. Hwang, and M.T. Suhartn. 2006. Bichemical characteristics f chitsanase frm the Indnesian Bacillus lichenifrmis MB-2. Ml. Bitechnl. 33, 93-102. 7. Feng, Y.Y., Z.M. He, L.F. Sng, S.L. Ong, J.Y. Hu, Z.G. Zhang, and W.J. Ng. 2003. Kinetics f ß-mannanase fermentatin by Bacillus lichenifrmis. Bitechnl. Lett. 25, 1143-1146. 8. Hng, I.P., H.K. Jang, S.Y. Lee, and S.G. Chi. 2003. Clning and
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