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1 The Korean Journal of Microbiology, Vol. 42, No. 4, December 2006, p Copyright 2006, The Microbiological Society of Korea Bacillus licheniformis WL-12 cellulase j x»y 1,2 w tá w l ƒ l cellulase ³ š WL-12 xk p, yw 16S rrna» w Bacillus licheniformis. B. licheniformis WL-12 cellulase j w» w cellulase (cela) 517 g w 1,551 j p.» w WL-12 cellulase y cellulose w, glycosyl hydrolase (GH) family 5 w B. licheniformis, B. subtilis B. amyloliquefaciens cellulase. j cela x vector w B. subtilis x g cellulase 7.0 units/ml. Key words ý Bacillus licheniformis, Bacillus subtilis, cellulase, expression, nucleotide sequence) Bacillus licheniformis B. subtilis z š, w ³ ƒ w w GRAS ³. B. licheniformis B. subtilis x» ƒ w p š, B. licheniformis DSM13» glucose w x» w x (19). w B. licheniformis š ƒ w z ³ ƒ w k w z w p š. B. licheniformis ƒ z ü α-amylase» w z w y š(8), w α-amylase z (4). w ü α-amylase ƒ T7 promoter w ³ x, z ƒ ³ ³ ƒ α-amylase signal peptide w (15). Protease w y y e protease w AP-1 (16) RSP (14) z p ƒ j, 2709 e portease B. subtilis WB600 x (17). w f p w³ PWD-1 (20) RG1 (12) ƒ keratinase e p. B. licheniformis ³ 77-2 xylanase (3), EGW039 β-1,3-1,4-glucanase (18), B endoglucanase (2), NBL420 cellulase (7), NK-27 ³ mannanase (6) *To whom correspondence should be addressed. Tel: , Fax: ykh@lion.woosong.ac.kr w z p ù ƒ. w chitosanase w Bacillus licheniformis MB-2ƒ (5), rp w z (13) (1) w. ü ƒ š B. licheniformis carboxymethyl cellulase (cellulase) wš B. subtilis w mw. Cellulase ³ k Cellulase w š w ƒ 1g 0.85% NaCl 10 ml xkwš xk w 0.5% carboxymethyl cellulose (CMC) trypan blue (70 mg/l) ƒw LB sq (5 g yeast extract, 10 g tryptone, 5 g NaCl, 15 g agar, per liter) w z 60 C o w. g ƒ x z CMC w y ùkù ³ w. ³ w ³ 16S rrna r Lee w (10) w ³ 16S rrna» primer w PCR swš» w. ³ v B. licheniformis WL-12 œ š w ³ Escherichia coli XL-1 blue B. subtilis WB700 (21) ƒƒ, v puc19 E. coli vector pj27 88U (9) B. subtilis vector w. LB (5 g yeast extract, 10 g bactotryptone, 5 g NaCl per liter) w, E. coli B. 313

2 314 Ki-Hong Yoon Kor. J. Microbiol subtilis x y w ampicillin (50 µg/ ml) ù kanamycin (10 µg/ml) ƒƒ ƒw LB ƒ. Cellulase w w E. coli x y w 0.5% CMC 70 mg/l tryphan blue ƒw LB sq w. CMCase y d Cellulase y CMC» w z z y 3,5-dinitrosalicylic acid (DNS) w d w. xk k 1.0% (w/v) CMC 0.5 ml 200 mm sodium citrate buffer (ph 5.5) 0.25 ml z 0.25 ml yww 50 o C 15 g. DNS 3 ml ƒw jš ò 5 ew k z 540 nm Ÿ d wš glucose t w w g w Ÿ w y w. z y 1.0 unit w 1 CMC l 1µmol glucose w y w z w. DNA LB w B. licheniformis ³ l DNA w» w Genomic DNA prep kit (Solgent, Daejeon, Korea), E. coli l v DNA w» w Plasmid Mini-prep kit (Solgent, Daejeon, Korea) w. v w E. coli B. subtilis x y competent cells ƒƒ w.» j wz w j» w r puc19 w w v w z, template DNA wš universal primer reverse primer w dideoxy-chain termination w. Cellulase y B. subtilis x y SDS-PAGEw z polyacrylamide gel SDS w» w 25% isopropanol sww 50 mm sodium citrate buffer (ph 5.5) w z 50 mm sodium citrate buffer (ph 5.5) ew. Polyacrylamide gel Éü w 70 mg/l tryphan blue, 0.3% CMC 1.5% agar w gel dw w z 50 C k z w o y w. š Cellulase ³ p CMC trypan blue ƒw LB w š CMCase y ³ 1 w. WL-12 w ³ 37 o C 60 o C w š, s x w ³ catalase y Bacillus ³ q. w WL-12 CMC w y skim mik, locust bean gum starch wy protease, mannanase amylase w. Vitek (biomerieux ) Bacillus e (BAC) w ³ WL-12 yw p w. w ³ LB w k w ³ 0.4% NaCl 600 nm Ÿ ƒ 0.3 ƒ w z BAC e w 12 w. ³ sucrose, glucose, inositol, arabinose, mannitol, salicin, amygdalin, maltose, trehalose, palatinose, N- acetyl-d-glucosamine k w esculin ƒ ww ùkû. VITEK BAC database ³ yw p w, WL-12 B. licheniformis 97% y. w PCR s DNA r» w 1,520 bp j» WL-12 16S rrna» NCBI BLAST w» w ³ w» w, B. licheniformis (Genbank accession No. AB039328, X68416, CP000002, AE017333, AJ582722, AB055006, DQ350834, AJ582721) 99.7%. ³ w p WL-12 B. licheniformis w ³ q. Cellulase j» B. licheniformis WL-12 DNA PstI wš j»ƒ 1.5 kb DNA r agarose gel w. PstI wš CIP puc19 ligation w z E. coli XL-1 blue w x y CMC wy x y 1 w. ³ x y v wš j DNA r wz w puc19 wš 2,162 bp» w.» w ORF w cellulase 2 ORF S ƒ. wù, ORF1 (» e 160~1,761) ORF2 (» e 211~1,761) q g g RBS (ORF1 -GGAG-, ORF2 -GGAGG-) š. w» signal peptide wš. Signal peptidase x e (A-X-A) ORF1 40» š, ORF A-A-A-A w š ORF2 cellulase w. ORF2 w w B. licheniformis WL-12 cellulase 517» g w 1,551 bp y (Fig. 1). Cellulase» NCBI»

Putative amino acid sequence corresponding to the signal peptide is italicized. The numbers at the end of each line correspond to the nucleotide positions. Fig. 1. Comparison of the B.

3 Vol. 42, No. 4 의 Cellulase 유전자 Bacillus licheniformis 315 Nucleotide sequence and deduced amino acid sequence of cellulase gene. The amino acid deduced from the open reading frame is shown with the one-letter code below the nucleotide sequence. Putative ribosomebinding site (SD) is underlined. Putative amino acid sequence corresponding to the signal peptide is italicized. The numbers at the end of each line correspond to the nucleotide positions. Fig. 1. Comparison of the B. licheniformis WL-12 cellulase with others. The amino acid sequences of six cellulases from B. licheniformis strains including WL-12 (WL12), F5 (CAJ), MD1 (CAE), ATCC (YP) and GXN151 (AAP), and Bacillus sp. CY1-3 (ABG) are given in the oneletter code and have been aligned by introducing gaps (hypens) to maximize similarities. Residues identical to amino acid sequence of the WL-12 cellulase are indicated by asterisks in other sequences. Numbers at the beginning of each line correspond to the amino acid position in the protein. Fig. 2. 교한 결과 여러종류의 B. licheniformis의 cellulase 유전자들 (Genbank accession No. AM183790, CP000002, AE017333, DQ841174, AJ616005, AY291583)과 그 염기서열이 상동성이 99 % 이상이었으며, 가수분해활성 영역을 코드하는 유전자 지역의

4 316 Ki-Hong Yoon Kor. J. Microbiol (» e 211~846) B. amyloliquefaciens (AJ576102) B. subtilis (DQ782954) cellulase 80% ùkû. wr ü e ³ cellulase y chitosanase y š B. licheniformis NBL420 cellulase 94% y (7). wr d cellulase GH family 5 w cellulase y ( e 49~303) cellulose w (CBM; e 374~455).» w B. licheniformis strains F5 (CAJ70710), MD1 (CAE82178), ATCC (YP_079251), GXN151 (11) Bacillus sp. CY1-3 (ABG91147) cellulases 97% (Fig. 2), B. subtilis 168 cellulase (CAA97610) 76% ùkþ. B. subtilis B. licheniformis WL-12 cellulase x B. licheniformis cellulase B. subtilis x j» w cellulase sww 1.8 kb DNA r B. subtilis x vector pj27 88U w w v pjhs1w w (Fig. 3). B. subtilis WB700 pj27 88U pjhs1w ƒƒ x y g x y kanamycin ƒ LB w ³ z w. Fig. 4 cellulase w w pjhs1w x y ƒ pj27 88U x y»ƒ ¼» ù, w. w cellulase y pjhs1w x y ƒ ³ w Fig. 3. Structure of recombinant plasmid pjhs1w containing the cellulase gene. Blank bar indicates the insert DNA containing cellulase gene. The arrows indicate the direction of transcription of genes. Abbreviations are as follows: P, BJ27 88 promoter; cela, cellulase gene; rrn, rrnbt 1 T 2 terminator; Amp, ampicillin resistance gene; Rori, replication origin of plasmid pbr322; Bori, replication origin (minus strand) of plasmid pub110; Km, kanamycin resistance gene; Bori+, replication origin (plus strand) of plasmid pub110. Fig. 4. Growth and cellulase production of the recombinant B. subtilis WB700 strains. Their growths and enzyme activities in the culture filtrates were observed periodically during growth in LB medium at 37 o C. The cell growth was determined by measuring absorbance of the cell culture at wave lenth of 600 nm. Enzyme activities were determined with the culture filtrate. Symbols: Growth ( ý ) and enzyme production ( þ ) by B. subtilis containing pj27 88U alone; Growth (ø) and enzyme production (ù) by B. subtilis containing pjshs1w. ƒw 16 7 U/ml. ù pj27 88U x y 0.1 U/ ml û z, ³ B. subtilis WB700 ƒ w z q. wr SDS-PAGE ³ w 8 z l B. subtilis (pjhs1w) B. subtilis (pj27 88U) ùkù bandƒ xw, wƒ ù ƒw (Fig. 5A). Cellulase y ww 8 1 y band ƒ 12, 16 2 y bandsƒ, ú y ùkû (Fig. 5B). w B. subtilisƒ w w cellulaseƒ w q, w cellulase y. B. subtilis cellulase B. subtilis DB104 x g cellulaseƒ w x š (9). w ü cellulase y U/ml, U/ml, U/ml w y CMC w z y j ùkû. ü w cellulase y CMC ww y gel CMC ww z d. š x 1. Berensmeier, S., S.A. Singh, J. Meens, and K. Buchholz

Vol. 42, No. 4 Bacillus licheniformis Cellulase 317 Fig. 5. SDS-PAGE and zymogram of the cellulase produced by B. subtilis transformants. B. subtilis WB700 containing pj27 88U and B.

5 Vol. 42, No. 4 Bacillus licheniformis Cellulase 317 Fig. 5. SDS-PAGE and zymogram of the cellulase produced by B. subtilis transformants. B. subtilis WB700 containing pj27 88U and B. subtilis WB700 carrying recombinant plasmid pjhs1w were grown at 37 o C in LB medium. Culture filtrates were applied to 10% (w/v) polyacrylamide gel. Lanes 1~4 indicate the 4 hr, 8 hr, 12 hr and 16 hr cultures of B. subtilis containing vector alone as a control, respectively. Lanes 5~8 indicate the 4 hr, 8 hr, 12 hr and 16 hr cultures of B. subtilis(pjhs1w), respectiely. (A) Coomassie blue-stained polyacrylamide gel, (B) Cellulase activity-stained gel. Cloning of the pela gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, highalkaline pectate lyase. Appl. Microbiol. Biotechnol. 64, Bischoff, K.M., A.P. Rooney, X.L. Li, S. Liu, and S.R. Hughes Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnol. Lett. 28, Damiano, V.B., R. Ward, E. Gomes, H.F. Alves-Prado, and R. Da Silva Purification and characterization of two xylanases from alkalophilic and thermophilic Bacillus licheniformis Appl. Biochem. Biotechnol , Declerck N., M. Machius, P. Joyet, G. Wiegand, R. Huber, and C. Gaillardin Hyperthermostabilization of Bacillus licheniformis α-amylase and modulation of its stability over a 50 o C temperature range. Protein Eng. 16, Ekowati, C., P. Hariyadi, A.B. Witarto, J.K. Hwang, and M.T. Suhartono Biochemical characteristics of chitosanase From the Indonesian Bacillus licheniformis MB-2. Mol. Biotechnol. 33, ? 6. Feng, Y.Y., Z.M. He, L.F. Song, S.L. Ong, J.Y. Hu, Z.G. Zhang, and W.J. Ng Kinetics of β-mannanase fermentation by Bacillus licheniformis. Biotechnol. Lett. 25, ? 7. Hong, I.-P., H.-K. Jang, S.-Y. Lee, and S.-G. Choi Cloning and characterization of a bifunctional cellulase-chotosanase gene from Bacillus licheniformis NBL420. J. Microbiol. Biotechnol. 13, Ikram-ul-Haq, H. Ashraf, J. Iqbal, and M.A. Qadeer Production of α-amylase by Bacillus licheniformis using an economical medium. Bioresour. Technol. 87, Jung, K.H., Y.C. Chun, J.-C. Lee, J.H. Kim, and K.-H. Yoon Cloning and expression of a Bacillus sp cellulase gene. Biotechnol. Lett. 18, Lee, E.-H., C.-J. Kim, and K.-H. Yoon Characterization and xylanase productivity of Streptomyces sp. WL-2. Kor. J. Microbiol. Biotechnol. 33, Liu, Y., J. Zhang, Q. Liu, C. Zhang, and Q. Ma Molecular cloning of novel cellulase genes cel9a and cel12a from Bacillus licheniformis GXN151 and synergism of their encoded polypeptides. Curr. Microbiol. 49, Ramnani, P. and R. Gupta Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology. Biotechnol. Appl. Biochem. 40 (Pt 2), Ryttersgaard, C., J. Le Nours, L. Lo Leggio, C.T. Jorgensen, L.L. Christensen, M. Bjornvad, and S. Larsen The structure of endo-β-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products. J. Mol. Biol. 341, Sareen, R., U.T. Bornscheuer, and P. Mishra Cloning, functional expression and characterization of an alkaline protease from Bacillus licheniformis. Biotechnol. Lett. 27, Shahhoseini, M., A.A. Ziaee, and N. Ghaemi Expression and secretion of an α-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene. J. Appl. Microbiol. 95, Tang, X.M., F.M. Lakay, W. Shen, W.L. Shao, H.Y. Fang, B.A. Prior, Z.X. Wang, and J. Zhuge Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis. Biotechnol. Lett. 26, Tang, X.M., W. Shen, F.M. Lakay, W.L. Shao, Z.X. Wang, B.A. Prior, and J. Zhuge Cloning and over-expression of an alkaline protease from Bacillus licheniformis. Biotechnol. Lett. 26, Teng, D., J.H. Wang, Y. Fan, Y.L. Yang, Z.G. Tian, J. Luo, G.P. Yang, and F. Zhang Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3). Appl. Microbiol. Biotechnol. 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. Gottschalk The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J. Mol. Microbiol. Biotechnol. 7, Wang, J.J., W.B. Greenhut, and J.C. Shih Development of an asporogenic Bacillus licheniformis for the production of keratinase. J. Appl. Microbiol. 98, Ye, R., J.H. Kim, B.G. Kim, S. Szarka, E. Sihota, and S.L. Wong High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis. Biotechnol. Bioeng. 62, (Received December 8, 2006/Accepted December 19, 2006)

6 318 Ki-Hong Yoon Kor. J. Microbiol ABSTRACT : Cloning and Expression of A Bacillus licheniformis Cellulase Gene Ki-Hong Yoon 1,2 ( 1 School of Food Science & Biotechnology, 2 Bioresource and Application Research Center, Woosong University, 17-2, Jayang-dong, Dong-gu, Daejeon , Korea) A thermophilic bacterium producing the extracellular cellulase was isolated from soybean paste, and the isolate WL-12 has been identified as Bacillus licheniformis on the basis on its 16S rrna sequence, morphology and biochemical properties. A gene encoding the cellulase of B. licheniformis WL-12 was cloned and its nucleotide sequence was determined. This cellulase gene, designated cela, consisted of 1,551 nucleotides, encoding a polypeptide of 517 amino acid residues. The gene product contained catalytic domain and cellulose binding domain. The deduced amino acid sequence was highly homologous to those of cellulases of B. licheniformis, B. subtilis and B. amyloliquefaciens belonging to the glycosyl hydrolase family 5. When the cela gene was highly expressed using a strong B. subtilis promoter, the extracellular cellulase was produced up to 7.0 units/ml in B. subtilis WB700.

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