The Krean Jurnal f Micrbilgy, Vl. 42, N. 2, June 2006, p. 142-148 Cpyright 2006, The Micrbilgical Sciety f Krea Bacillus amylliquefaciens MJ-1 chitsanase j p 1 *Á w 1 *Áy Ÿ 2 Á 3 Áx 1 **Á ** w w w œ l w w p w y ƒ š chitsan ligsaccharides z w» w», m z t chitsan w w ³ w. w ³ xkw, yw w w w, Bacillus amylliquefaciens MJ-1 w. B. amylliquefaciens MJ-1 l chitsanase sww 1,049 bp DNA r j w, chitsanase 825» 274 š, 30.9 kda. j w chitsanase hmlgy search, glucside hydrlase family 46 w chitsanase. B. amylliquefaciens MJ-1 chitsanase E. cli BLR (DE3) w, 1 mm IPTG chitsanase x wš w z, ph w p w. z y 60 C, 80 C 75% y ùkü ü ƒ z. wr, ph 5.0, ph 5~7 80% y w. Key wrds ý Bacillus amylliquefaciens MJ-1, chitsanase, gene expressin Chitsan q, š w Ë w chitin k py, chitsan w chitsan ligsaccharides ü, w³z, w z, x g l w, w y ƒ š» t,» t, y t w š (4, 7, 12, 20, 21). Chitsan ligsaccharides w chitsan - e ww yw chitsanase w z. yw w œ w s w y j», chitsanase w w z l w chitsanase w z w š (5, 14). ¾ l w chitsanaseƒ š(3, 8, 10, 15, 22, 24, 25), j (6, 9, 16, 26), z chitsan» w chitsan ligsaccharides w. chitsanase 10~50 kda j»ƒ w, ph 4.0~8.0, 30~60 C š š (8, 10, 12, 22, 25). *These authrs cntributed equally t this wrk **T whm crrespndence shuld be addressed. Dae-Kyung Kang (Tel: 041-550-3655, Fax: 041-550-3604, E-mail: dkkang@dankk.ac.kr) Yung Hyun (Tel: 041-585-2881, Fax: 041-585-2887, E-mail: yhyun@eddram.net) chitsan ligsaccharides ww chitsanase w» w», m z t l chitsan w Bacillus sp. MJ-1 w š, chitsanase j w p w. w, j w chitsanase ³ x k z, w ph p w. Chitsan w³ Chitsan w ƒ w» w m, š, Ë,, w l w. xk, g, 0.5% cllidal chitsan w sq (0.05% MgSO 4 Á7H 2 O, 0.03% KH 2 PO 4, 0.07% K 2 HPO 4, 0.001% FeSO 4, 0.0001% ZnSO 4, 0.0001% MnCl 2, 1.5% agar, ph 7.0) w z 30 C 37 C ƒƒ w. 3-5 z n y x w g chitsan w ³, w z ³ w. Cllidal chitsan Cllidal chitsan Yabuki (24) xw w. Chitsan (Sigma, USA) 20 g 2 l 2% acetic acid ƒw 6 chitsan 142
Vl. 42, N. 2 Bacillus amylliquefaciens MJ-1 chitsanase 143 z, 8 l ƒwš w w. 5 N NaOH w ph 5.0¾ w z, ph 9.0¾ 0.1 N NaOH w w cllidal chitsan x g. g xk» w w» w w. cllidal chitsan d»(kett Electric Lab., Japan) w w d w z w. w ³ w ³ w» w 16S rdna xkw, yw p ww. ³ 16S rdna w» w Pavlva (17) w. Genmic DNA extractin kit (Qiagen) w ³ l genmic DNA w, Table 1 primer w plymerase chain reactin (PCR) ww. 50 pmle primer, 50 ng template DNA, 10 Taq DNA plymerase buffer 5 µl, 2.5 mm dntp mixture 4 µl, Taq DNA plymerase (TaKaRa-Krea, Krea) 1 Uƒ sw PCR mixture 95 C 5 k z 95 C 1, 55~60 C 1, 72 C 1 30 cycle w PCR ww. PCR pstblue-1 (Nvagen, USA) j w, BigDye TM -terminatr sequencing kit ABI PRISM377 sequencer (Perkin-Elmer, USA)» w. w ³ xkw p x (Scanning Electrn Micrscpy) w, Shn (19) w. w, API 50CHB kit (bimerieux C, France) w 49 k w w w. Chitsanase j w ³ l genmic DNA w z,» š chitsanase z w cnserved regin šw primer (CSB-1 : 5'-CTT RTT TTC ACC ATG TTT TT-3', CSB-2 : 5'-ACR TGA ATC GGY CCG TTT A-3')w,» w PCR ww. PCR 1% agarse gel (TAE buffer) w 100V 40» ww w, QIA quick gel extractin kit (QIAGEN) w agarse gel l DNA r Table 1. Olignucletide primers fr amplificatin f 16S rdna Primer 1) Sequence (5-3 ) Target site 2) F1 AGAGTTTGATCCTGGCTCAG 27 F2 GTGCCAGCAGCCGCGG 530 F3 AAACTCAAAGGAATTGACGG 926 R1 TCTACGCATTCCACCGCTAC 685 R2 GGGTTGCGCTCGTTG 1,100 R3 AAGGAGGTGATCCAGCC 1,525 1) F1, F2 and F3: frward primers; R1, R2 and R3: reverse primers. 2) Accrding t the numbering f the Escherichia cli 16S rdna. w. w DNA r pgem-t easy vectr j w z, DNA» ww. Chitsanase full sequence» w DNA walking speedup premix kit (Seegene, Krea) w. DNA walking kit w» chitsanase» 5' 3' w sw pgem-t vectr ƒƒ j w. ƒ j DNA» w chitsanase full sequence y w,» GenBank w (GenBank accessin number DQ683023).»»» g w, GenBank database (Natinal Center fr Bitechnlgy Infrmatin, USA) BLAST algrithm w» w. multiple sequence alignment Clustal W v w w. Recmbinant chitsanase x j w chitsanase E. cli x j» w N- His-tag pet21b x l w. Frward (ME-1 : 5'-AAA GGA TCC GAT GAA GGC AAA AGT AGA TTC-3') reverse (ME-2 : 5'-AAA ACT CGA GCT TGA TTA CGA AAT CAC-3') primer w PCR ww, PCR r pet21b l w z (BamHI / XhI) w z x l j w. E. cli BLR (DE3) w z, x y ³ w. x y ³ ampicillin 50 µg/ml ƒ w Luria-Bertani (LB) O.D.(600 nm) 0.5 ¾ w z, IPTG (isprpyl-β-d-thi-galactpyranside) 1mM ƒw x w. 37 C 5 w z s yw. x w» w, cell pellet lysis buffer (50 mm Na-Pi (ph 7.0), 300 mm NaCl, 1 mm DTT) xkw z lyszyme (1 mg/ml) ƒw. Snicatr (Snics & Materials, Inc) s q w, 14,000rpm 30 w w. Ni 2+ -NTA affinity clumn (Qiagen) ladingwš 50 mm imidazle buffer w z 250 mm imidazle buffer w, 10% SDS-PAGE mw w x y w (11). w chitsanase y d» w cllidal chitsan» w w chitsanase z chitsan w d w. 1% cllidal chitsan 250 µl, 1 M ptassium phsphate (ph 6.5) 50 µl, z 1 ml 37 C w. 30 k z, 100 C 10 ƒ w g.
144 Chan-S Park et al. Kr. J. Micrbil (13,000 rpm, 10분)하였으며, 상등액에 존재하는 환원당을 DNS (dinitrsalicylic acid)법(13)으로 정량하여 효소 활성을 측정하였 다. Chitsanase 활성단위(unit)는 상기 서술한 조건에서 분당 1 µmle의 환원당(D-glucsamine)을 생성하는 효소의 양으로 정의 하였다. 재조합 의 최적 온도 및 chitsanase ph 효소 반응의 최적 온도를 조사하기 위해, 50 mm ptassium phsphate 완충용액(pH 6.5)에서 1% cllidal chitsan 용액을 기질로 사용하여 20 C에서 80 C까지 10 C 구간별로 반응시킨 후, 상기 서술한 효소활성 측정법으로 효소 활성을 비교하였다. 효소의 최적 ph를 조사하기 위해서는, 정제된 효소액을 아래의 각 ph 별 완충용액에 넣고 반응시킨 후, 활성을 비교하였다. 사 용한 완충용액으로서, ph 2.0-4.0 사이는 50 mm glycine-hcl buffer, ph 5.0~6.0 사이는 50 mm sdium acetate buffer, ph 7.0~8.0 사이는 50 mm ptassium phsphate buffer, ph 9.0~ 10.0 사이는 50 mm Tris-HCl buffer, ph 11.0~12.0 사이는 50 mm glycine-naoh buffer를 각각 사용하였다. 재조합 Scanning electrn micrscpy (SEM) micrgraph ( 15,000) f B. amylliquefaciens MJ-1. Fig. 1. DNA walking kit (Seegene, Krea)을 이용하여 chitsanase 유전 자의 전체 염기서열을 클로닝하였다(Fig. 2). 클로닝한 chitsanase 유전자의 크기는 825 bp로서 274 개의 아미노산으로 구성되어 의 온도 및 안정성 chitsanase ph 효소의 온도 안정성을 검토하기 위해, 50 mm ptassium phsphate buffer (ph 6.5)에 효소액을 첨가하여 20 C에서 80 C 까지의 각 온도에서 30분간 처리한 후, 효소의 잔존 활성을 측 정하였다. 효소의 ph 안정성을 조사하기 위해서는, ph 2.0에서 ph 12.0까지의 각 완충액에 정제된 효소액을 첨가하여 4 C에서 24시간동안 보관한 후, 잔존 활성을 측정하였다. 결과 및 고찰 Chitsan 분해균주의 분리 및 동정 전통 발효식품인 메주로부터 chitsan 분해능이 우수한 미생물 인 MJ-1 균주를 분리하였다. 전자현미경(XL30-CP, Philips)으로 관찰한 결과 (Fig. 1), Gram 양성의 간균이었으며 포자를 형성하 였다. 또한, 호기성이고 catalase test에서는 양성으로 나타나 Bacillus 속의 세균으로 추정할 수 있었다. 분리한 MJ-1 균주를 보다 정확하게 동정하기 위하여 API 50CHB kit (bimerieux C, France)를 이용하여 49개의 탄소원에 대한 이용성을 조사한 결과, 98.9%의 확률로 Bacillus amylliquefaciens와 일치하였다 (Table 2). 또한, 16S rdna 서열을 분석한 결과에서도 Bacillus amylliquefaciens와 99% 일치하였으므로 (data nt shwn), 본 균주를 B. amylliquefaciens MJ-1으로 명명하였다. B. amylliquefaciens 의 클로닝 MJ-1 균주로부터 chitsanase 유전자 Chitsanase 유전자 염기서열의 상동성 비교를 통해 얻어진 cnserved regin primer를 이용하여 PCR을 수행한 결과, 636 bp 크기의 키토산 분해효소의 부분 염기서열을 얻을 수 있었다 (data nt shwn). PCR에 의해 얻어진 부분 염기서열을 토대로, Nucletide and deduced amin acid sequences f the chitsanase frm B. amylliquefaciens MJ-1. Cding regin starts at psitin 143 and ends at psitin 967. The putative Shine-Dalgarn (SD) sequences fr ribsme-binding site are underlined. Fig. 2.
Vl. 42, N. 2 Bacillus amylliquefaciens MJ-1 chitsanase 145 Table 2. Characteristics f the islated strain, MJ-1 Characteristics Result Characteristics Result Mrphlgical characterizatin Manitl + Shape Rd Srbitl + Gram stain + 1) α Methyl-D-mannside - Mbility + α Methyl-D-glucside + Spre frmatin + N-acetyl glucsamine + Physilgical characterizatin Amygdalin + Catalase + Arbutine + VP-reactin + Esculine + Ph 5.7 agar + Salicine + Utilizatin f prpinate - Cellbise + Utilizatin f citarte + Maltse + Egg-ylk lecthinase - Lactse - Decmpsitin f casein + Melibse - Starch hydrlysis + Sucrse + Carbhydrate degradatin 2) Trehalse + Glycerl + Inuline - Erythritl + Melezitse - D-Arabinse - D-Raffinse - L-Arabinse + Starch - Ribse + Glycgen - D-Xylse - Xylitl - L-Xylse - β Gentibise + Adnitl - D-Turannse - β Metyl-D-xylside - D-Lyxse - Galactse - D-Tagatse - D-Glucse + D-Fucse - D-Fructse + L-Fucse - D-Mannse + D-Arabitl - L-Srbse - L-Arabitl - Rhmnse - Glucnate + Dulcitl - 2-Ke-glucnate - Insitl - 5-Ket-glucnate - + : Psitive result, - : Negative result API 50CHB kit was used. 1) 2), 30.9 kda. w, g 9 bp ribsme-binding site AAGG wš. B. amylliquefaciens MJ-1 chitsanase k d BLASTP,» chitsanase z ùkü (Fig. 3)., Bacillus subtilis chitsanase precusr (18) 87%, Bacillus sp. thermstable chitsanase (26) 68% ùkü š, Streptmyces sp. N174 chitsanase (2) 39%, Bacillus ehimensis EAG1 chitsanase (1) 25% ùkü. p, glycside hydrlase family 46 chitsanase œm ùkù E-[DNQ]-x(8)-Yx(7)-D-x[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G-x(5)-D (23) š, w catalytic activity v Glu-22 Asp- 40 (Streptmyces sp. strain N174 chitsanase ) (2), j w B. amylliquefaciens MJ-1 chitsanase glucside hydrlase family 46 w chitsanase. B. amylliquefaciens MJ-1 chitsanase x B. amylliquefaciens MJ-1 l j w chitsanase E. cli BLR (DE3) wš, 1 mm IPTG chitsanase x 10% SDS-PAGE y w (Fig. 4, lane 3). ù, x w chitsanase inclusin bdy ( 80%) ( 20%) z ƒƒ x. w chitsanase w» w x y s q wš, Ni 2+ -NTA clumn ladingw z 50 mm imidazle buffer w, 250 mm imidazle buffer elutinw w chitsanase w. w z 10% SDS-PAGE w, 30 kda
146 Chan-S Park et al. Kr. J. Micrbil Fig. 3. Amin acid sequence cmparisn f chitsanases frm B. amylliquefaciens MJ-1 (BAC_MJ-1), B. subtilis 168 (BAC_SUB), Streptmyces sp. N174 (STR_N174) and B. ehimensis EAG1 (BAC_EHI). Sequence alignment was dne using the CLUSTAL W prgram. Identical residues in all sequences are indicated by asterisks. Gaps are indicated by bars. The amin acid residues that seem t be essential fr the chitsanase activity are shwn n a black backgrund (2). chitsanaseƒ y w (Fig. 4, lane 4). Bradfrd w, w z z p w. Ni 2+ -NTA clumn ladingw» z y 0.4 unit/mg, Ni 2+ - NTA clumn z y 120 unit/mg ùkû. w chitsanase ph w p Ni 2+ -NTA clumn w chitsanase 50 mm ptassium phsphate (ph 6.5) 4C 24 n w z, ph w z p w. w chitsanase z y e w» w, 20 C 80 C¾ ƒ z y d w, z y 60 C ùkû (Fig. 5A)., B. subtilis chitsanase(18), Jang (6) Kim (9) šw Bacillus sp. chitsanase Yn (27) šw thermstable chitsanase w. z 80 C 75% z y ùkü. wr, z w» w, 20 C 80 C Fig. 4. SDS-PAGE analysis f chitsanase prtein frm the recmbinant E. cli. Lane 1, prestained prtein marker; Lane 2, E. cli cell lysate befre IPTG inductin; Lane 3, E. cli cell lysate after IPTG inductin; Lane 4, purified chitsanase using Ni 2+ -NTA clumn. ¾ ƒ z 30 wš þ ƒ k z, 37 C z y d w. Fig. 5
Vl. 42, N. 2 Bacillus amylliquefaciens MJ-1 chitsanase 147 (A) ùkù, 50 C 30 ¾ z y 90% ù 60 C y, 80 C 30 ew y 50% ¾ w.», Yn (27) šw Bacillus sp. š chitsanase û r ù, Rivas (18) šw B. subtilis chitsanase z, Jang (6) šw B. cereus chitsanase z r., mw y w B. amylliquefaciens MJ-1 chitsanase ü z w. z y e ph w w» w, z ph 2.0~12.0¾ ƒ ƒwš 37 C» k z, z y d w. Fig. 5 (B) ph 5.0 z y ƒ w ùkû, ph 7.0 y w., Kim (9) šw Bacillus sp. chitsanase ph w. wr, ph z w» w, ph 2.0 ph 12.0¾ phƒ Fig. 5. Enzyme prperties f the purified recmbinant chitsanase. (A) temperature effect ( ø ) and thermstability ( ù ), (B) ph effect ( ý ) and ph stability ( þ ). w z ywwš 4 C 24 w z, z y d w. Fig. 5 (B), ph 5 ~7 80% y w. mw y w chitsanase m z t w ³ l w z ü ü p ùkü. z y w, chitsan z» p, z w chitsan ligsaccharide, z y e» w w ƒ w. š x 1. Akiyama, K., T. Fujita, K. Kurshima, T. Sakane, A. Ykta, and R. Takata. 1999. Purificatin and gene clning f a chitsanase frm Bacillus ehimensis EAG1. J. Bisci. Bieng. 87, 383-385. 2. Bucher, I., T. Fukamiz, Y. Hnda, G.E. Willick, W.A. Neugebauer, and R. Brzezinski. 1995. Site-directed mutagenesis f evlutinary cnserved carbxylic amin acids in the chitsanase frm Streptmyces sp. N174 reveals tw residues essential fr catalysis. J. Bil. Chem. 270(52), 31077-31082. 3. Chi, Y.J., E.J. Kim, Z. Pia, Y.C. Yun, and Y.C. Shin. 2004. Purificatin and characterizatin f chitsanase frm Bacillus sp. strain KCTC 0377BP and its applicatin fr the prductin f chitsan ligsaccharides. Appl. Envirn. Micrbil. 70(8), 4522-4531. 4. Hiran, S., M. Hayashi, K. Miura, H. Tsuchida, and T. Nishida. 1988. Chitsan and its derivatives as activatrs f plant tissues and seeds. Plym. Sci. Technl. 88, 45-60. 5. Hrwitz, S.T., S. Rseman, and H.T. Blunenthal. 1957. The preparatin f glucsamine ligsaccharide I. separatin. J. Am. Chem. Sc. 79, 5064-5049. 6. Jang, H.-K., J.-H. Yi, J.-T. Kim, K.-E. Lee, and S.-G. Chi. 2003. Purificatin, characterizatin, and gene clning f chitsanase frm Bacillus cereus H-1. Kr. J. Micrbil. Bitechnl. 31(3), 216-223. 7. The Japanese Sciety fr chitin and chitsan. 1990. Applicatin f chitin and chitsan. p. 71-98. Kibdang Publisher, Tky, Japan. 8. J, Y.-Y., K.-J. J, Y.-L. Jin, W.-J. Jung, J.-H. Kuk, K.-Y. Kim, T.- H. Kim, and R.-D. Park. 2003. Characterizatin f endchitsanases-prducing Bacillus cereus P16. J. Micrbil. Bitechnl. 13, 960-968. 9. Kim, P.-I., T. -H. Kang, K.-J. Chung, I.-S. Kim, and K.-C. Chung. 2004. Purificatin f a cnstitutive chitsanase prduced by Bacillus sp. MET 1299 with clning and expressin f the gene. FEMS Micrbil. Lett. 240, 31-39. 10. Kurakake, M., S. Yu, K. Nakagawa, M. Sugihara, and T. Kmaki. 2000. Prperties f chitsanase frm Bacillus cereus S1. Curr. Micrbil. 40, 6-9. 11. Laemmli, U.K. 1970. Cleavage f structural prtein during the assembly f the head f bacteriphage T4. Nature. 227, 680-685 12. Matsuda, Y., Y. Idia, T. Shingi, K. Kakutani, T. Nnmura, and H. Tyda. 2001. In vitr suppressin f mycelial grwth f Fusarium xysprum by extracellular chitsanase f Sphingbacterium multivrum and clning f the chitsanase gene csnsm1. J. Gen. Plant Pathl. 67, 318-323. 13. Miller, L. 1987. Use f dinitrsalicylic acid reagent fr determinatin f reducing sugar. Anal. Chem. 31, 425-431.
148 Chan-S Park et al. Kr. J. Micrbil 14. Pantalene, D., M. Yalpani, and M. Scllar. 1992. Unusual susceptibility f chitsan t enzyme hydrlysis. Carbhydr. Res. 237, 325-332. 15. Park, R.-D., Y.-Y. J, H.-C. Lee, C.-S. Ch, and D.-H. J. 1998. Endchitsanase prduced by Bacillus sp. P21 as a ptential surce fr the prductin f chitligsaccharides. Kr. J. Appl. Micrbil. Bitechnl. 26(4), 345-351. 16. Park, Y.-M., H.-L. Chang, T.-L. Hur, and S.-Y. Ghim. 2004. Mlecular clning f chitsanase gene and quantitative prductin f chitsan ligmer. Kr. J. Micrbil. Bitechnl. 32(1), 16-21. 17. Pavlva, S.I., A.O. Kilic, S.S. Kilic, J.-S. S, M.E. Nader-Macias, J.A. Simes, and L. Ta. 2002. Genetic diversity f vaginal lactbacillus frm wmen in different cuntries based n 16S rrna gene sequences. J. Appl. Micrbil. 92, 451-459. 18. Rivas, L.A., V. Parr, M. Mren-Paz, and R.P. Mellad. 2000. The Bacillus subtilis 168 csn gene encdes a chitsanase with similar prperties t a Streptmyces enzyme. Micrbil-SGM. 146, 2929-2936. 19. Shn, J. H., J.H. Lee, H.Yi, J. Chun, K.S. Bae, T.Y. Ahn, and S.J. Kim. 2004. Krdia algicida gen. nv., sp. nv., an algicidal bacterium islated frm red tide. Int. J. Syst. Evl. Micrbil. 54, 675-680. 20. Smashekar, D., and R. Jseph. 1996. Chitsanase-prperties and applicatins: a review. Biresurce Technl. 55, 35-45. 21. Sugan, N., K. Yshida, M. Hashimt, K. Enmt, and S. Hiran. 1992. Hyperchlestrlemic activity f partially hydrlyzed chitsan. Advances in chitin and chitsan. p472-478. In C. J. Brine. P. A. Standfrd and J. P. Zikakis(eds). Elsevier. 22. Tanabe, T., K. Mrinaga, T. Fukamiz, and M. Mitsutmi. 2003. Nvel chitsanase frm Streptmyces griseus HUT6037. Bisci. Bitechnl. Bichem. 67, 354-364. 23. Tremblay, H., J. Blanchard, and R. Brzezinski. 2000. A cmmn mlecular signature unifies the chitsanases belnging t families 46 and 80 f glycside hydrlases. Can. J. Micrbil. 46(10), 952-955. 24. Yabuki, M., M. Hiran, A. And, T. Fujii, and Y. Amemiya. 1987. Islatin and characterizatin f a chitsan degrading bacterium and frmatin f chitsanase by the islate. Tech. Bull. Fac. Hrt. Chiba Univ. 39, 23-27. 25. Yi, J.-H., H.-K. Jang, S.-J. Lee, K.-E. Lee, and S.-E. Chi. 2004. Purificatin and prperties f chitsanase frm chitinlytic β-prtebacterium KNU3. J. Micrbil. Bitechnl. 14(2), 337-343. 26. Yn, H.-G., H.-Y. Kim, Y.-H. Lim, H.-K. Kim, D.-H. Shin, B.-S. Hng, and H.-Y, Ch. 2000. Thermstabe chitsanase frm Bacillus sp. strain CK4: Clning and expressin f the gene and characterizatin f the enzyme. Appl. Envirn. Micrbil. 66(9), 3727-3734. 27. Yn, H.-G., H.-Y. Kim, H.-K. Kim, B.-S. Hng, D.-H. Shin, and H.-Y. Ch. 2001. Thermstable chitsanase frm Bacillus sp. strain CK4: its purificatin, characterizatin, and reactin patterns. Bisci. Bitechnl. Bichem. 65(4), 802-809. 28. Zhu, X.-F., X.-Y. Wu, and Y. Dai. 2003. Fermentatin cnditin and prperties f chitsanase frm Acinetbacter sp. C-17. Bisci. Bitechnl. Bichem. 67, 284-290. (Received May 22, 2006/Accepted June 13, 2006) ABSTRACT : Mlecular Clning and Characterizatin f Chitsanase Gene frm Bacillus amylliquefaciens MJ-1 Chan-S Park 1, Hae-Geun Oh 1, Sn-Kwang Hng 2, Byung Chul Park 3, Yung Hyun 1 *, and Dae-Kyung Kang* (Department f Animal Resurce and Science, Dankk University, Chenan 330-714, Krea, 1 Bi-Resurces Institute, EASY BIO System, Inc, Chenan 330-812, Krea, 2 Department f Bilgical Sciences, Myngji University, Yngin 449-728, Krea, 3 NUTRABIO, Yksam-dng, Seul 135-080, Krea) In rder t develp chitsanase fr the prductin f chitsan ligsaccharides, a chitsanase-prducing bacterium was islated frm the traditinal fermented sybean, Meju, and identified as Bacillus amylliquefaciens MJ-1. The clned chitsanase gene, 825 bp in size, encded a single peptide f 274 amin acids with a estimated mlecular mass f 30.9 kda. The deduced amin acid sequence shwed significant hmlgy with micrbial chitsanases. The recmbinant chitsanase was expressed in Escherichia cli upn inductin with isprpyl-d-thigalactpyranside, and purified using Ni 2+ -NTA agarse clumn chrmatgraphy. The maximal activity f the recmbinant chitsamase is at ph 5.0 and 60 C. The recmbinant chitsanase is stable between ph 5.0 and ph 7.0 at 37 C fr 30 min, and mre than 75% f the activity still remain at 80 C fr 30 min incubatin.