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1 The Krean Jurnal f Micrbilgy, Vl. 42, N. 2, June 2006, p 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: , Fax: , dkkang@dankk.ac.kr) Yung Hyun (Tel: , Fax: , 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, % ZnSO 4, % 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

2 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.

3 144 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 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.

4 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

5 146 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

6 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 Purificatin and gene clning f a chitsanase frm Bacillus ehimensis EAG1. J. Bisci. Bieng. 87, Bucher, I., T. Fukamiz, Y. Hnda, G.E. Willick, W.A. Neugebauer, and R. Brzezinski 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), Chi, Y.J., E.J. Kim, Z. Pia, Y.C. Yun, and Y.C. Shin 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), Hiran, S., M. Hayashi, K. Miura, H. Tsuchida, and T. Nishida Chitsan and its derivatives as activatrs f plant tissues and seeds. Plym. Sci. Technl. 88, Hrwitz, S.T., S. Rseman, and H.T. Blunenthal The preparatin f glucsamine ligsaccharide I. separatin. J. Am. Chem. Sc. 79, Jang, H.-K., J.-H. Yi, J.-T. Kim, K.-E. Lee, and S.-G. Chi Purificatin, characterizatin, and gene clning f chitsanase frm Bacillus cereus H-1. Kr. J. Micrbil. Bitechnl. 31(3), The Japanese Sciety fr chitin and chitsan Applicatin f chitin and chitsan. p 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 Characterizatin f endchitsanases-prducing Bacillus cereus P16. J. Micrbil. Bitechnl. 13, Kim, P.-I., T. -H. Kang, K.-J. Chung, I.-S. Kim, and K.-C. Chung Purificatin f a cnstitutive chitsanase prduced by Bacillus sp. MET 1299 with clning and expressin f the gene. FEMS Micrbil. Lett. 240, Kurakake, M., S. Yu, K. Nakagawa, M. Sugihara, and T. Kmaki Prperties f chitsanase frm Bacillus cereus S1. Curr. Micrbil. 40, Laemmli, U.K Cleavage f structural prtein during the assembly f the head f bacteriphage T4. Nature. 227, Matsuda, Y., Y. Idia, T. Shingi, K. Kakutani, T. Nnmura, and H. Tyda 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, Miller, L Use f dinitrsalicylic acid reagent fr determinatin f reducing sugar. Anal. Chem. 31,

7 148 Chan-S Park et al. Kr. J. Micrbil 14. Pantalene, D., M. Yalpani, and M. Scllar Unusual susceptibility f chitsan t enzyme hydrlysis. Carbhydr. Res. 237, Park, R.-D., Y.-Y. J, H.-C. Lee, C.-S. Ch, and D.-H. J Endchitsanase prduced by Bacillus sp. P21 as a ptential surce fr the prductin f chitligsaccharides. Kr. J. Appl. Micrbil. Bitechnl. 26(4), Park, Y.-M., H.-L. Chang, T.-L. Hur, and S.-Y. Ghim Mlecular clning f chitsanase gene and quantitative prductin f chitsan ligmer. Kr. J. Micrbil. Bitechnl. 32(1), Pavlva, S.I., A.O. Kilic, S.S. Kilic, J.-S. S, M.E. Nader-Macias, J.A. Simes, and L. Ta Genetic diversity f vaginal lactbacillus frm wmen in different cuntries based n 16S rrna gene sequences. J. Appl. Micrbil. 92, Rivas, L.A., V. Parr, M. Mren-Paz, and R.P. Mellad The Bacillus subtilis 168 csn gene encdes a chitsanase with similar prperties t a Streptmyces enzyme. Micrbil-SGM. 146, Shn, J. H., J.H. Lee, H.Yi, J. Chun, K.S. Bae, T.Y. Ahn, and S.J. Kim Krdia algicida gen. nv., sp. nv., an algicidal bacterium islated frm red tide. Int. J. Syst. Evl. Micrbil. 54, Smashekar, D., and R. Jseph Chitsanase-prperties and applicatins: a review. Biresurce Technl. 55, Sugan, N., K. Yshida, M. Hashimt, K. Enmt, and S. Hiran Hyperchlestrlemic activity f partially hydrlyzed chitsan. Advances in chitin and chitsan. p In C. J. Brine. P. A. Standfrd and J. P. Zikakis(eds). Elsevier. 22. Tanabe, T., K. Mrinaga, T. Fukamiz, and M. Mitsutmi Nvel chitsanase frm Streptmyces griseus HUT6037. Bisci. Bitechnl. Bichem. 67, Tremblay, H., J. Blanchard, and R. Brzezinski A cmmn mlecular signature unifies the chitsanases belnging t families 46 and 80 f glycside hydrlases. Can. J. Micrbil. 46(10), Yabuki, M., M. Hiran, A. And, T. Fujii, and Y. Amemiya Islatin and characterizatin f a chitsan degrading bacterium and frmatin f chitsanase by the islate. Tech. Bull. Fac. Hrt. Chiba Univ. 39, Yi, J.-H., H.-K. Jang, S.-J. Lee, K.-E. Lee, and S.-E. Chi Purificatin and prperties f chitsanase frm chitinlytic β-prtebacterium KNU3. J. Micrbil. Bitechnl. 14(2), Yn, H.-G., H.-Y. Kim, Y.-H. Lim, H.-K. Kim, D.-H. Shin, B.-S. Hng, and H.-Y, Ch Thermstabe chitsanase frm Bacillus sp. strain CK4: Clning and expressin f the gene and characterizatin f the enzyme. Appl. Envirn. Micrbil. 66(9), Yn, H.-G., H.-Y. Kim, H.-K. Kim, B.-S. Hng, D.-H. Shin, and H.-Y. Ch Thermstable chitsanase frm Bacillus sp. strain CK4: its purificatin, characterizatin, and reactin patterns. Bisci. Bitechnl. Bichem. 65(4), Zhu, X.-F., X.-Y. Wu, and Y. Dai Fermentatin cnditin and prperties f chitsanase frm Acinetbacter sp. C-17. Bisci. Bitechnl. Bichem. 67, (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 , Krea, 1 Bi-Resurces Institute, EASY BIO System, Inc, Chenan , Krea, 2 Department f Bilgical Sciences, Myngji University, Yngin , Krea, 3 NUTRABIO, Yksam-dng, Seul , 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.