(H yaluronic acid, HA ) , HA,. HA,, HA. N T G (S trep tococcus zooep id em icus J 18), 50 g
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1 V ol. 25 N o CHEM ICAL JOU RNAL O F CH IN ESE UN IV ERS IT IES , 1, 1, 1, 2, 1 (1., ; 2., ) N 2 2N 2 2N 2 (N TG),., Savage D EA E2 (D E52) Sephadex G275., (S trep tococcus zooep id em icus J 18),., ; ; ; O 657 A (2004) (H yaluronic acid, HA ), [1 ]. [2 ]. [3 5 ].,, HA HA, HA, HA [6 9 ]. HA. HA [2 ].,, HA , HA,, K im [10 ] N T G, Β2, 6 7 ggl. HA,, HA, HA ggl [11 ]. N 2 2N 2 2N 2 (N T G), (S trep tococcus zooep id em icus J18) [12 ], 2. 0 ggl.,,, HA, S trep tococcus zooep id em icus J18 [12 ]. D EA E2 (D E52) SephadexG275, HA, Sigm a,. B ruker IFS66V ; B ruker AV 400 ; J2810 ; Sh im adzu UV ; Ch rist alpha N T G (S trep tococcus zooep id em icus J 18), 50 g : : ( : 2002, ) ( : ). : (1942 ),,,,. E2m ail: ying@public. cc. jl. cn
2 854 V ol g N ac l 5 g, K 2H PO g, KH 2PO g, M gso g SD S g [12 ]. 250 ml 25 ml, 200 rgm in h rgm in 10 m in,, 2. 3,, 10 m in, HA. [13 ] HA. [14 ], m g, 5 ml 6 molgl HC l, 2 h, N aoh ph 7. 0, 50 ml. E lson2m organ [15 ] D m g, 100 ml, [13 ] C NM R M H z, Λs, 5 Λs,, 5 000, D 2O, 5%, KB r, B ruker IFS66V, cm - 1. HA 1 m ggml, nm, 200 nm gm in molgl, HA 5 m ggml. N aoh ( molgl ). [ 16 ] HA 1 1, N aoh, 15 m in, N aoh ( ), nm HA. [ 17 ], 0. 5 mm, 25, 0. 2 molgl N ac l, HA ggml, t0 ( ) ti ( ), Γr, Γsp [Γ], Savage HA, HA 2. 0 ggl ml 500 ml, Savage [18 ]. 500 ml g [V ( ) V ( ) = 4 1 ],, rgm in 20 m in,,, 3,. 2. 3, rgm in 10 m in,, P 2O 5, g ml, 4% (CPC ),. 1 h, rgm in 10 m in, 100 ml 1. 5 molgl N ac l,. 2. 3, P 2O g D EA E2 100 ml, D EA E2 (D E52) (2. 5 cm 20 cm ) molgl N ac l, 1 ml gm in, ( 5 ml ),, 1.,,
3 N o. 5 : 855, g. F ig. 1 Elution curve of polysacchar ide on F ig. 2 Elution curve of polysacchar ide af ter gel D EAE-cellulose column f iltra tion on Sephadex G-75 column Sephadex G %, Sephadex G2 75 (1. 5 cm 120 cm ), 2. 0 ml,, 6 ml gh,, 2., g. Savage D EA E2 SephadexG275, %, % ΛggmL, ΛggmL, w 1 (% ) = [ (c1 V 1) gm ] 100% (w 1 ; c1 ; V 1 ; m ), 45. 8%. E lson2m organ N ΛggmL, (10 ΛggmL ) 525 nm, w 2 (% ) = [ (A c1) g(a c2) ] 100% (w 2 ; A ; c2 ; A ; c1 ; ) 45. 9%. N 1gN 2 = [ (w 1gM r 1 ) g(w 2g M r 2 ) ] (N 1gN 2 ; M r 1 ; M r 2 ), , HA , 3. COO , COO ; O (O )CH ; > CHOH ; > CHN H ; CH 2OH , CH C NM R,. F ig C NM R spectrum of the pur if ied polysacchar ide F ig. 4 IR spectra of the pur if ied polysacchar ide(a) and standard HA (b)
4 856 V ol cm - 1 OH ; cm - 1, CH 2 CH 3 C H ; cm - 1 C O C N, ; cm - 1 COO C O OH 2,. HA , Cotton. ( ) ( ), 230 nm. Χ2Π ( nm ), Cotton, Cotton Π2 Π 3. O 24 Π2Π 3 Cotton, O 23 Π2Π 3 Cotton [19 ]. HA, 5., nm Cotton, nm Cotton, O 23, HA,. F ig. 5 C ircular dichro ic spectra of the pur if ied HA and standard HA a. Purified polysaccharide; b. standard HA. F ig. 6 Change in the absorption max ium of the Congo red-ha com plex a. Congo red2ha comp lex; b. Congo red (Congo red), C 32H 22N 6O 6S2N a2,, N aoh,. [20 ]. HA 6,,,. HA COO -,,. [21 ], HA. HA, HA HA [Γ]= , [Γ]= M r HA ,, HA,, [ 1 ] L ap ik L. J ṙ, L ap ik L., De Sm edt S. et al.. Chem. Rev. [J ], 1998, 98 (8) : [ 2 ] ZHOU Rong2Q ing ( ), L IL in ( ), GUO Si2Yuan ( ). Sugarcane and Canesugar ( ) [J ], 2001, (6) : [ 3 ] A kim a K., Ito H., Iw ata Y. et al.. J. D rug Target[J ], 1996, 4 (1) : 1 8 [ 4 ] M uckenschnabel İ, Bernhardt G., Sp russ T. et al.. Cancer Chemotheṙ Pharm acol. [J ], 1996: 38 (1) : 88 94
5 N o. 5 : 857 [ 5 ] Pouyani T., P restw ich G. D.. B ioconjug. Chem. [J ], 1994, 5 (4) : [ 6 ] A laniz L., Cabrera P. V., B lanco G. et al.. Cell Comm un. A dheṡ [J ], 2002, 9 (3) : [ 7 ] Sy M. S., L iu D., Schiavone R. et al.. Curṙ Top M icrobiol. Imm unol. [J ], 1996, 213: [ 8 ] Knudson W.. Am. J. Pathol. [J ], 1996, 148 (6) : [ 9 ] Entw istle J., Hall C. L., Turley E. A.. J. Cell B iochem. [J ], 1996, 61 (4) : [ 10 ] Kim J. H., Yoo S. J., O h D. K. et al.. Enzym e and M icrob. Technol. [J ], 1996, 19: [ 11 ] LUO Rui2M ing ( ), GUO M ei2j in ( ), CHU Ju ( ) et al.. Journal ofw uxiu niversity of L ight Industry ( ) [J ], 2003, 22 (1) : [12 ] M EN G Q ing2fan ( ), TEN G L i2rong ( ), HON G Shui2Sheng ( ) et al.. Journal of J ilin U niversity, Science Edition (, ) [J ], 2004, 42 (1) : [ 13 ] B itter T., M uir H. M.. A nal. B iochem. [J ], 1962, 4: [ 14 ] B radford M. M.. A nal. B iochem. [J ], 1976, 72: [ 15 ] E lson L. A., M organ W. T. J.. B iochem. J. [J ], 1933, 27: [ 16 ] B lunhm T. L., Sarko A.. B iopolym ers[j ], 1977, 16 (9) : [ 17 ] L aurent T. C., Ryan M., P ietruszkiew icz A.. B iochim. B iophyṡ [J ], 1960, 42: [ 18 ] Staub A. N.. M ethods in Carbohydrochem. [J ], 1956, 40: [ 19 ] WU Dong2Ru ( ). Saccharide B iochem istry ( ) [M ], Beijing: H igher Education P ress, 1987: [ 20 ] O hno N., Yadom ae T.. Carbohydṙ Reṡ [J ], 1987, 159 (2) : [ 21 ] Kulicke W. M., L ettan A. İ, Thielking H.. Carbonhydṙ Reṡ [J ], 1997, 297 (2) : Pur if ication and Structure Character ization of the Hyaluron ic Ac id Produced by a Streptococcus Zooep idem icusm uta tion Stra in HON G Shui2Sheng 1, CH EN J ia 1, T EN G L i2rong 1, M EN G Q ing2fan 1, ZHAN G J ian2guo 2, L IU L an2ying 13 (1. Colleg e of L if e S cience, J ilin U niversity, Chang chun ; 2. S tate K ey L aboratory of E lectroanaly tical Chem istry, Chinese A cad am y of S ciences, Chang chun Institute of A p p lied Chem istry, Chang chun , China) Abstract A m utated strain of S trep tococcus zooep id em icus capable of p roducing the polysaccharide in a h igh yield w as obtained after being m utated by the chem ical agent N 2m ethyl2n 2nitro2N 2 nitrosoguanidine (N T G). Polysaccharide p roduced by the m utated strain of S trep tococcus zooep id em icus w as purified by the p rocedure including Savage m ethod, quaternary amm on ium com pound p recip itation, D EA E2cellulo se (D E52) colum n ch rom atograph and Sephadex G275 gel filtration. T he purified polysaccharide w as p roved to be hyaluronic acid (HA ) by m eans of chem ical composition analysis, 13 C NM R, Infrared spectra, and circular dich ro ism. T he single helix confo rm ation of the purified HA w as studied by Congo red experim enṫ m ethod. T he molecular w eigh t of the HA w as about , w h ich w as m easured by viscosity Keywords S trep tococcus zooep id em icus; H yaluronic acid; Separation and purification; Characterization (Ed. : H, J, Z)
PUR IF ICATION AND CHARACTER IZATION OF EXTRACELL ULAR PROTEASE ECPASE 54 FROM A EROM ONAS H YD RO PH ILA
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