枯草桿菌屬細菌群體計畫---枯草桿菌屬細菌發酵系統之研究(II)
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(4) . ! "#$ %&'()*+,-./ Bacillus subtilis.
(5) ! " #$ % & Bacillus subtilis '()* +,./0123456789:;< =>?@A:B CDE+F G:;HIJKLMNOPQ'(R STUV:WXYZ[\(]^_ )`#abAc(]^_)defgh ijklmn# NaCl 0.3g/lo K2HPO4 0.4g/l o CaCl20.2g/l o MgSO4 0.5g/l o FeSO4 30mg/lo MnSO4 20mg/l8cpG '(qYLrAst89u*RS >vwxyz{|o(]^_)d}Bg h8~ D.O.G 10~20% A Gxyt89~RS -e&:Emn F OD600 , 154 :E(biomass), 54 g/lc #$ DNA recE <=;3 4 Kmr d Cmr b ¡ recE Y+F¢£¤¥¢£ recE ¦ DB430 Y9 double crossover recombinant ¡ recE §¨ -© Cmr recE <=;ªY ª«#¬®¯°A VP1 d VP3 b±*²# 25kDaN v³´ VP1 d VP3 ² FY VP3 G DB430 Yµ~Q'( -¶·M 1.4 ¸ª¹ Restriction - Modification system ºE IJ 34¹N E coli RY13 EcoRI A R-M system1,2,3,4 »¼-E ½TUV:o~Q'( . . Abstract Bacillus subtilis has many advantages for biotechnology that including nonpathogenic, easy to cultivate, store and secrete proteins. Therefore, it has been used to product the recombinant DNA proteins. The goal of this study is to improve the biomass as will as recombinant proteins production ability in Bacillus subtilis. In order to obtain the high cell density, the optimization of fed-batch culture was studied. The experiment was carried out by using glucose as carbon source and yeast extract as nitrogen source. The parameters of control fermentation have been examined which includes how to manipulate carbon and nitrogen sources feeding stratagem, agitation and aerate, as well as trace elements supply. The results showed that the optimal glucose concentration is 0.5%(w/v), and the yeast extract using rate is 1%(w/v) when O.D.600 value increased 8 g. When Bacillus subtilis DB430 grew in 2.5L fermenter at 37 and 700rpm of stir rate and agitation of 50% of oxygen into culture, the cell density was as high as 154 (O.D.600) and the cell dry weight (biomass) achieved to 54g/l. The highest expression of recombinant VP3 protein was 1.4g/l in fed-batch culture at 37, 700rpm of stir rate. To protect the recombinant proteins, a recE mutant was also constract in Bacillus subtilis DB430. Keywords: Bacillus subtilis, fermentation
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(7) . RSY 89bùQRS Gû[b TUV:>z{|o | (fructose) d| (sucrose) RS6 ñ±óòÑ5Z[ Y> soytoneo polypeptone d yeast extract RS DB428 >-&: Emn >Bû[ïZ[ ýßRS¤89:ERSGR S6'5Zoû :EAð öu !FY> soytone dz{|#û[ïZ[ :Emn&G>WX Y "> soytone d yeast extract #ç# Z[>89Gè(qYRSWX. Bacillus spp. #¾¿ÀÁKVÂà :Ä Å
(8) 5 Bacillus spp. ¹ Æ
(9) B 1 Ç ( È É amylase, protease, isomerase Ê#TUV:Ë. LrÊÌKdbÍΪ(ÈÏÐÑ Ò)ÓÔÕÖ >!×"Ø ÙÚÛ.ÜÝ 'Þ_)ßà á â:; ,ã>TUV:89 A #$äTUV:å# :; 34>TU V:æçè(éê#!× ëìíîæ çè(estïEGðÜYA IJK >dñ±òãóò Ò recE TUV:<=;>ôõª« =ö(5)÷ø>bùQè(ï~ Qè(RSTUV: eú!GÆ? &:* >äÆu* ª#$+TUV: (Bacillus subtilus) '(Ï,-./012 G¹e f û[ dZ [ü ý «þ x' ( s tÉ5'(Qoo *oij}BghmndûZ[mn Ê áävwå«,-0xy& n'(!!×"%& DNA ï* >Ï ,-12 1. B. subtilis . 2. !"# pH $%&' B. subtilis ()*+ B. subtilis #c$%:; 89 bùQRS6 RSY&%'( ÷)*Fð GWXYC=RS: E6+,>C=RSY&% DB428 b> 200 rpm d 100 rpm B»89bùQRS » # 200 rpm 6 DB428 ðóò »# 100 rpm 6$ &% -& DB428 ðó.-$ ªRS6/n0'1:E ð'Dã)* WX DB428 b> 252o 302 d 372 B5/n89 bùQRS 3>'G 372 RS DB428 6 Fðó.f Ñ> 25 2 6e 3G> soytone d yeast extract #Z[6"5ðG pH 7 RSY:Emn& ÑG pH 6.5 RSY:Eðe . #$ ÇG Bû[ïZ[R S B. subtilis >-e&:Em n G ã DB428 RSÆ>z{ |#û[üý5Z[ d> yeast extract
(10) #Z[ ¤üýF û[ã 2.
(11) `C= >,-e&:En! . 3. ,-./0/' B. subtilis 123 45 (plasmid stability) *+. > DB428 # aRSG 600 rpm d 700 rpm >5-ð öGB» O.D.600 e&""_ Gbc6 !G:* 700 rpm = 600 rpm d&_$ 20%ef eúg* D 700 rpm #¶·M 22 ¸= 600 rpm #¶·M 16 ¸
(12) $ 30%ef Ûh 1Ã:ð u)L. 4E«5 G5SbRS E pUB110 IJKe& Ñ pE194 I JKe ªZ[Y> tryptone RS: E6 EIJK6789 IJKG 50% > Ñ5:RS 60 ; E pUB110 d pC194 I J < G Æ æ ç DB430 =RSÆ DB428 &. Ûi«
(13) #89~'(6Z [ijjk3l O.D.600 Ni« mGbndbc6bj 1% ( W/ V )(]^_) 100 ef %&$ 150 ef 7N 28 ¸LM- 54 ¸ Ñ:D7LM$ 30%o . 4. 6789:; DB428 DB430 <=> ?@() DB430 > DB428 G?@AAð B !c8CDA( log phase ) DB430 ðEÆ DB428 ÑÒ Fð6 7GÛWXHJ DB430 I #
(14) < =c J Ñ1ð K)L MÑ+ð @E. 6. BC recE DEF GWXpY ¹ãq © Cmr ï Kmr # marker recE <= ¹F»¼- pUB110 L W X Y > double cross-over recombinant Ýãq© Cmr recE <=r§^s DB430 :; tuEL. 5. 89:;A?@%& 34bþ4cNMOst FYP © ooE© %*oûoZ[Q~Qo6 odm noRoSÊTUV±*WX> O.D.600 ï6 WXå´ «YZþ4} stA5F +RS# [RS\ ûoZ[klmnb# 2%( W/V )d 5%( W/V )Az{|d(] ^_) RSst# 372opH7od÷ ];RS^CDAB: +'(kl stÛ O.D.600 # 0.1 _l F st. 7. GHIJ"' DB430 ?@*K 34äz{|LJv«xy '( +'(ÜYz{|mnc J YZ&9z{|mn1Ã:ð )*WX>1.5·M'(q89N3 YYZ-z{|mnwG¶·M5¸ 3.
(15) PQ . åO.D.600 #140o:E# ¶·M42¸ z{|mnwG¶·M10 ¸O.D.600 #118o:E#¶·M29 ¸ jD.O.=0~jz{|'( 3O.D.600#133o:E#¶·M43 ¸. 1. ,-0/#./' B. subtilis () *+ DB428 RSÆ5Zû[ '> soytoneo yeast extract ý F û[-:Emn&> GbùQï~QRSY Jb> yeast extracto soytone dz{|#eR Sã«RS B. subtilis >-e &A:Emn. 8. ,-?@%&' DB430 ?@*K 5'(stïQ1:En. ø u öùQ'(Ï-: Emn=~Q'(x$abAyc z {|+z{|d(]^_)A g*"c:}ó.7_G+ab Ac%L 5:">~Q'(! e:Emn+abAc% 6=~&_$abAc:Em n. 2. !"# pH $%&' B. subtilis ()*+ ëì&%*b G+,RS Y> 200 rpm +,nRS DB428 6 F ðóò 100 rpm #fGRS/n > 372 # B. subtilis Ae ð/n ªG5 pH RS z{ RS\ pH G:Ï N Å=0 !uL> pH 7.0 ef#e fRSst. 9. LM1( VP1 # VP3 )NO WX+E «[ c" pUB110 ¬®(VP3) c " pQE30/VP1 d pDH87-VP1-R |" ¬® (VP1)5RSQ d5E89RS Ô:E> lysozym TUV:&> 12000 xg V ^>JÝJ _¶:g* Ê*^ 89 SDS-PAGE Av³b Û Æ VP3 b±*d 25 kDa Ñ VP1 b±*7ud"d 25 kDaNv ³b3> DB430 > DB428 G5RSQüý5E" *d 25 kDa A VP1 ï VP3 . 3. ,-./0/' B. subtilis RS (Sporulation) *+ Û3'>z{|#û[6 G ij5Z[RS B. subtilis 6 F ñóò" Ï"#z{|< G 6 ' y ñ ± ò ã (catabolic repression) (6)Ñ> yeast extract #Z[ 6 j5û[RS > maltose ï (starch) #û[RS6 ãñ ±óò# !ïg:D= ñ 4.
(16) ±ã=9. xyjz{|A( O.D.600 # 133 :E# 43 ¸=A ö & mn z{ |' )* :E ð +eú*¬&mn z{|'©TUV:yð ) :Emn-&*-
(17) } By®O"G O.D.600 # 70 ef¯_ !G89&mnz{| 1% ( w / v )' (6 Ã:ð?@A=° ±8 CDA }lG~Q'(²A³ > 1%~2%( w / v )z{| G O.D.600 M^ 50 ef6z{|mn¬^ 0.5%( w / v )> >-=G@&*. 4. ,-./0/' B. subtilis 123 45 (plasmid stability) *+ NWX3E pUB110 B GRS 30 á 60 ; FE<G= G 50% >L ^u b ² G 80-100% A $> tryptone #Z[ AIJK9>ª GFRS ãYIJK & 3 "> DB428 á DB430 #æç6 5WL Û E pUB110 B BÈ<G RS 80 ; G: EY© 50 copy number (7) #cIJ<GÆ B. subtilis :E YAE. 6. VP3 # VP1 LM1NO G VP3 d VP1 Û3>~QRSG5R SstA:E yb±*A !34´# VP3 d VP1 Eå8cp westem blotting «µWd ¶·* >b±* 25kDa Av³ ´d¸¹¶¶c·M'(\ 1.4 ¸ VP3 UVWX. 5. <=T>'89:; DB430 ?@* + z{|~"> D.O.d pH =0«xy Ã:89å6; eE"z{| ÑÒ'z {|6å D.O.'E LM pH 7 ¡LM>G D.O. ELMA¢£¤¥L¦§z{| É ¦§¨©Ã:ðªk3 34«G~Q'(Y z{|e9 mn#abA 0.3 -abA 0.5 ( w / v )A äLz{|Jv z{ |mnxyG 5 g/L d 10 g/L L89 '( ª9mnz{|'(ã=& mnz{|$'(ã9mnz{ |'( O.D.600 # 118o:E # 29 ¸ >&mnz{|'( O.D.600 # 140o:E# 41 ¸ D.O.. [1] Kulakauskas,S.,A. Lubys, and S.D.Ehtlich., J.Bacteriol, 177(12) :3451- 3454, 1995. [2] Lubys, A., A. Janulaitis, Gene. 157:25-29, 1995. [3] Naito, T., K. Kusano, and I. Kobayashi, Science, 267(10):897-899, 1995. [4] Yarmolinsky, M.B.,.Science, 267(10):836-837, 1995. [5] Ceglowski, P., G. Luder, and J. C. Alonso. 5.
(18) 1990. Genetic analysis of rec E activities in Bacillus subtilis. Mol. Gen. Genet. 222:411-445. [6] Kobayashi, Y. 1989. Sporulation of B. subtilis. Bacillus subtilis:Molecular Biology and Industrial Application. p.43-54. [7] Lovett, L. M., JR., P. E. Love, and R. E. Yasbin. 1989. Competence-specific induction of the Bacillus subtilis recA protein analog: evidence for dual regulation of a recombination protein. J. Bacteriol. 171:2318-2322. . 6.
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