BL21 (D E3)2p E T28a ( + )2bgl 2
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1 Journal of Food Science and Biotechnology 28 No. 2 Vol. Mar : (2009) BL21 (D E3)2p E T28a ( + )2bgl 2,, 3, (, ) : 2E. coli BL21 (DE3)2p ET28a ( + )2bgl LB, p H 2 ;IP T G :p H 710, (OD ) ( ) 10 %,37,200 r/ min 3 h,od 110, mmol/ L IP T G 10 mmol/ L,24 6 h, ( U/ ml), 1112 g/ L, U/ ml, 6162, TB,10 h, U/ ml, U/ ml, , : ; ;;; 2 :Q : A Optimization of Fermentation Conditions of Recombinant E1 coli BL21( DE3)2pET28a( + )2bgl Producing 2Glucanase L I Yong2xian, ZH EN G Fei2yun, L I Qi 3, GU Guo2xian ( The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi , China) Abstract : In t his manuscript, t he optimum co nditio ns for b - glucanase p roductio n f ro m t he reco mbinant st rain : :863 (2006AA020204) E1 coli BL21 ( D E3) 2Pet28a ( + ) 2bgl were caref ully determine and list s as follows : 10 % seed cult ures (OD600 = 0. 35) was supplemented to t he culture medium (initial p H 7. 0) and incubated for 3h when t he cell concentration reaching at OD600 1 under 37 and 200 r/ min. Then mm IP T G and 10 mm lactose were fed as inductor to the E1 coli BL21 (DE3)2 Pet28a ( + )2bgl cult ure brot h and t he temperat ure was shifted to 24 and maintained for 6 h. t he total activity of 2glucanase achieved at U/ ml, and U/ ml 2glucanase was fo und in supernatant liquor, was higher times t han t hat of t he parent st rain. : (19642),,,, 3 :(19712),,,,,, edu. cn In TB
2 2 : BL21 (DE3)2p ET28a ( + )2bgl medium, t he values can achieve at ( U/ ml in supernatant and U/ ml in total). The result s p resented here indicated t hat t he reco mbinant st rain exhibited t he potential application in industrial production. Key words : reco mbinant E. coli, fermentatio n co nditio ns, optimizatio n, inducing, 2glucanase 21,321,4 ( EC ), 2, 2 21,3 21,4, 2, [ 1-2 ] 2,, 2 ( bgl) [3-4 ] [5 ] E1 coli J M1092 pl F3,,30 h U/ ml ; [6 ] EGs2 2, U/ ml B acill us am y loliquef aciens SYB2001 ( 2) 2, E1 coli BL21 (D E3)2p ET28a ( + )2 bgl, laci T7, IP T G 2 2, Tac T7, [7 ] 2,, p H,, IPTG, E1 coli BL21 (D E3)2 p ET28a ( + )2bgl, ( Sigma ),IPTG(BIO BASIC INC), 2( ), (BIO BASIC INC) LB (g/ L) : 10,5, NaCl 10, 0103 ;p H 710,20 g LB TB ( g/ L ) : 12, 24,6,NaCl 10, KH2 PO4 214, K2 HPO4 3 H2 O 1215 ;, 0103 g/ L (g/ L) IP T G 20, : 10 ml LB 100 ml,37,200 r/ min 11 h, :110 ml 25 ml LB 250 ml, 37, 200 r/ min 2 3 h, 11212,,,,37,200 r/ min,25 ml 5 L IP T G 250 L 2,27,200 r/ min 5 h, ( ) IP T G 5 h r/ min 2 min,,,,, 600 nm OD ;10 ml, r/ min 2 min,,,, OD, OD ml LB 250 ml 110 ml,37,200 r/ min, 1 h 600 nm OD ,52 (DNS) :1 ml 40 p H 615, 2 1 mol 1
![coli, fermentatio n co nditio ns, optimizatio n, inducing, 2glucanase 21,321,4 ( EC31211173), 2, 2 21,3 21,4, 2, [ 1-2 ] 2,, 2 ( bgl) [3-4 ] [5 ] E1 coli J M1092 pl F3,,30 h 2 297171 U/ ml ; [6 ]](/docs-images/41/22700183/images/page_2.jpg "EGs2 2, 321156 U/ ml B acill us am y loliquef aciens SYB2001 ( 2) 2, E1 coli BL21 (D E3)2p ET28a ( + )2 bgl, laci T7, IP T G 2 2, Tac T7, [7 ] 2,, p H,, IPTG, 1 1.")
3 252 28, U/ ml ; :,, ; :, 400 W, 1 s 3 s,60 s, 11216,IP T G, 1, L 16 (4 5 ) 1 Tab. 1 Factors and levels of orthogonal experiment A : IPT G / L B : / L C: / D : / h 2 OD Fig. 2 Curve of cell quantity and OD value p H p H , OD 0135, 5 %,37,200 r/ min 3 h, 5 L IP T G 250 L 5 h, r/ min 2 min,, 3,p H 710,, p H , OD,1, OD, OD 1 OD Fig. 1 Relationship between dry weight of cell and OD of value 212 OD 2 2-4, 0-1, 1-2, 2-4,4 h 3 ph Fig. 3 Effect of initial ph on 2glucanase activity OD, 215 % 510 % 10 % 15 % 20 % 25 %,4 4, 10 %, ,,37,200 r/ min 3 h, 5 L IP T G
4 2 : BL21 (DE3)2p ET28a ( + )2bgl L5 h, r/ min 2 min,, 2,,, 3 h 3 Tab. 3 Effect of initial cell concentration on 2glucanase ac2 tivity / h OD / (g/ L) / (U/ ml) Fig. 4 Effect of inoculation volume inoculation on 2 glucanase 2 Tab. 2 Effect of secondary seed age on 2glucanase expressed / h OD / ml LB / ml / (U/ ml) ,1 h 3,5 L IP T G 250 L 5 h, r/ min 2 min,, 3,, OD = 11266,,,,,,,,,,,,, 21413, 1 h 3,, 4 :,, 4,, 4 h,, 4 Tab. 4 Effect of additiontime of melucer on 2glucanase ac2 tivity / h / (g/ L) / (U/ ml) ,,,, OD 0135,10 %,37,200 r/ min 3 h,od 110, 5, 6 5,, : > IP T G > >, : mmol/ L IP T G 10 mmol/ L, 24,
5 h, IP T G / L 5 Tab. 5 Results of orthogonal experiment / L / / h / (U/ ml) 1 A 1 5 B1 125 C1 24 D A 1 5 B2 250 C2 27 D A 1 5 B3 375 C3 30 D A 1 5 B4 500 C4 33 D A 2 10 B1 125 C2 27 D A 2 10 B2 250 C3 30 D A 2 10 B3 375 C4 33 D ,57 8 A 2 10 B4 500 C1 24 D A 3 15 B1 125 C3 30 D A 3 15 B2 250 C4 33 D A 3 15 B3 375 C1 24 D A 3 15 B4 500 C2 27 D A 4 20 B1 125 C4 33 D A 4 20 B2 250 C1 24 D A 4 20 B3 375 C2 27 D A 4 20 B4 500 C3 30 D K K K K R , 6,,, U/ ml, 4413 %, U/ ml, TB, TB 27, TB, 6 h, LB,, 10 h, U/ ml,, U/ ml, 19173,,,,,,,, 6 Tab. 6 Comparison of 2glucanase of pro2optimization and optimization IPT G / L / L / / h / (g/ L) / (U/ ml) / (U/ ml) ( ) ( )
6 2 : BL21 (DE3)2p ET28a ( + )2bgl / h 7 TB Tab. 7 Results of recombinant strain s expression in TB medium / (g/ L) / (U/ ml) / (U/ ml) ( / ) / % , LB p H 710, OD 0135, 10 %,37,200 r/ min 3 h, OD 110, mmol/ L IP T G 10 mmol/ L, 24 6 h,, U/ ml, 1112 g/ L, U/ ml, 6162, TB, 10 h, U/ ml, U/ ml, 19173, ( References) : [1 ],,. [J ]., 2002, 29 (2) : L I Yong2xian, GU Guo2xian, YU Zhong. The application of thermal2 2glucanases in mshing[j ]. Liquor Making, 2002, 29 (2) : (in Chinese) [2 ] Flint H J, Pherson E C, Bisset J. Molecular cloning of genes from rum inococcus flavefaciens encoding xylanase and (1, 321,4)2glucanase activities[j ]. J Appl Environ Microbiol, 1989, 55 : [3 ] Antoni P. Bacterial 1,321,42 2glucanases : structure, f unction and protein engineering[j ]. Biochimica et Biophysica Acta, 2000, 543 (1) : [4 ],,,. 21,321,42[J ]., 2005, 25 (3) : L Β Wen2ping,XU Zi2rong, L I Wei2fen,et al. Cloning and expression of (21,321,42glucanase gene f rom bacillus amylolique2 faciens[j ]. Chinese Journal of Veterinary Science,2005, 25 (3) : (in Chinese) [5 ],,,. 21,321,42[J ]., 2006, 36 (2) : CH EN G Zhi2jing,DEN G Xu,L U Ying2hua,et al. Optimization of recombinant escherichia coli fermentation medium affect2 ing 123,1242glucanase production[j ]. Industrial Microbiology, 2006, 36 (2) : (in Chinese) [6 ],,,. EGs2 2 [J ]., 2007, 15 (4) : L I Qing2qing,CH EN Qi2he,J IAN G Xiao2yan,et al. Optimization of recombinant eschreichia coli EGS2 fermentation codi2 tion affecting 2glucanase production and it s characteristics of enzyme [J ]. Journal of Agricultural Biotechnology, 2007, 15 (4) : (in Chinese) [7 ],,,. [J ]., 2006, 29 (10) : YAN G Rui, WAN G Shu2xiu, CH EN Zheng2yue,et al. Enhancement of expression efficiency and solubility of recombinant protein in gene engineering by a new mthod[j ]. Chinese Journal of Clinical Rehabililation, 2006, 29 (10) : (in Chi2 nese) ( :, )
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