BL21 (D E3)2p E T28a ( + )2bgl 2

28 2 2009 3 Journal of Food Science and Biotechnology 28 No. 2 Vol. Mar. 2009 :167321689 (2009) 0220250206 BL21 (D E3)2p E T28a ( + )2bgl 2,, 3, (, 214122) : 2E. coli BL21 (DE3)2p ET28a ( + )2bgl LB, p H 2 ;IP T G :p H 710, (OD600 0135) ( ) 10 %,37,200 r/ min 3 h,od 110, 01033 6 mmol/ L IP T G 10 mmol/ L,24 6 h, (336133 U/ ml), 1112 g/ L, 459132 U/ ml, 6162, TB,10 h,1 090131 U/ ml, 1 570183 U/ ml, 191 73, : ; ;;; 2 :Q 939. 97 : 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 214122, 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 :2008205201 :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 010336 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 45913 U/ ml, and 33613 U/ ml 2glucanase was fo und in supernatant liquor, was higher 61 62 times t han t hat of t he parent st rain. : (19642),,,, 3 :(19712),,,,,, Email : liqi @jiangnan. edu. cn In TB

2 : BL21 (DE3)2p ET28a ( + )2bgl 2 251 medium, t he values can achieve at 19173 (1 09013 U/ ml in supernatant and 1 57018 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 ( 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 ] 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. 1 11111 E1 coli BL21 (D E3)2 p ET28a ( + )2bgl, 11112 2( Sigma ),IPTG(BIO BASIC INC), 2( ), (BIO BASIC INC) 11113 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 11114 (g/ L) IP T G 20, 2180 112 11211 : 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, 11213 ( ) IP T G 5 h 10 000 r/ min 2 min,,,,, 600 nm OD ;10 ml,10 000 r/ min 2 min,,,, OD, OD 11214 25 ml LB 250 ml 110 ml,37,200 r/ min, 1 h 600 nm OD 11215 2 3,52 (DNS) :1 ml 40 p H 615, 2 1 mol 1

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: / 1 5. 0 125 24 4 2 10. 0 250 27 5 3 15. 0 375 30 6 4 20. 0 500 33 7 2 D : / h 2 OD Fig. 2 Curve of cell quantity and OD value 213 21311 p H p H 515 610 615 710 715 810 815, OD 0135, 5 %,37,200 r/ min 3 h, 5 L IP T G 250 L 5 h, 10 000 r/ min 2 min,, 3,p H 710,, p H 211 11213, 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 2131 2 0135 OD, 215 % 510 % 10 % 15 % 20 % 25 %,4 4, 10 %, 214 21411,,37,200 r/ min 3 h, 5 L IP T G

2 : BL21 (DE3)2p ET28a ( + )2bgl 2 253 250 L5 h,10 000 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) 1. 3 0. 100 0. 880 0. 000 98. 57 0. 94 2. 0 0. 328 1. 135 0. 005 168. 24 1. 87 3. 0 1. 266 1. 160 0. 010 240. 25 7. 48 4. 0 2. 992 1. 470 0. 010 110. 73 0. 47 5. 0 3. 650 2. 045 0. 005 35. 21 0. 70 4 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 0. 35 1. 250 0. 00 158. 89 0. 47 2 1. 07 0. 780 0. 47 198. 63 0. 94 3 2. 64 0. 180 1. 07 241. 65 4. 68 4 3. 55 0. 122 1. 13 232. 07 4. 91 5 3. 54 0. 123 1. 13 216. 17 0. 70 21412 1,1 h 3,5 L IP T G 250 L 5 h,10 000 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) 1 0. 680 0. 010 27. 73 0. 70 2 1. 055 0. 005 29. 13 0. 70 3 1. 270 0. 010 83. 84 2. 57 4 1. 325 0. 005 189. 52 2. 10 5 1. 285 0. 005 210. 32 2. 81 6 1. 235 0. 005 276. 72 0. 47 215,,,, OD 0135,10 %,37,200 r/ min 3 h,od 110, 5, 6 5,, : > IP T G > >, : 01033 6 mmol/ L IP T G 10 mmol/ L, 24,

254 28 6 h, IP T G / L 5 Tab. 5 Results of orthogonal experiment / L / / h / (U/ ml) 1 A 1 5 B1 125 C1 24 D1 4 145. 33 12. 63 2 A 1 5 B2 250 C2 27 D2 5 144. 86 0. 94 3 A 1 5 B3 375 C3 30 D3 6 127. 09 2. 81 4 A 1 5 B4 500 C4 33 D4 7 102. 08 1. 17 5 A 2 10 B1 125 C2 27 D4 7 205. 18 0. 94 6 A 2 10 B2 250 C3 30 D1 4 112. 60 0. 47 7 A 2 10 B3 375 C4 33 D2 5 104. 88 2,57 8 A 2 10 B4 500 C1 24 D3 6 266. 90 6. 08 9 A 3 15 B1 125 C3 30 D3 6 111. 66 4. 21 10 A 3 15 B2 250 C4 33 D4 7 115. 40 2. 34 11 A 3 15 B3 375 C1 24 D1 4 236. 04 0. 47 12 A 3 15 B4 500 C2 27 D2 5 221. 78 4. 91 13 A 4 20 B1 125 C4 33 D2 5 112. 83 2. 10 14 A 4 20 B2 250 C1 24 D3 6 250. 54 9. 35 15 A 4 20 B3 375 C2 27 D4 7 255. 45 3. 97 16 A 4 20 B4 500 C3 30 D1 4 160. 29 1. 40 0 0 27 5 0. 00 K1 519. 36 575. 00 898. 81 654. 26 2 672. 90 K2 689. 56 623. 39 827. 26 584. 35 K3 684. 88 723. 46 511. 64 756. 19 K4 779. 10 751. 05 435. 19 678. 10 R 259. 75 176. 05 463. 62 171. 84, 6,,, 336133 U/ ml, 4413 %,459132 U/ ml, 6162 2. 6 TB, TB 27, TB, 6 h, LB,, 10 h, 1 093131 U/ ml,,1 570183 U/ ml, 19173,,,,,,,, 6 Tab. 6 Comparison of 2glucanase of pro2optimization and optimization IPT G / L / L / / h / (g/ L) / (U/ ml) / (U/ ml) ( ) 5 250 27 5 1. 25 233. 00 1. 169 291. 25 ( ) 10 500 24 6 1. 12 336. 33 2. 572 459. 32 0 0 24 30 69. 35 0. 705

2 : BL21 (DE3)2p ET28a ( + )2bgl 2 255 / h 7 TB Tab. 7 Results of recombinant strain s expression in TB medium / (g/ L) / (U/ ml) / (U/ ml) ( / ) / % 2 2. 13 128. 73 0. 7 666. 02 9. 72 19. 33 3 2. 10 312. 02 3. 5 971. 76 23. 15 32. 11 4 2. 25 487. 89 8. 84 1 068. 98 6. 94 45. 64 5 2. 28 511. 62 3. 72 1 125. 69 17. 31 45. 45 6 2. 47 551. 17 4. 19 1 202. 08 10. 41 45. 85 7 2. 51 655. 33 5. 99 1 358. 52 3. 71 48. 24 8 2. 75 845. 15 12. 2 1 411. 34 9. 03 59. 88 9 3. 30 936. 86 3. 48 1 508. 33 5. 26 62. 11 10 3. 60 1 093. 31 17. 63 1 570. 83 2. 31 69. 41 11 3. 75 1 259. 25 2. 78 1 492. 13 6. 94 84. 39 79. 6 3. 65 3, LB p H 710, OD 0135, 10 %,37,200 r/ min 3 h, OD 110, 01033 6 mmol/ L IP T G 10 mmol/ L, 24 6 h,, 336133 U/ ml, 1112 g/ L, 459132 U/ ml, 6162, TB, 10 h, 1 093131 U/ ml, 1 570183 U/ ml, 19173, ( References) : [1 ],,. [J ]., 2002, 29 (2) : 81-83. L I Yong2xian, GU Guo2xian, YU Zhong. The application of thermal2 2glucanases in mshing[j ]. Liquor Making, 2002, 29 (2) : 81-83. (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 : 1230-1233. [3 ] Antoni P. Bacterial 1,321,42 2glucanases : structure, f unction and protein engineering[j ]. Biochimica et Biophysica Acta, 2000, 543 (1) : 361-382. [4 ],,,. 21,321,42[J ]., 2005, 25 (3) : 263-265. 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) : 263-265. (in Chinese) [5 ],,,. 21,321,42[J ]., 2006, 36 (2) : 26-30. 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) : 26-30. (in Chinese) [6 ],,,. EGs2 2 [J ]., 2007, 15 (4) : 708-712. 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) : 708-712. (in Chinese) [7 ],,,. [J ]., 2006, 29 (10) : 92-93. 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) : 92-93. (in Chi2 nese) ( :, )