[1]张锋,王期,李桂兰,等.溶氧反馈分批补料高密度培养枯草杆菌谷氨酰胺合成酶工程菌[J].南京师大学报(自然科学版),2010,33(02):96-103.
　Zhang Feng,Wang Qi,Li Guilan,et al.High Density Fed-Batch Culture of Escherichia coli BL21/pET28b-glnA With DO Feed-back Control of Nutrient Feeding[J].Journal of Nanjing Normal University(Natural Science Edition),2010,33(02):96-103.
点击复制

溶氧反馈分批补料高密度培养枯草杆菌谷氨酰胺合成酶工程菌()
分享到：

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第33卷
期数:
2010年02期
页码:
96-103
栏目:
生命科学
出版日期:
2010-06-20

文章信息/Info

Title:
High Density Fed-Batch Culture of Escherichia coli BL21/pET28b-glnA With DO Feed-back Control of Nutrient Feeding
作者:
张锋;王期;李桂兰;徐健红;洪素丽;岳玲;冯志勇;殷志敏;
南京师范大学生命科学学院, 江苏南京210046
Author(s):
Zhang Feng; Wang Qi; Li Guilan; Xu Jianhong; Hong Suli; Yue Ling; Feng Zhiyong; Yin Zhimin
School of Life Science,Nanjing Normal University,Nanjing 210046,China
关键词:
谷氨酰胺合成酶;  重组大肠杆菌;  分批补料培养;  高密度培养
Keywords:
G lutam ine synthetase;  recomb inan tE. co li;  fed -batch cu lture;  h igh cell dens ity cu lture
分类号:
TQ925
摘要:
为了建立枯草杆菌谷氨酰胺合成酶基因重组工程菌BL21/pET28b-glnA的高密度培养工艺,首先以摇瓶培养为基础,对影响工程菌生长及目的蛋白表达的因素如培养基、pH值、诱导剂浓度及时间等进行优化,再扩大至B IOTECH-5BG(5 L)自动玻璃发酵罐培养,利用溶氧反馈补料策略进行分批补料,并在培养过程中保持20%~50%的溶解氧,7.0~7.5的pH,以及1.0 g/L乳糖诱导12 h,成功地进行了工程菌的高密度培养,最终细菌干重达到48.1 g/L,目的蛋白的表达量占菌体蛋白总量的55%,粗提物酶比活为10.35 U/mg,整个补料过程细菌比生长率稳定的控制在0.1 h-1左右.
Abstract:
To investigate the optim a l h igh cell density culture conditions of recomb inant BL21 /pET28b-g lnA to produce recom b inant G lutam ine Synthetase ( GS) of Bac illus Subtilis, based on the exper im enta l resu lts obtained in shake flask cu ltivation, the effects of the composition o f the cu lturem edium, the range o f pH, induce r concen tration and induced tim e on the g row th o f bac teria and the expression leve l o f GS we re ana lyzed. Then the bacter ia we re transferred to B IOTECH- 5BG( 5 L) DO feedback fed ba tch cu lture system. By keep ing dissolved oxygen at 20% ~ 50%, pH at 7.0~ 7.5, cultu ring in Optim izedM9M edium and induc ing for 12 h in the presence o f 1 g /L lactose at 37℃ during fedbatch cu ltiva tion, we perform ed a three tim e repeated culture. Ce ll density of recomb inan t E. co li w as reached to 48.1 g /L under constant ( 0.10 h - 1 ) specific g row th rates. The express ion leve l o f recom binant prote in GS w as over 55% o f the to tal pro te in in E. coli. Specific ac tiv ity o f crude ex trac t enzyme w as 10.35 U /mg. A h igh expression and stab ilized cu lture process o f recomb inan t Glutam ine Synthe tase of Bac illus Subtilis has been established, wh ich founds the basis for further larg e sca le produc tion o f G lutam ine Synthe tase.

参考文献/References:

[ 1]殷志敏, 陈群英, 司马健, 等. 耐热嗜酸古细菌Su lfo lobus ac idocaldar ius谷氨酰胺合成酶的表达调控和性质研究[ J]. 生物化学与生物物理进展, 2003, 30( 5): 767-771.
[ 2]韩娜, 葛荣朝, 赵宝存, 等. 植物谷氨酰胺合成酶研究进展[ J]. 河北师范大学学报: 自然科学版, 2004, 28( 4): 407-410.
[ 3]Labow B I, SoubaW W, Abcouw er S F, et a.l M echanism s govern ing the expression of the enzym e o f g lutam ine m etabo lism?? g lutam inase and g lutam ine synthe tase[ J]. J Nutr, 2001, 131: 2 467-2 474
[ 4]Reeds P J, Burr in D G. G lutam ine and the bow el[ J]. J Nutr, 2001, 131: 2505-2508.
[ 5]Renn ieM J, Ahm ed A, Ehog ali S E, et a.l G lu tam inem etabo lism and transpor t in skeleta lm uscle and heart and the ir c linical relevance[ J]. J Nutr, 1996, 126: 1 142-1 149.
[ 6]Ka rinch A M, PanM, L in CM, et a .l G lutam ine m etabo lism in sepsis and infection[ J]. J Nutr, 2001, 131: 2535-2538.
[ 7]N ew sho lm e P.W hy is L g lutam inem etabo lism important to cells o f the imm une system in health, post in jury, surg ery or infec?? tion[ J]. J Nutr, 2001, 131: 2 515-2 522.
[ 8]Neu J. G lutam ine in the fetu and cr itica lly iII low b irth we ight neona te: m etabolism and m echan ism of action [ J]. J Nu tr, 2001, 131: 2 585-2 589.
[ 9]陈群英, 陈国安, 薛彬, 等. 基因工程酶法结合酵母能量耦联高效合成L-谷氨酰胺的研究[ J]. 生物工程学报, 2004, 20 ( 3): 456-460.
[ 10]Co rchero J L, V illaverdeA. Plasm id m a intenance in Escherich ia co li recom binant cu lture is dram atica lly, stead ily, and spec if  ically in fluncened by features o f the encoded pro te ins[ J]. B iotechno l B ioeng, 1998, 58( 60): 625-632.
[ 11]Jacques N, Gu illerez J, Drey fusM, et a.l Culture conditions d iffe rentia lly a ffect the transla tion of ind iv idua l E scher ichia co li mRNA[ J]. JMo l B io,l 1992, 226: 597-608.
[ 12]Sambrook J, Fr itsch E F, M an iatis T, et a.lM o lecular C lon ing: A Laborato ryM anua l[M ]. 3rd ed. NewYork: Co ld Spring H arbor Laboratory Press, 2001: 1 595.
[ 13]Korz D J, R inas U, H e llm uth K, et a.l Sim ple fed -batch techn ique for h igh ce ll density cultiva tion of E scher ichia co li[ J]. Journa l of B iotechno logy, 1995, 39: 59-65.
[ 14]Bender R, Janseen K, Resnickn, e t a.l B io chem ica l param ete rs of g lutam ine synthetase from K lebsiella aerogenes[ J]. J Bac  terio,l 1977, 129: 1 001-1 009.
[ 15]吴一凡, 张双全, 高秀玉, 等. 乳糖诱导pET载体表达重组蛋白的研究[ J]. 南京师大学报: 自然科学版, 2002, 25( 1): 89-93.

相似文献/References:

[1]刘顺谊,殷志敏.重组枯草芽孢杆菌谷氨酰胺合成酶蛋白在改良M9培养基中的诱导表达[J].南京师大学报(自然科学版),2007,30(04):74.
　Liu Shunyi,Yin Zhimin.Induced Expression of Recombinant Bacillus Subtilis Glutamine Synthetase in Optimized M9 Medium[J].Journal of Nanjing Normal University(Natural Science Edition),2007,30(02):74.
[2]刘俊红,刘顺谊,殷志敏,等.乳糖诱导大肠杆菌中重组谷氨酰胺合成酶的表达[J].南京师大学报(自然科学版),2006,29(03):66.
　Liu Junhong~.Expression of Recombinant Glutamine Synthetase in Escherichia coli Induced by Lactose[J].Journal of Nanjing Normal University(Natural Science Edition),2006,29(02):66.
[3]白 婧,吴桐思雨,王 期,等.利用谷氨酸棒状杆菌高效表达枯草芽孢杆菌的谷氨酰胺合成酶发酵生产L-谷氨酰胺[J].南京师大学报(自然科学版),2015,38(02):78.
　Bai Jing,Wu Tongsiyu,Wang Qi,et al.Glutamine Synthetase of Bacillus subtilis is Applied to Corynebacterium glutamicum Fermentation System to Produce L-Glutamine[J].Journal of Nanjing Normal University(Natural Science Edition),2015,38(02):78.

备注/Memo

备注/Memo:
通讯联系人: 殷志敏, 博士, 教授, 研究方向: 生物化学及细胞生物学. E-mail:yinzhimin@ njnu. edu. cn
更新日期/Last Update: 2013-04-08