研究了茶氨酸生物合成基因工程菌重组质粒pET32a-GGT的稳定性。结果表明,该基因工程菌在连续传代100代后,质粒的目的基因片断没有缺失,具有结构稳定性;但在无抗生素选择压力下,连续传代20代后开始出现质粒丢失的细胞,连续传代100代后18%的细胞含有正常质粒,因此,表现出一定的分裂不稳定性。该基因工程菌在优化培养基和优化条件下的整个发酵过程中,始终表现出良好的结构和分裂稳定性,γ-谷氨酰转肽酶的活性为4.64 U/ml,催化合成茶氨酸的产量为35.18 g/L。
The stability of the recombinant plasmid pET32a-GGT in the genetically engineered E. coli bacteria strain for the biosynthesis of theanine was in investigated this article. Result showed that the recombinant plasmid didn’t appear obvious gene deletion, showed structural stability after 100 generations. However, the plasmid is showed segregational instability, the plasmid-free cells appeared after 20 generations, and 18% cells was plasmid-harboring cells after 100 generation under no Amp selection pressure. It was structural and segregational stability under optimizing culture medium and condition during the fermentation process, with activity ofγ-GGT 4.64 U/ml and yield of theanine 35.18 g/L.
[1] 吕毅, 郭雯飞, 倪捷儿, 等. 茶氨酸的生理作用及合成[J]. 茶叶科学, 2003, 23(1): 1~5.
[2] 高小红, 袁华, 喻宗沅. 茶氨酸的研究进展[J]. 化学与生物工程, 2004(1): 7~9.
[3] 李平, 宛晓春, 张正竹. 生物合成茶氨酸的研究进展及构建基因工程菌合成荼氨酸的可行性探讨[J]. 食品与发酵工业, 2004, 30(1): 71~75.
[4] 王贤波, 成浩, 王丽鸳, 等. 茶氨酸生物合成工程菌构建[J]. 茶叶科学, 2007, 27(1): 61~66.
[5] M Karbasi, T Keshavarz.Effect of dilution rate on the stability of TOL plasmid pTK0 in Pseudomonas putida PPK1[J]. Biotechnology Letters, 1997, 19(10): 985~988.
[6] 吴乃虎. 基因工程原理第2版(M). 北京: 科学出版社, 1998.
[7] Nayak D P, Vyas V V.Improved stability and expression of a recombinant shuttle plasmid in Escherichia coli during fedbatch cultivation[J]. World journal of Microbiology, 1999, 15: 65~71.
[8] Xiaoli Zhang, Zhaojie Xia, Binxia Zhao.Enhancement of plasmid stability and protein productivity using multi-pulse, fed-batch culture of recombinant Saccharomyces cerevisiae[J]. Biotechnology Letters, 2002, 24: 995~998.
[9] GC Gurakan, A. Bayindirli, ZB Ogel, et al. Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis[J]. World Journal of Microbiology & Biotechnology, 1998, 14:293~295.
[10] 邱英华, 沈益, 王玉海, 等. Cecropin-X发酵过程中工程菌质粒稳定性的研究[J]. 微生物学报, 2004, 44(3): 390~392.
[11] J 萨姆布鲁克, D W拉塞尔. 分子克隆实验指南第3版[M], 黄培堂, 王嘉玺, 朱厚基, 等译. 北京: 科学出版社, 2002.
[12] 张泉, 薛方明, 于可响, 等. 质粒pcDNKLYZ在发酵过程中的稳定性[J]. 农业生物技术学报, 2006, 14(1): 113~116.
[13] 林智, 杨勇, 谭俊峰. 茶氨酸提取纯化工艺研究[J]. 天然产物研究与开发, 2004, 16(5): 442~447.
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