为了高效合成茶氨酸,本研究通过将荧光假单胞菌GS基因转接入pET32a质粒中,再将重组质粒转化到E. coli BL21中,构建了一种生物合成茶氨酸的基因工程菌。工程菌株经0.1βmmol/L IPTG,28℃诱导表达,湿菌体的酶活达到41.79 U/mg prot,大约是出发菌株E. coli BL21的126.64倍。工程菌催化L-谷氨酸钠和盐酸乙胺反应生成茶氨酸的产量达到6.2 g/L,其催化L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的能力较出发菌株E. coli BL21有显著提高。
An experiment on the construction of E. coli recombinant strain for theanine biosynthesis with GS gene embedded was reported. In this experiment, Glutamine Synthetase gene in E. coli BL21 was ligated with the pET32a vector in order to produce theanine. The ligation product was transformed to E. coli BL21 and the engineered E. coli strain was constructed successfully. The glutamine Synthetase was expressed with induction of 0.1 mmol/L IPTG in 28℃. The activity of γ-glutamyl transpeptidase of the engineered strain of fresh E. coil engineered strain reached 41.79 U /mg prot, and was 126.64 times higher than that of the original strain one and the yield of theanine from L-Gln and ethylamine was 6.3 mg/ml. The ability of theanine biosynthesis of recombinant was enhanced obviously compared with that of original E. coli BL21.
[1] Sadzuka Y, Sugiyama T, Hirota S.Modulation of cancer chemotherapy by green tea[J]. Clin Cancer Res, 1998, 4(1): 153~156.
[2] Sadzuka Y, Yamashita Y, Sugiyama T, et al. Effect of dihydrokainate on the antitumor activity of doxorubicin[J]. Cancer Lett, 2002, 179(2): 157~163.
[3] Yokogoshi H, Kobayashi M.Hypotensive effect of gamma-glutamethylamide in spontaneously hypertensive rats[J]. Life Sci, 1998a, 62(12): 1065~1068.
[4] 成浩, 高秀清. 茶叶悬浮细胞茶氨酸生物合成动态研究[J]. 茶叶科学, 2004, 24(2): 115~118.
[5] Matsuura T, Kakuda T, Kinoshito T, et al. Theanine formation by tea suspension cells[J]. Biosci Biotech Biochem, 1992, 56: 1179~1181.
[6] Matsuura T, Kakuda T, Sakane I, et a1. Effects of plant growth regulators and carbon sources on theanine formation in callus cultures of tea(Camellia sinensis)[J]. Biosci Biotachnol Biochem, 1994, 58: 2529~1521.
[7] 高秀清. 茶氨酸工业化生产途径的研究发展[J]. 潍坊学院学报, 2004, 4(4), 19~21.
[8] 钱绍松, 陈然, 刘毅, 等. 中试规模制备L-茶氨酸及其衍生物[J]. 精细化工, 2005, 22(11), 845~847.
[9] Yamamoto S, Uchimura K, Wakayama M, et a1. Purification and characterization of glutamine synthetase of Pseudomonas taetrolens Y-30, an enzyme useable for production of theanine by coupling with the alcoholic fermentation system of baker’s yeast[J]. Biosci Biotechnol Biochem, 2004, 68: 1888~1897.
[10] Yamamoto S, Uchimura K, Wakayama M,et a1. Cloning and expression of Pseudomonas taetrolens Y-30 gene ecoding glutamine synthetase:an enzyme available for theanine production by coupled fermentation with energy transfer. Biosci Biotechnol Biochem, 2006, 70(2): 500~507.
[11] Kumnada Y, Benson D R, Hillemann D, et al. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes[J]. Proc Natl Acad Sci USA, 1993, 90(8): 3009~3013.
[12] Prusiner S, Stadtman E R. The terminal quinol oxidase of the hypertherruophilic Archaeon Acidianus ambivalens exhibits a novel subunit structure and gene organization[J]. J Bacteriol, 1973, 179(4): 1344~1353.
[13] A1ef K, Burkardt H J, Horstmann H J, et a1. Molecular characterization of the glutamine synthetase from the nitrogenfixing phototrophic bacterium Rhodopseudomonas palustris[J]. Z Naturforsch Sect, 1981, C36: 246~254.
[14] Streicher S L, Tyler B.Purification of glutamine synthetase from avariety of bacteria[J]. J Bacteriol, 1980, 142(1): 69~78.
[15] Sampaio M J, Rowell P, Stewart W D P. Purification and some properties of glutamine synthetase from nitrogen fixing cyanohacteria Anabaena cylindrical and Nostoc sp[J]. J Gen Microbiol, 1979, lll(1): 181~191.
[16] Stacey G, van Baalen C, Tabita F R. Nitrogen and ammonia assimilation in the cyanobacteria: regulation of the glutamine synthetase[J]. Arch Biochem Biophys, 1979, 194(3): 457~467.
[17] 张雪雁, 李琳琳. 一种提取肠道细菌总基因组DNA的方法[J]. 新疆医科大学学报, 2007, 30(7): 722~724.
[18] J.萨姆布鲁克, D W, 拉塞尔. 分子克隆实验指南[M]. 黄培堂, 王嘉玺, 朱厚基, 等译. 北京: 科学出版社(第3版), 2002.
[19] 林智, 杨勇, 谭俊峰, 等. 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.
[20] 杨海君, 谭周进, 肖启明, 等. 假单胞菌的生物防止作用研究[J]. 中国生态农业学报, 2004, 12(3): 158~161.
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