生物技术通报 ›› 2019, Vol. 35 ›› Issue (6): 221-226.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0977

• 技术与方法 • 上一篇    下一篇

ATP合成菌株的构建及用于联合生产S-腺苷甲硫氨酸

江林林, 吴磊, 许海霞, 黄坚丽, 张永进, 徐期, 杨勇   

  1. 浙江海正药业股份有限公司,台州 318000
  • 收稿日期:2018-11-13 出版日期:2019-06-26 发布日期:2019-07-08
  • 作者简介:江林林,女,硕士研究生,研究方向:酶催化;E-mail:jiangll@hisunpharm.com

Construction of ATP Synthetic Strain and Its Application in the Production of S- adenosylmethionine

JIANG Lin-lin, WU Lei, XU Hai-xia, HUANG Jian-li, ZHANG Yong-jin, XU Qi, YANG Yong   

  1. Zhejiang Hisun Pharmaceutical Co. Ltd.,Taizhou 318000
  • Received:2018-11-13 Published:2019-06-26 Online:2019-07-08

摘要: 为降低S-腺苷甲硫氨酸的生产成本,构建了同时表达腺苷激酶、腺苷酸激酶和乙酸激酶3种酶的重组大肠杆菌菌株用于ATP的合成,并对ATP的转化条件进行了优化,优化后的反应体系为:腺苷30 mmol/L,乙酰磷酸二锂盐135 mmol/L,硫酸镁5 mmol/L,硼砂50 mmol/L,菌体2 g/L(湿重),反应液初始pH7.5,反应温度为35℃,反应时间为3 h,反应转化率可以达到99%以上。按照上述反应体系进行5 L放大,反应结束后再投入65 mmol/L D,L-甲硫氨酸和50 g/L(湿重)表达腺苷甲硫氨酸合成酶的重组大肠杆菌菌体,并补加15 mmol/L硫酸镁,转化18 h S-腺苷甲硫酸浓度能达到8.7 g/L,转化率达到72.5%。

关键词: ATP合成, 腺苷激酶, 腺苷酸激酶, 乙酸激酶, S-腺苷甲硫氨酸

Abstract: In order to achieve ATP production as well as reduce the cost of producing S-adenosylmethionine,a recombinant Escherichia coli strain expressing adenosine kinase,adenylate kinase and acetate kinase simultaneously was constructed for ATP synthesis. The conditions of transforming ATP were optimized,and the optimized reaction system was as:adenosine 30 mmol/L,acetylphosphate dilithium 135 mmol/L,magnesium sulfate 5 mmol/L,borax 50 mmol/L,E. coli strain 2 g/L(wet weight),initial pH 7.5,the reaction temperature 35℃,and reacting for 3 h,the conversion rate was over 99%. Based on the above result,the reaction system was enlarged in 5 L. After reaction ended,50 g/L(wet weight)recombinant E. coli strain expressing adenosylmethionine synthase,65 mmol/L D,L-methionine and 15 mmol/L magnesium sulfate were added to the reaction system,the concentration of S-adenosylmethionine was up to 8.7 g/L and the conversion rate reached 72.5% after 18 h transformation.

Key words: ATP synthesis, adenosine kinase, adenylate kinase, acetate kinase, S-adenosylmethionine