重组大肠杆菌全细胞催化合成S-苷甲硫氨酸

Biosynthesis of S-adenosylmethionine by Whole Cell of Recombinant E. coli.

从大肠杆菌(E.ColiK12)基因组DNA中克隆出甲硫氨酸腺苷转移酶基因,构建了能高效表达甲硫氨酸腺苷转移酶的重组大肠杆菌E.ColiJM109(pBR322-MAT)。将重组大肠杆菌细胞用于催化合成S-腺苷甲硫氨酸(SAM)的研究中。实验发现,用φ(甲苯)=2%的水溶液对重组细胞进行通透化处理后,能大幅提高SAM的产率。探讨了底物浓度、温度、pH、反应时间以及菌体密度对反应转化率的影响。最佳反应条件为:底物浓度(ATP)30 mmol/L,反应温度35℃,pH=7.0,反应时间8 h,细胞密度80 g湿细胞/L反应液。在此条件下,底物ATP的转化率超过95%。

The methionine adenosyltransferase gene was cloned from the genome of E. Coli K12, and the recombinant E. Coli JM109 （pBR322 -MAT） strain, which can highly express methionine adenosyhransferase was constructed. The optimal reaction conditions for the synthesis of S- adenosylmethionine（SAM） by the recombinant cells were investigated. The SAM yield can be greatly improved by using the recombinant cells treated with 2% toluene （volume fraction, water solvent） as the catalyst. The optimal conditions for the synthesis of SAM were investigated as follows： substrate concentration（ATP） 30 mmoL/L, the reaction temperature 35 ℃, the pH of Tris-HCl buffer solution 7.0. The reaction time was 8 h. The cell density 80 g wet weight cells/L reaction solution. Under the above optimal conditions, the conversion rate based on the amount of ATP was above 95 %.