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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 adenylyltransferase gene was cloned from E.coli K12 genomic DNA and recombinant E.coli JM109 (pBR322-MAT) was constructed to express methionine adenosyltransferase efficiently. Recombinant E. coli cells were used in the study of catalytic synthesis of S-adenosylmethionine (SAM). The experiment found that, with φ (toluene) = 2% aqueous solution of the permeabilized recombinant cells, can significantly improve the yield of SAM. The effects of substrate concentration, temperature, pH, reaction time and cell density on the reaction conversion were discussed. The optimal reaction conditions were as follows: substrate concentration (ATP) 30 mmol / L, reaction temperature 35 ℃, pH = 7.0, reaction time 8 h, cell density 80 g wet cells / L reaction solution. Under these conditions, the substrate ATP conversion rate exceeds 95%.