生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。

L-asparagine Production by Biotransformation Method

The novel biotransformation method is developed for preparing L-asparagine. Asparagine synthetase gene A (asnA) from Escherichia coli (E. coli) JM109 is amplified and transformed into E. coli BL21 (DE3) using molecular biological methods, and then the ASNA is expressed. After analyzing the ASNA activity, the constructed strain E. coli BL21 (DE3)/pET28a (+)-asnA is applied on conversion from L-aspartic acid to L-asparagine. The substrate and product are detected by PITC column derivatization-high performance liquid. The molecular weight of expressed protein is of about 37kDa by SDS-PAGE, which is consistent with the expected size. The enzyme activity is 1 786.6U/g wet cell. The conversion ratio of L-aspartic acid is 95.8%, the yield of L-asparagine is up to 126.5g/L, and the production rate is 15.81g/(L·h). E. coli BL21 (DE3)/pET28a (+)-asnA engineered strain is constructed successfully. Then it is used to catalyze the conversion of L-aspartic acid to L-asparagine, and ATP is not necessarily in the production process, which results in the lower cost and provides a novel green method for preparing L-asparagine.