重组a-氨基酸酯水解酶合成头孢曲嗪

Synthesis of cefatrizine by recombinant a-amino acid ester hydrolase

摘要:

为了探索酶法合成头孢曲嗪的产业化工艺路线，从红纹黄单胞菌Xanthomonas rubrillineans中克隆a-氨基酸酯水解酶基因全序列，转化入大肠杆菌中表达。以头孢曲嗪的合成转化率为指标，分别考察纯化的重组a-氨基酸酯水解酶合成头孢曲嗪的最适温度、最适pH和最佳底物摩尔比。经聚丙烯酰胺凝胶电泳分析，重组a-氨基酸酯水解酶的单体分子量为70 kDa。催化合成头孢曲嗪的最适pH为 (6.0±0.1)，最适温度为36 ℃。底物浓度约为7-ATTC 30 mmol/L、HPGM×HCl 120 mmol/L，酶用量22 U/mL时，头孢曲嗪的转化率达到64.3％。结果为优化酶法合成头孢曲嗪的产业化工艺奠定了基础。

Abstract:

To explore the enzymatic route of cefatrizine synthesis, a-amino acid ester hydrolase (AEH) gene was cloned from the whole genome of Xanthomonas rubrillineans, and expressed heterologously in Escherichia coli BL21 (DE3). The effects of temperature, pH and substrates’ molar ratio upon the transformation yield of cefatrizine by purified recombinant AEH were investigated. The monomer of AEH was determined as 70 kDa by SDS-PAGE. The optimal pH and temperature reaction were (6.0±0.1) and 36 °C for cefatrizine synthesis. The transformation yield was 64.3% under 36 °C, pH (6.0±0.1), when the concentrations of two substrates were about 30 mmol/L (7-ATTC) and 120 mmol/L (HPGM×HCl), respectively, and the enzyme consumption was 22 U/mL. The results pave the way for optimization of the industrial enzymatic synthesis of cefatrizine.