α-氨基酸酯水解酶突变体催化合成头孢丙烯

Enzymatic synthesis of cefprozil by mutant of α-amino acid ester hydrolase

目的通过α-氨基酸酯水解酶突变体合成头孢丙烯的工艺条件实验,获得相关实验数据,为推行头孢丙烯的绿色生产技术打下基础。方法通过定向进化对野生型α-氨基酸酯水解酶进行改造,用突变体酶催化合成头孢丙烯,考察了包括pH、温度、酶浓度、母核/侧链投料比及有机溶剂等多个因素对酶法合成头孢丙烯的影响。结果对野生型AEH酶进行分子改造,筛选出一株突变体。动力学数据分析表明,突变体酶比野生型更适合用于头孢丙烯的合成。在突变体酶催化合成头孢丙烯的过程中,体系最适合的pH为6.0;最适温度为30℃;底物浓度为50mmol/L;最适的母核侧链比为1:2;最适的反应体系为15%异丙醇-水溶液。结论相对于野生型的α-氨基酸酯水解酶,该突变体更适合用于头孢丙烯的合成,实验数据为头孢丙烯酶法合成的实际应用打下基础。

Objective Obtain the relevant experimental data through experiment of process conditions of synthesis of cefprozil by m-amino acid ester hydrolase mutants, to lay the foundation for the implementation of the green production technology of cefprozil. Methods Molecular transformation of the wild-type amino acid ester hydrolase through directed evolution, and synthesize cefprozil by the mutant. Investigated the influence to the synthesis of cefprozil by several factors including pH, temperature, enzyme concentration, nucleus/side chain feed ratio and adding an organic solvent. Results Obtain a mutant of AEH through molecular modifying and High Throughput Screening of the wild-type enzyme. The mutant enzyme is more suitable for the synthesis of cefprozil than the wild-type according to the analysis of kinetic data. We also found that during the synthesis of cefprozil by mutant enzyme, the most suitable pH for the system is 6.0, the most suitable temperature is 30~C, the concentration of substrate is 50mmol/L, the most suitable nucleus/side chain feed ratio is 1：2, and the most suitable organic system is 15% of isopropanol. Conclusion The mutant m-amino acid ester hydrolase is more suitable for the synthesis of cefprozil than wild-type enzyme. The experimental data lay the foundation for the practical application of the cefprozil enzymatic synthesis.