青霉素G酰化酶突变体酶催化合成顺式头孢丙烯工艺优化

Process optimization for enzymatic synthesis of cis-cefprozil by a mutant penicillin G acylase

头孢丙烯作为第二代头孢菌素类抗生素,广泛应用于治疗敏感菌所致的上、下呼吸道感染,皮肤和皮肤软组织感染.酶法合成头孢丙烯与化学法相比更加绿色环保.本研究利用来源于大肠杆菌的青霉素G酰化酶(EC 3.5.1.11)野生型WT、突变体βF24A和αF146Y/βF24A合成顺式头孢丙烯(cis-cefprozil).通过比较不同突变体催化顺式头孢丙烯的合成效率,发现突变体βF24A具有较高合成活性及低水解活性.以顺式7-氨基-3-丙烯基-4-头孢烷酸(cis-7-APRA)和对羟基苯甘氨酸甲酯(D-HPGME)为底物合成顺式头孢丙烯.基于水相体系中合成条件优化,最适温度为25℃,最适pH为6.0,底物配比M_(D-HPGME):M_(cis-7-APRA)=3:1,酶用量2.6 U/mL,在此最佳条件下转化率可高达99%.突变体βF24A的固定化酶合成顺式头孢丙烯的转化率为99%,固定化酶连续使用60批次后,活性仍保持52%.本研究对突变体的筛选和酶催化条件的优化,为酶法合成顺式头孢丙烯奠定了基础.

Cefprozil is a second generation cephalosporin antibiotics widely used in the treatment of upper and lower respiratory tract infections as well as skin and soft tissue infections caused by sensitive bacteria. Because of the great advantages of biocatalysis over the conventional chemical synthesis by avoiding toxic reagents, solvents and harsh reaction conditions, it is desirable to develop a feasible enzymatic synthesis of cefprozil. In this work, we chose the key sites flF24. αF146 of pecicillin G acylase from Escherichia eoli for site-directed mutagenesis. By comparing the synthesis efficiency of WT, βF24A and aF146Y/βF24A, the obtained mutant flF24A which has high synthesis/hydrolysis ratio was used in the enzymatic synthesis of cis-cefprozil from cis-7-APRA and d-HPGME. Based on the aqueous-phase synthesis system, the yield was dependent on the multiple factors. The most suitable temperature and pH was 25 ℃ and 6.0, respectively, the optimum molar ratio of D-HPGME/cis-7-APRA 3：1 and the enzyme loading 2.6 U/mL. At the optimized conditions, 99% yield was attained with 100 mmol/L of cis-7-APRA. The maximum yield was 99% with immobilized flF24A at the optimized conditions with 100 mmol/L cis-7-APRA. Yields remained 52% after 60 consecutive batch reactions. This paper studies on the selection of mutants and optimization for enzyme catalysis conditions, which provides useful information for enzymatic synthesis of cis- cefprozil.