固定化青霉素酰化酶在光-pH敏感可回用两水相中裂解青霉素G为6-APA  

Phase Transfer Bioconversion of Penicillin G into 6-APA by Immobilized Penicillin Acylase in Recycling Aqueous Two-phase Systems With Light-pH Sensitive Copolymers

作　　者：金科铭[1] 曹学君[1] 庄英萍[1] 储炬[1] 张嗣良[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《中国生物工程杂志》2007年第10期53-58,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(20474016)

摘　　要：在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,通过优化,在1%NaCl存在下,6-APA的分配系数可达5.78。催化动力学显示,达平衡的时间近7h,反应最高得率约85.3%(pH7.8,20℃)。较相近条件下的单水相反应得率提高近20%。在反应过程中,通过底物及产物的分配系数检测,发现底物分配系数变化不大,而产物6-APA及苯乙酸的分配系数发生很大变化,从而引起产物的得率变化。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。此外,采用固定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相。因此,在两水相中进行固定化酶的催化反应具有明显的优越性。形成两水相的高聚物PNBC通过488nm的激光照射或经滤光的450nm光源照射得到回收;pH敏感型成相聚合物PADB可通等电点4.1沉淀可实现循环利用,高聚物的回收率在95%～98%之间,按此回收率计算,聚合物可使用60次以上。Abstract Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB. Partition coefficients of 6-APA was found to be ： about 5.78, in the presence of 1% NaCl. Enzyme kinetic showed that reaction reached equilibrium at 7h or so. The 6-APA mole yields were 85.3% （ pH 7.8, and 20 ℃ ） and this value was about 20% higher than control in reaction of single aqueous phase buffer. Partition coefficient of penicillin G （Na） washardly changeable, while partition coefficient of product,6-APA and significantly changeable. Reason is due to Donnan effect of phase systems andhydropho change of partition systems, substrate, phenylacetate acid was bicity of products. The coefficients of products also affects bioconversion yield of products. In the aqueous two-phase penicillin G, products 6- APA and phenylacetate acid are biased in top phase, while immobilized penicillin acylase is, completely partitioned in bottom. Substrate, penicillin G enters into bottom phase, and it is catalyzed into 6-APA and phenylacetate acid, then the products enter into top phase. Finally, inhibition of substrate and products is removed to result in improvement of products yield. Moreover, immobilized enzymehashigher efficiency than immobilized cells and occupy smaller volume. Comparing with free enzyme, immobilized enzymehashigher stability, longer use life, completely partitioned in bottom phase and recycle. Bioeonversion in two-phase systems using immobilized penicillin aeylase showed outstanding advantage. The light- sensitive eopolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light,while pH-sensitive polymer PADB could be recovered by isoeleetrie point （ pH 4. 1 ） . The recovery of the two eopolymers was 95% -99%.

关 键 词：可回用两水相 光敏感聚合物 pH敏感聚合物 6-APA 固定化酶青霉素酰化酶 

分 类 号：Q599[生物学—生物化学]