中国生物工程杂志

The Key problem in application of aqueous two-phase systems is polymer recycling. The phase systems should be recycled and reused due to reasons of production cost and reducing environmental pollution. The most feasible way is that using environmental sensitive and reversible dissolution copolymers to form aqueous two-phase systems. In this study, 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) was hardly changeable, while partition coefficient of product, 6-APA and phenylacetate acid was significantly changeable. Reason is due to Donnan effect of phase systems and hydrophobicity of products. The change of partition coefficients of products also affects bioconversion yield of products. In the aqueous two-phase systems, substrate, 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 enzyme has higher efficiency than immobilized cells and occupy smaller volume. Comparing with free enzyme, immobilized enzyme has higher stability, longer use life, completely partitioned in bottom phase and recycle. Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage. The light-sensitive copolymer 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 isoelectric point (pH 4.1) . The recovery of the two copolymers was 95-99%.