Aqueous two-phase systems (ATPS) are potential industrial technology in bioseparation engineering and biocatalysis engineering areas. In industry, the main problem is that ATPS could not be recycled efficiently to result in a high cost and pollution of the environment. In this study, a new thermo-sensitive copolymer, poly(NIPA-co-BA) (abbreviated P-NB), has been synthesized by using N-isopropylacrylamide and N-butyl acrylate as monomers. It can form ATPS with another novel pH-sensitive copolymer poly(AA-co-DMAEMA-co-BMA) (abbreviated P-ADB). Based on the property of pH-thermo sensitivity, the copolymers could be recycled with over 97% recovery. Bioconversion of Penicillin G was carried out in the P-NB-P-ADB ATPS. In the systems, optimized partition coefficients of substrate, Penicillin G, products 6-APA and phenylacetate acid were found to be: 3.87, 3.54 and 1.76, respectively. In the way, substrate and products 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. The yield of product 6-APA could reach 95.4% and the value is higher 10% than in single aqueous phase. (C) 2010 Elsevier B.V. All rights reserved.