Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 106 and, presumably, cross-linked protein-polymer aggregates of MW > 107. Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 M) was found to be slightly higher than that of the free enzyme (12 M), and evaluation of Vmax testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from D-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of -lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).