Immobilization of whole-cell penicillin G acylase by entrapping within polymethacrylamide beads.

Abstract

Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide and N,N'-methylenebisacrylamide. A solution of monomer that was made up from methacrylamide and N,N'-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013-0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45 degrees C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.