The conversion of penicillin G into 6-aminopenicillanic acid (6-APA) is a very important reaction, 6-APA being the basic raw material for the industrial production of semisynthetic penicillins. Bulk 6-APA can be achieved either by chemical' or enzymatic hydrolysis. Each system has its advantage. However, because of improvements in immobilization techniques that promote stability and prolonged high activity, enzymatic hydrolysis has become a major alternative to chemical hydrolysis. The industrial route for 6-APA bioproduction was developed ca. 1960 and it can be assumed that almost all 6-APA is produced today by the immobilized enzymatic route.Numerous immobilization techniques for penicillin acylase have been tried on a laboratory scale. For instance, adsorption, crosslinking, covalent and physical attachment, and entrapment methods have been described.' However, more efficient procedures to obtain 6-APA are still being investigated and the immobilization of penicillin G acylase on a polysaccharide matrix, such as chito~an,~ by an economical and simple procedure could undergo an interesting biocatalyst .We have prepared different physical forms of chitosan (powder, particules, and beads), glyoxal reticulated or not, and used them as carriers to immobilize penicillin G acylase either by adsorption followed by reticulation with glutaraldehyde or by direct crosslinking to the matrix pretreated with glutaraldehyde. The first results of our experiments as well as the catalytic evaluation of the resins obtained are described in the present communication. MATERIALS AND METHODSPenicillin G and penicillin G acylase were a gift from Rhone-Poulenc; penicillin G acylase (0.25 U/mg protein) from E . coli was an acetone precipitate where no detectable p-lactamase activity was found (see below). Crab-shell chitosan was supplied by Rousselot (France); the degree of deacetylation was estimated by titrimetry4 to be more than 64%. Different reagents were synthetic grade products and were * To whom all correspondence should be addressed used without further purification: buffer A -0.005M phosphate buffer, pH 7.5, and buffer B -0.1 M phosphate buffer, pH 8 .00. Spectrophotometric measurements were made with a DU-8 Beckman spectrometer and the pH was regulated either with a Metrohm pH-stat or with a home-made regulation unit connected to a microcomputer (Apple IIe). Preparation of SupportsDifferent physical forms of matrices were prepared from the same chitosan solution (1 g chitosan dissolved in 15 mL of 5% acetic acid in water and filtered to remove insoluble materials). Powder was obtained by pouring dissolved chitosan into excess solution of 0.5M sodium hydroxide; it was collected by centrifugation and thoroughly washed with distilled water until neutrality.Particles were prepared at 25°C by addition of 0.1M sodium hydroxide to a stabilized (0.1-0.5% Tween SO) emulsion obtained by mixing the acetic acid solution of chitosan with cyclohexane (1 : 1). The particles were then washed in distilled water until neutrality and sized (0....