The conversion of benzyl penicillin to 6‐aminopenieillanie acid using an insoluble derivative of penieillin amidase

Abstract

SummaryPenicillin amidase was extracted from Escherichia coli ATCC 9637, grown on phenylacetic acid and glutamate, and purified by fractionation with streptomycin sulphate, ammonium sulphate and polyethylene glycol, followed by chromatography on DEAE-cellulose. The purification factor was 100-200X and the overall yield was about 357,. The enzyme was chemically attached to derivatives of cellulose and the kinetics of these insolubilized penicillin amidase preparations was investigated.

Application of the fluorescamine reaction with 6-aminopenicillanic acid to estimation and detection of penicillin acylase activity

“…were similar to those obtained from the Lineweaver-Burk plot. The Km obtained was lower than that previously reported by workers who were not able to use initial reaction rates (10). The activation energy between 15 and 43°C was found to be 54.05 kJ mol-1.…”

Immobilised enzymes

“…The overall operation costs of the immobilised enzyme process were reported to be about 60% of that of the conventional batch process using native enzyme. Furthermore, a process yielding 6-aminopenicillinic acid for the preparation of semisynthetic penicillins from penicillin using immobilised penicillin amidase is in operation [63,64]. Immobilised whole ceils entrapped in polyacrylamide gel networks are used on an industrial scale for the production of L-aspartic acid which involves passing a solution of ammonium fumarate through a column containing entrapped Escherichia coli cells [65].…”

Stability and stabilisation of penicillin acylase

“…However, the K M,app value for the soluble enzyme from E coli ATCC 9637 under similar conditions (pH 7.0 and 37°C) was found to be equal to 7.7 mmol dm À3 . 20 Consequently, as the Michaelis constant is a measure of the af®nity of the enzyme for its substrate (as K M,app decreases, the enzyme's af®nity for substrate increases), 21 these values could suggest that penicillin acylase has more af®nity for the synthetic substrate NIPAB than for penicillin G. Nevertheless, a direct comparison between these ®gures is not possible since they were obtained with different enzyme preparations.…”