Production of heterologous Providencia rettgeri penicillin acylase in Escherichia coli

In this work, we constructed several expression plasmids for the production of Providencia rettgeri penicillin acylase (EC 3.5.1.11; PAC) in Escherich ia coli. DNA fragments containing the pac gene from P. rettgeri ATCC31052 w ere PCR-amplified and cloned in to the expression vectors so that the pac g ene expression was controlled by the tac or trc promoter system. The effect s of culture conditions, such as IPTG concentration, temperature, and carbo n source, on the native or heterologous expression were investigated. Among a selection of expression systems, JM109 harboring pUTKnPAC2601 gave the h ighest PAC activity and could be of interest for industrial application. Cu ltivation should be performed at a temperature ranging from 28 degrees C to 33 degrees C and the medium could be supplemented with glycerol. The host/ vector system offers an opportunity for high-temperature-oriented PAC produ ction, which is usually conducted at a low temperature. Volumetric PAC acti vity at more than fiftyfold (similar to 820 U/L) that of the native express ion in ATCC31052 (similar to 15 U/L) could be reached by optimization of th e host/vector system and culture conditions.