Heterologous production of Kluyvera citrophila penicillin G acyalse (KcPGA) was optimized in Escherichia coli (E. coli). Several factors, including the effect of different medium and culture performance were identified to be crucial for the enzyme overproduction. The KcPGA yield was significantly and conveniently increased by more than 2.4-fold in TB medium compared to that in LB medium, which need inducing by isopropylthio-beta-D-galactoside (IPTG). The results shown the lower temperature (28 degrees C) was more feasible for the overproduction of KcPGA compared to the higher temperature (37 degrees C). The highest enzyme activity was obtained at 28 degrees C in TB medium only when culture pH was closed to its original pH value and the basic condition was more optimal for the production of KcPGA than that of acidic condition. On the other hand, the higher temperature (37 degrees C) culture condition resulted in poor enzyme activity although the optimum growth temperature for most of the E. coli strains is approximately 37 degrees C. It was possible due to the limitation by translation or posttranslational steps of the heterologous production of KcPGA in E. coli. The results suggest that culture temperature and pH should be precisely controlled for overproduction of KcPGA in E. coli. (c) 2005 Elsevier Inc. All rights reserved.