Abstract
Penicillin G acylase (Benzylpenicillin amidohydrolase, EC 3.5.1.11, PGA) is an industrial enzyme that catalyzes the deacylation reaction of penicillin G to give 6-APA (6-aminopenicillanic acid). The 6-APA is an important intermediate in production of many semisynthetic penicillins. In this study, fermentation of the recombinant E. coli HB101 (pGL5) for production of penicillin G acylase is the system to investigate the effects of temperature and plasmid stability on cell growth and penicillin G acylase production.From the literatures of penicillin G acylase production, incubating temperature is one of the key factors affecting the activity of the acylase. Therefore, we focused on the effect of incubating temperature, and designed a series of batch cultures at 28℃, 32℃, and 37℃, respectively. Thereafter, two temperature-shift experiments were carried out. One was 32℃ to 28℃ after 12 hours fermentation, and the other was 37℃ to 28℃. In the incubating temperature experiments, the results showed that higher temperature gave higher growth rate but less enzyme activity. This was similar to the previous results. The recombinant strain had the same properties as the parent strain. During 40 hours of fermentation period, the plasmid exhibited high segregational stability.Similarly, in the temperature-shift experiments, the plasmid also exhibited high stability. However, the activity of penicillin G acylase was affected by the temperature-shift. Before changing the temperature, the activity of penicillin G acylase exhibited similar to that in the previous temperature runs. After temperature shift at 12th hour, the results depended on degree of the temperature-shift. For instance, when temperature-shift was from 32℃ to 28℃ (the temperature decreased 4 degree), the activity of penicillin G acylase decreased 20% (from 380 U/g to 300U/g). When temperature-shift was from 37℃ to 28℃ (the temperature decreased 9 degree), the activity of penicillin G acylase decreased 70% (from 380 U/g to 120U/g). It should that the effect of temperature-shift on the activity of penicillin G acylase was irreversible.