Maintenance of penicillin G acylase expression by B. megaterium: Preservation methods and activity recovery

Abstract

This work reports the influence of different culture preservation methods on the production of penicillin G acylase (PGA) by Bacillus megaterium ATCC 14945. The initial stock culture, presenting PGA activity of 97 IU 1 -1, was preserved for two years using different procedures: monthly subculturing and storage in refrigerator (S), freeze-drying using skim milk 10%, plus inositol 5% as cryoprotectors (L1), freeze-drying using sucrose 7%, plus peptone 7% (L2), and freezing with glycerol 10% (F). After cultivations at standard operational conditions, different values of enzyme activity were obtained: 56 IU 1-1 for monthly subculturing (S), 15-41 IU 1 -1 for freeze-dried cells and frozen spores. All the tested methods have failed in preserving the PGA expression. Among all tested cultures, S presented the highest specific activity, and was used to prepare a standard inoculum in cryovials (-50°C, frozen spores in a 20% glycerol solution). By cultivation of this inoculum under different conditions, it was found that PGA activity raised to 128 IU 1-1 when 0.4 g 1-1 of salts was added to the medium.